Polyclonal Antibodies

In the past few decades immunological methods utilizing antibodies have become indispensable research tools in cell biology. They have broad application and they are successful largely due to high specificity, cross reactivity, affinity and sensitivity. The major advantage with antibodies is that they can be induced against virtually any desired macromolecule if they are presented to the immune system in an appropriate way. Immunoglobulins perform the task of eliminating foreign molecules that have invaded an individual. They are water soluble glycoproteins and comprise about 20% of total protein in blood serum with a concentration of 15-20 mg/ml. All immunoglobulins are composed of two heavy chains and two light chains linked by interchain disulphide bonds. The two major immunoglobulin classes found in serum are IgG (75%) and IgM (10%). The variable region contains antigen antibody binding sites and consist of approximately 120 amino acids at the N terminus of both the heavy and light chains. The process by which an individual can generate different variants of immunoglobulins has been clarified to large extent. Antibodies are secreted by plasma cells that arise from B lymphocytes. Each individual contains many different subtypes of B cells (10⁶-10⁸), as immunoglobulins with different antigen specificities can be generated. Most proteins are complex antigens containing two or more epitopes (antigenic determinants) that will cause stimulation of different B lymphocytes clones and resulting response is polyclonal antibodies.

Polyclonal antibodies (pAbs) are those antibodies which are produced in the body by diverse B cell lineages on the contrary to the monoclonal antibodies which come from the lineage of a single cell. They are a group of molecules (immunoglobulins) that binds to a specific antigen based on the identification of different epitopes. These types of antibodies are of extreme importance in a wide number of applications. Polyclonal antibodies are regarded as heterogeneous in nature and therefore has the ability to bind to more than a few diverse epitopes of an antigen. Even though the use of the polyclonal antibody decreases specificity and thereby possibly adding up to non-specific reactions, in case of successful binding to a target antigen the polyclonal antibody is observed to be more proficient in the action. It is used in virtually all disciplines of the sciences to identify and quantitate individual molecular species, The speed and ease with which polyclonal antibodies can be generated makes them invaluable reagents for the research community.Thereby the polyclonal antibodies production is and will be an important area of research practice involving the use of vertebrate animals.

Polyclonal Antibodies Versus Monoclonal Antibodies

Most of the mammalian systems are known to be constituted by lymphocytes having clonal populations of roughly one thousand which is characterised based on the antigen-receptor target specificity. The diversity in the population confers the advantage of the generation of specific immune responses to a number of immunogens ranging from proteins, saccharides, peptides and bacteria/virus. A wide range of antibody production takes place at the lymphoid organs/tissues as and when stimulation by B-cells are provided. One antibody is able to bind to a specific epitope on the antigen and a polyclonal humoral reaction initiated by the whole group of polyclonal antibodies increase the binding capacity to a foreign antigen and thereby provides immunity to the organism against the pathogen. The first B-cell hybridomas were constructed by Köhler & Milstein via a fusion method utilising the Sendai virus that allows the immortalisation of lymphocyte. Subsequently, there were efforts towards the development of efficacious fusions by implementing other reagents like polyethylene glycol. The hybridoma technique based on monoclonal antibody production leads to the formation of hybridoma cell clones resulting from a single B lymphocyte making them identical and imparts specificity towards only one particular epitope. However, in some scenario where due to some unknown reasons like mutations if the antigen site is changed the extremely specific monoclonal antibody will fail to bind. This is why monoclonal antibodies are sometimes used in combinations to increase chances of successful binding. On the contrary, due to the reason that polyclonal antibody’s specificity is governed by the grouping of a number of clones and thereby providing them the ability to bind to different antigenic sites, the binding of the polyclonal antibodies may not be significantly affected by the change of antigenic sites due to mutations. Therefore, when it is required to be chosen between polyclonal or monoclonal antibodies one has to decide on the basis of the application and the time that can be devoted for the process as well as the budget constraints involved in the process. Usually, the monoclonal antibody production takes a considerable amount of time upto 3 to 6 months with the involvement of cell cultures technique along with immunisation of animals but the induction of production of polyclonal antibody is comparatively faster and can be completed in a stipulated time of approximately 4–8 weeks. The hybridoma that is immortalised becomes an unlimited source of quality antibody. In many cases, the polyclonal antibody sera may differ from batch-to-batch because it is generally produced in high concentration and subjected to dilution before use. Nevertheless, a polyclonal antibody can be produced in a short interval of time with a less amount of cost involved which makes them more convenient for application.

The laboratory animals are immunised for induction of a humoral immune response as a general procedure for the production of antibodies. This necessitates the significant use of animals without proper census as scientists across the disciplines require antibodies for different purposes. Most often there is a lack of sufficient knowledge of the procedures involved in immunisation and production of antibodies involving animals. Hence, there is a requirement to establish guidelines for the above processes. Commonly used species for production of antibodies involves mice and rabbits wherein mice are preferred for monoclonal and rabbits for polyclonal antibody production. Importantly, procuring animals which can produce specific antibodies and are devoid of any pathogenic infection will guarantee that the could be animal devoted towards eliciting an anticipated immune response against the target immunogen presented. The rabbit is generally preferred for the production of polyclonal antibody to a particular immunogen as it is proficient in escalating a concurrent immune response towards several immunogens and in turn generating an end product with multiple antigens. This approach also allows for conservation of resources and time along with improving competence. However, it must be ensured that the multiple antibodies produced should not hamper the isolation and the subsequent use of specific one among them via affinity purification practices that are mandatory for the technique to be successfully applied. The rabbit depicts advantages over other vertebrates in terms of suitable body size and offers convenience in reaching out the marginal ear vein and the central auricular artery that in turn simplifies the procedure for collection of a large volume of blood. Moreover, the rabbit has the ability to elicit a first-rate immune response to a number of antigens and is bestowed with the existence of only one type of isotype of the primary Immunoglobulin G. Also, for the production and purification of immunoglobulins from rabbit there is an elaborate volume of information existing which in turn simplifies the procedure. Considering animal protection and welfare some of the procedures of immunisation are under perusal. For instance, most of the adjuvants applied to augment the immune response, in turn, maximising the antibody production are identified as potential causes of discomfort in animals arising due to inflammation and pain. Keeping this in mind, some of the countries as well as organisations/academic institutes have framed guidelines on the route as well as the number of times administration is executed along with the maximum volume of injection permitted. These guidelines are aimed at ensuring proper immunisation protocols are implemented and generation of reliable immunological outcomes with minimum distress for animals. The immunisation protocol followed can have a considerable impact on the end results as well as animal welfare.

Selection of host

Polyclonal antibodies have been raised in numerous species including mice, rats, hamsters, guinea pigs, rabbits, goats, chickens, horses, donkeys, cattle, sheep, and even emus. The use of the resulting antibody decides the choice of host. If a large volume diagnostic product is required or extensive research utilizing the resultant antiserum is the objective, immunizing mice is not a viable approach because of the extremely small volume of serum that is produced. On the other hand, if the antiserum is to be used for the analysis of a dozen western blots, it doesn’t make much sense to immunize a horse or a cow, which can provide several liters of antiserum from a single bleeding. For small volumes of antiserum (i.e., <100 ml), the rabbit is the most common species for polyclonal production. Goats and sheep are the species of choice for large-scale antiserum production. One other consideration regarding the host is the number of animals required for immunization. Several animals should be used for any immunization program and the animals should be assessed separately to identify those that provide the desired antibodies. With goats or rabbits, a minimum of two animals should be used, with three to four preferred where antigen availability is not a great concern. For mice and rats, groups of ﬁve are commonly used.

The other related issues are the source of the antigen and the expected immunogenicity in a particular host species. Most proteins with molecular weights greater than 6,000 daltons are immunogenic to some degree. Small haptenic molecules can be rendered immunogenic for most species by conjugation to an appropriate carrier molecule, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or ovalbumin (OVA). These carrier proteins provide the T-cell epitopes necessary for a successful immune response. Unfortunately, it is not possible to determine prior if a given protein will be immunogenic or not.

Types of immunogen/antigen

A type of molecule that is bestowed with the capacity of generation of an immune response when present inside an animal through injection is attributed as an immunogen. They supposedly have a molecular weight in the range above 10000 and are mostly molecules like polysaccharides, glycoproteins, lipoproteins polypeptides and glycolipids as these are well known to be present on pathogenic surfaces. Molecules that contain one or more than one sites on their surface against which antibodies are produced and can bind to them are known as immunogens. All kinds of immunogens can be called as antigens excepting some which can harbour a specific epitope but due to their less molecular weights (<10000) they remain incompetent of bringing about an antibody response. In these cases, antibodies against such molecules can be produced by injecting them in presence of haptens which develop immunogenicity when in the bound state with carrier complexes.

Thymus-Independent Antigens

The antigens that are able to induce a humoral antibody response through the direct activation of B cells devoid of the participation of CD4 T helper cells are known as T-independent antigens. They are mostly comprised of molecules accompanying pathogenic infections that include LPS, ﬂagella proteins etc. They act by either binding to the receptors in the B-cells or induce cross-linking of multiple receptors of B-cells that consequentially activates the B cells. Out of these many are probable B cell mitogens that are non-specific in nature and commonly denoted as polyclonal B-cell activators. This type of a humoral immune response is independent of the antigen processing and presentation mediated via CD4 T helper cells making it qualify the criteria of an immune response than can be created in short interval of time. But this type of a response is limited and holds no mechanism for affinity enhancement of antibodies as predominantly IgM production takes place through this route. However, this kind of a response is highly beneficial in the first line of defence against the invading pathogens as the specific immune response induced by the association of CD4 T cells takes substantial time to develop. Though this kind of response is essential for protection against pathogens in case polyclonal antibody production it is of little importance.

Thymus-Dependent Antigens

The antigens that produce antibodies via the specific interaction of antigen with B-cells, APCs and CD4 T cells in the in the secondary lymphoid tissues are the thymus dependent antigens. In this case, the antigen is processed and presented to the specific CD4 T-cells together with the MHC class II molecules which results in the initiation of specific helper T-cells towards the antigen. The B-cells then interact with the specific T helper cells in the areas consisting of T-cells in the secondary lymphoid tissue and gets activated. As a consequence of this, there is a rapid proliferation of B lymphoblasts in the specific region of the lymphoid tissue. These proliferating B lymphoblasts transfer and develop to form the plasma cells that are responsible for antibody production. The other part of the B lymphoblasts moves towards increasing the antigen affinity and continue to produce the plasma cells giving more antibody and memory B-cells that are supposed to exist for a longer time.

Dosage of immunogen

The doses of immunogen highly contribute to the enhancement of antibody response level (quantity) as well as improves antigen-antibody affinity (quality). Introduction of lower doses of immunogen may result in inadequate immunoglobulin production. But at the lower immunogen dose, memory B-cells production is activated by a subsequent immunogen exposure. The outcome of excess doses of immunogen is a yield of low-affinity antibodies or the development of tolerance having no production of antibody. Numerous researches have been conducted where comparative studies have been made between antibody response and varying doses of immunogen thereby revealing prominent variations. In a comparison study conducted by a group of scientists where the antibody response was compared with the varying doses of dinitrophenylated bovine γ-globulin (DNP-γG) at a range of 0.05–50mg blended in Freund’s Complete Adjuvant (FCA). The entire study conducted in rabbits suggested the optimum response of antibody to be at a dose concentration of 0.5-mg. When Immunized at a dose concentration of 5 and 50 mg, there observed a higher initial antibody response at 13 days post immunization. But during 41 days post-immunization the dose concentration at 0.05-mg elevated the response of antibody three times higher. Another study conducted by researchers it was recommended that upon immunizing guinea pigs via DNP-bovine serum albumin (DNP-BSA) at a dose concentration of 50µg or 1mg, there observed a quantitative increment in antibody response at a time period of 2 weeks. At a time duration of 2 months post-immunization, an enhanced antibody affinity was observed in those animals after being immunized with DNP-BSA at a dose concentration of 50µg. An attempt was made by a group of researchers where they have aimed to carry out enzyme immunoassay by developing antibodies against viomycin. By their study, the optimized initial doses of immunisation for FCA as the adjuvant, Freund’s Incomplete Adjuvant (FIA) and aluminium hydroxide (alum) were 10 µg, 200µg and 10µg respectively. The approach to optimize immunogen dose is complicated which involves various parameters. The event of immunogen dose optimization is governed by various notable factors such as specificity of immunogen used (immunogenicity, purity, pH, contaminants, etc.), the adjuvant introduced, a number of immunizations and its frequency, and immunization route. When proteins which are highly immunogenic are used in conjugation with robust adjuvants the optimum concentration of immunogen dose would be as lower as 10 µg. On the other hand, when proteins which are less immunogenic such as small peptides, or polysaccharides are used in combination with adjuvants which are less robust, there demand much greater doses in the generation of an optimal immunological response. Our primary interest lies in the production of functional polyclonal antibody for multi-purpose and not the explicit determination of optimal immunogen dose. The estimation of immunogen dose is usually carried out by long term skill and experience of the investigator, survey on literature that have detailed the data with similar compounds, and the suspected immunogenicity from the compound of interest. It is suggested that at a concentration of 100–250 µg of proteins contained in adjuvant, an elevated titre of antibody can be observed in rabbits (Cooper and Paterson, 2009; Harlow, 1988).

Route of immunization

Depending on the route of immunization of the desired target antigen/immunogen against which antibodies are to be generated its delivery and presentation vary effecting the class/type of immune response produced. Amongst all immunization routes, the ideal would be the one which would allow a number of antigen-presenting cells (APCs) to interact with the antigen followed by secondary exposure to lymphoid tissues through lymphatic as well as blood circulation. In addition to this, the site of injection should be chosen based on the fact that it should allow the minimal suffering of the animal in question due to inflammation and pain after exposure to adjuvant along with antigen. There is a description of widely available immunization sites like intralymph node, intrasplenic and intra-articular for polyclonal antibody production, however, these routes suffer from drawbacks that limit their successful use. Rabbits are devoid of true foot pads and therefore because of their substantial body weight the foot pad route of administration is out of the question in rabbits. Intraperitoneal injections too are generally observed to be accompanied by pain and discomfort in animals. Though intravenous injection is implemented for immunogens that are particulate in nature and also for the soluble immunogens in case of booster injections complications related to anaphylaxis stand a chance along with the appearance of lesions significantly in organs. Therefore, in case of rabbits, the suitable primary routes of injection for polyclonal antibody formation are intradermal, transdermal, subcutaneous and intramuscular. Each of the routes has their own merits and demerits thus the choice exclusively depends on the type of immunogen/antigen used, adjuvant as well as immunisation period. Going by the terms of technicality the intramuscular and subcutaneous injections are mostly favoured in the rabbit as in contrast to the intradermal route it also favours larger injection volumes in each site. Subcutaneous injections generally release the injected immunogen at a distance from the site of injection via fstulous tract formation. In rabbit subcutaneous injections on its dorsal back are generally performed as a mixture of immunogen and adjuvant and in reducing the possible problems/complications administration of several low volume injections at each site is practised. Most of the intramuscular injected immunogen-adjuvant mixtures are generally performed in a single site either in the quadriceps or in the thigh (biceps femoris). In case of the intradermal route, it offers advantages like minimising the diffusion, protection of antigen against rapid degradation and improvement in the depot effect even though its execution is challenging in nature. For efficient processing of the immunogen and widespread spreading of the stimulated immune cell populations in the lymph nodes contact with the Langerhans cells, dendritic cells and the professional antigen-presenting cells in high concentration is necessary. The intradermal route suffers from drawbacks such as the restricted volume of injection allowed at a particular site, episodes of pain associated with injection into a sealed space and the possibilities of production of ulcerative lesions. Transdermal gene gun mediated DNA vaccine plasmids delivery and jet injection technology is implemented currently for immunogen delivery into the skin epidermis. Once inside the targeted site, the introduced DNA undergoes active processing followed by the subsequent presentation of its proteins to immunological T-cells. Both methodologies permit the addition of adjuvants to the immunogen that potentially enhances immunological response with negligible side effects. Both routes for intradermal injection produces an immune response against DNA based immunogens. DNA immunization is considered advantageous than traditional protein based immunization due to the easy implantation of plasmid DNA at a lesser cost together with avoiding the requirement of generation and purification of specific peptides and proteins dedicated to immunization. DNA vaccination via transdermal route is evolving to a greater extent whose developments are complicated due to the prominent behavioural differences between species and injection sites Though promising approaches implemented during DNA vaccination are in their nascent stages of development and have restricted application in routine polyclonal antibody production.

Schedule of immunization

The production of polyclonal antibodies involves re-immunization or booster doses after intervals of time that is significantly different depending on the situation. The primary aim of the booster injection during immunisation is to reactivate the immune response and also simultaneously amplify the antibody levels. To have maximum action it is necessary that the booster dose is subjected after the effect of the initial immunisation has subsided that in turn allows for the assortment of antibody produced from the B-cells that have a higher affinity towards the antigen. With the maintenance of correctly scheduled booster injections doses, it is possible to stimulate the production of more amount of antibody with higher affinity via development of memory B-cells with higher affinity resulting. If the booster injection is administered right after the original immunization it is bound to uphold high levels of exposure to the immunogen, in turn, effecting the selection process of B-cells with higher affinity thereby weakening the possibilities of induction of an anticipated secondary antibody response. Like in the other cases of polyclonal antibody production this determination of an appropriate time gap in between booster dose and the primary immunisation is governed by a variety of factors like immunogen, adjuvant type, dose, route of injection applied which complicates the process even more. In case of an adjuvant with a depot forming ability, the antibody concentration reaches its peak levels after 2–3 weeks of primary immunization which consecutively shows a marked decline thereafter. Whereas in case of a depot forming adjuvant the antibody levels usually remain high for several weeks which depends on the adjuvant used and also the immunogen involved.

It should be noted that the characteristics of the immune response are changing during the early phase. The primary immunoglobulin response can be detected by typical serological reactions within 5 to 7 days following the initial exposure to the antigen. The antibody titer gradually increases for several days to 2 weeks and afterward begins to drop. The general shape of the primary response is a bell-shaped curve with an extended decay phase, but the exact shape of the response is inﬂuenced by many of the same variables mentioned previously. When booster injections are given, the immune response is characterized by a rise in antibody titer for a period of 10 to 14 days to levels much higher than the primary response. The decay phase is extended because more cells are involved in antibody production and the predominant class of antibodies produced during the secondary immune response is longer lived. The primary immune response is characterized by relatively high levels of IgM antibody, which has a half-life of 8 to 10 days. The IgG class of antibodies makes up the major proportion of the secondary response, and this class has a half-life of 25 to 35 days. Here too, the nature of the antigen plays an important role in determining the best strategy for generating a good immune response. For most analytical work, IgM antibodies are undesirable because they tend to produce more nonspeciﬁc binding than IgG antibodies. For this reason, it is advisable to utilize a protocol with at least three or four booster injections to maximize the IgG response. One note of caution should be mentioned: minor impurities in the antigen will begin to exert their inﬂuence on the immune response after multiple immunizations. Unless the immunogen is highly puriﬁed, there is a risk of inducing unwanted antibody responses during a prolonged immunization protocol. These undesirable responses can be eliminated by several methods that follow, but in some instances, these remedies are not practical. Keep in mind the fact that a polyclonal immune response is always changing, and that careful evaluation of the quantitative and qualitative aspects of the resulting antibodies is crucial to the generation of an optimal antiserum.

Collection of blood

While collection of blood samples it should be ensured that the animal is subjected to less pain and stress. For avoided complications arising from stress induced vasoconstriction and the welfare of the animal, it is necessary that the animals are habituated with the staff who will carry put the procedure. The conditions under which blood collection is to be executed should keep the animlas warm that would ensure proper blood supply and organic solvents are to be avoided for induction of vasodilation as they are known to be potential carcinogens. Preparation of plasma instead of serum from the blood is recommended during antibody production as it can substantially increase the fluid content. Blood collection should be done only from the recommended sites preferably through a route where the application of anaesthesia is not necessary. This, in turn, makes it favourable for choosing a species where blood can be collected from conscious animals thereby making rabbits and small ruminants more beneficial in contrast to mice. But keeping in mind for commercial production, workers mostly tend to apply sedatives for maintenance stress levels under control when accomplishing the quick collection of blood. Also, to enable rapid and smooth blood collection the needle size should be optimum and vacutainers can be implemented keeping in mind that the vein collapse should not happen. A type of needle called butterfly needles is generally preferred as it permits the motion of the head of the animal during sample collection. The volume of the blood collected each time should be below 15% of the total volume of blood and 1% of the total body weight of the animal and the rate of recurrence of blood collection should be between (1–4 weeks). General anaesthesia should be entertained during generally achieved exsanguination through heart puncture followed by displacement of the cervical region or euthanasia by anaesthesia overdose.

Assessment of Side-effects

Post immunisation it is important that the investigators monitor the animals for any signs of side-effects due to the immunisation regime for minimising the pain and discomfort. Some of the assessment techniques for analysing the uneasiness in animals are proposed and systems which include measuring the alteration in physical activity and behavioural changes for a specified time period. Regular routine check-up of animals should be done which comprises of observation of the over-all appearance, food and water consumption and monitoring of the injection site. When performing the assessment of side effects implementing new antigen adjuvant mixtures the tissues of the animals after the end of the experiment should be subjected to pathological investigation for comparison. It sis to be taken care that the pathological changes though present sometimes may not at all times be apparent during a clinical investigation which relie on the injection route like in case of intraperitoneal injection. Moreover, the pathological changes may not always be limited to the to the site of injection and can spread occasionally. Also, blood collection under anaesthesia may also lead to side-effects, although blood samples are to be preferably collected from animals without anaesthesia in many cases still require the process to be concluded in presence of anaesthesia. Animals should be checked after blood sampling as the bleeding may tend to continue in some cases and also to ensure wound closure and healing after the procedure for sampling is over. In case the blood is drawn from the ear artery of rabbit one must be careful to ensure that the artery is closed and there is no leakage which can be achieved by the compression of the artery for a period of time.

Developing polyclonal sera from immunized animals is well established and has been optimized for many antigens. To produce sera with desired properties over a long period of time animals must be immunized repeatedly. Therefore, preparation of large quantities of antigen is necessary, which may be costly and time consuming. Furthermore, production of polyclonal sera is complicated by the requirement of animal house facilities. Hybridoma technology has provided a solution but it also suffers from several disadvantages. However a new technology called recombinant antibody may overcome many of the problems associated with producing antibodies by immunization of animals or by hybridoma cultures. Genes of antibody fragments which may be expressed in E coli are developed either by sub-cloning antibody genes from hybridoma cells or by library selection utilizing phage display technology. Insertion of an antibody fragment gene into an appropriate expression vector and transformation into E coli provides a source of a well-defined reagent which can be sequenced and characterized. Bactria containing the construct can be stored at -70ºC and the reagent may be re-expressed reproducibly at any time. Bacterial cells are easier and less time consuming to handle than animal cells and require in comparison little specialized equipment. Two very important post translational modifications for complete immunoglobulin molecule are intermolecular disulphide binding and glycosylation, which bacteria do not carry out. So recombinant antibody technology deals only with the production of antibody fragments. The redox condition in the periplasmic space enables the appropriate folding and correct formation of disulphide bonds. Proteins are harvested from periplasm or culture supernatant. After synthesis of polyclonal sera or recombinant antibodies it needs to analysed for potency and further purification.

Potency of polyclonal protein

To develop high quality polyclonal sera it is very important to assess the progress of an immunization program. Because the immune response is constantly undergoing change, it is imperative to monitor both quantitative and qualitative aspects of the antibody response. The technique to be used for evaluating the antibodies developed depends on the ultimate use of antibody because a polyclonal antiserum that is ideal for ELISA assays may not work very well for immunoprecipitation or immunohistochemistry. The ELISA method is, by far, the most widely used for the initial evaluation of potency, or titer.

Enzyme -Linked Immunosorbent Assay

The easiest method for testing the resulting antiserum, is enzyme linked immunosorbent assay (ELISA). Most antigens can be detected when bound directly to the solid phase (typically an 8 × 12 96-well polystyrene plate). Antigen is allowed to bind passively to the plate overnight at 4°C (100 µl, 0.1 to 1.0 µg per well in carbonate buffer. Followed by washing 3-4 times using PBS-Tween 20. Then blocking solution containing excess protein (1 to 2% BSA, gelatin, or casein in PBS) 0.05% Tween 20 is added (150 to 200 µl) to eliminate the remaining protein binding sites and reduce non-specific binding. At room temperature it is incubated for 1 to 2 h. 3. Again washing 3-4 times with wash solution as mentioned in the first washing step. Primary antibody (i.e. the serum which is developed in the immunization protocol) is titrated across the plate (100 µl of a 1:50 dilution is a good starting point) and incubated for 30 to 60 min followed by washing of the plate to remove unbound antibody. Secondary (anti-species) antibody conjugated to horseradish peroxidase (100 µl per well, dilution depends on speciﬁc lot, needs to be in excess) is incubated for 30 to 60 min followed by washing of the plate to remove unbound antibody. Enzyme substrate (100 µl of 10 mg/ml OPD in citrate-phosphate buffer containing 0.1% H₂O₂) is added and incubated for 10 to 30 min, after which the reaction is stopped by the addition of sulfuric acid (50 µl). The plate is read in a microtiter plate reader, and the amount of color developed is directly proportional to the amount of primary antibody in the well. Using this technique, the relative titers of the various serums collected can be compared and ranked.

For small antigens and haptens, the antigen is conjugated to a different protein. This antigen conjugate is then coated on the solid phase and presents the antigen in manner similar to the immunogen, thereby increasing the potential for antibody–antigen interaction. It is best to use different conjugation chemistries for the solid-phase antigen and immunogen because some linker molecules elicit strong immune responses. If the same chemistry is used, much of the observed reactivity may be directed at the linker and not the hapten. The antigen can be biotinylated and bound to streptavidin-coated plates. This binds the antigen in an oriented fashion so that detection is relatively straightforward. One of the drawbacks to this ELISA format is that it requires the antigen to be bound to the solid phase. This can result in conformational changes in the antigen that reduce the afﬁnity of the antibody for the antigen. Conjugation of the antigen to an inert carrier protein and immobilization via biotin–avidin, as mentioned above, are two remedies for this. Another remedy is to utilize a “sandwich” assay format. The limitation to this approach is that you must have two antibodies that are directed at different epitopes and the antibodies must be derived from different species if the indirect method is to be used (anti-species conjugate), or one of the antibodies must be puriﬁed and labeled if the direct method is used.

Radial Immunodiffusion

This technique is commonly used to quantitate antisera directed against serum proteins. Because it relies on the formation of immune complex precipitates, it is not particularly useful for antisera raised against haptens. If a source of antigen is available for which the concentration is reliably known, this technique can provide an absolute titer of a polyclonal antiserum (mg antigen consumed per ml antiserum) This protocol assumes the use of 8-cm circular Plastic radial immunodiffusion (RID) plates with 4.6-cm center wells. Pouring 8 ml of agarose into this conﬁguration provides a layer 1.0 mm thick. Use of alternative conﬁgurations will require a different volume of agarose or a correction factor in the equation. In the protocol agarose is heated to 68°C, at this temperature it wouldn’t denature the antibody to be subsequently added. In a disposable mixing cup appropriate volume of antiserum is added and total volume is made to 10 ml using agarose. This combination is quickly mixed to produce a homogeneous solution. Then 8 ml of agarose/antibody mixture is added evenly into the RID plate. The use of a circular rotatory bed facilitates the even distribution of agarose. After agarose cools and solidifies holes are punched in the agarose to provide wells for the addition of antigen solution. This can be facilitated by attaching a piece of tubing and applying a slight vacuum. Distorted holes will result in inaccurate measurements so care should be taken while applying pressure. Pipet 2 µl of each antigen solution into separate wells. A minimum of three different concentrations should be used, and ﬁve standards seem to give consistently reliable results. The antibody–antigen reaction is allowed to come to equilibrium and incubated for a period of 24 h. However larger molecules, which diffuse more slowly (e.g., IgM), require additional time. The reaction can be monitored by reading the diameters of the precipitin rings at various times and selecting a time after which no further changes take place. The square of the diameter versus the antigen concentration is plotted. From the slope of the resulting line, the antiserum titer (T, mg/ml) can be calculated.

ELISA and RID methodologies provide measurements of potency. If highly puriﬁed antigens are utilized in those assays, it is possible to get a good estimate of the speciﬁc antibody titer, but the nature of the other antibodies present in the serum cannot be easily determined with those techniques. Instead, methods that employ a separation step for the antigen prior to introduction of the antiserum provide valuable information regarding the speciﬁcity of the antibodies.

Western blot

Western blots are performed in a three-stage procedure. Following separation of an antigenic mixture by SDS-PAGE, the proteins are transferred to a nitrocellulose or PVDF membrane in the second stage. At this point, the membrane-bound antigen mixture is treated in much the same way as an ELISA plate. The excess binding sites are blocked with a solution containing an irrelevant protein and some detergent. Primary antibody is diluted into the blocking buffer and incubated with the membrane. Following washing to remove excess antibody, secondary antibody conjugate is incubated with the membrane to allow binding to any primary antibody captured by the immobilized antigens. After another wash cycle, enzyme substrate is added to disclose where conjugate has been localized. The substrates used for Western blots differ from those used in the ELISA protocol because the ﬁnal colored product is insoluble and binds to the membrane wherever enzyme conjugate is found. This results in a pattern of colored bands indicating what molecular weight species in the antigenic mixture reacts with the primary antibody mixture. A complex pattern of reactivity indicates that either the antiserum contains undesirable antibodies or that the epitopes recognized by the antiserum are present on a heterogeneous mixture of antigens (e.g., degradation fragments). A single band indicates a monospeciﬁc antibody relative to the electrophoresed antigen preparation.

Immunoelectrophoresis

In this process, antibody diffuses into the gel and the antibody–antigen reaction is visualized by staining the resulting immunoprecipitates. It is a two-stage procedure. The ﬁrst stage separates the antigenic material in biological ﬂuids by their differential migration in an electric ﬁeld. The second stage of this technique is the immunological characterization of the separated proteins by the immunodiffusion procedure. This method is commonly utilized to evaluate the speciﬁcity of antisera raised against serum proteins. The protocol is as followed – Pipet 1 to 2 µl of bromphenol blue dye into well #1 and allow this to be adsorbed into the gel. Pipet 4 µl normal serum into well #1, and 4 µl of the appropriate antigens into the other wells. Fill the electrophoresis chambers with barbital buffer (the same volume on both sides to prevent capillary migration due to hydrostatic pressure). Place the slide into the electrophoresis chamber and make contact with the running buffer. Turn on the power supply and electrophorese the antigens at 170 volts (5 to 7 volts/cm) for 60 to 90 min or until the bromphenol blue tracking dye has migrated 5 to 10 mm from the end of the antiserum trough. Remove the gel from the precut trough and dispense 90 µl of antiserum into the appropriate troughs and place the slide in a humidiﬁed chamber for the immunodiffusion portion of the assay (18 to 24 h at room temperature). Direct analysis of the plate can be performed prior to staining and drying. This is recommended because faint immunoprecipitant lines can be abolished during the subsequent washing step prior to staining. This is accomplished by viewing the plate against a black background using a point source of light held behind the plate at an angle of approximately 45°. If staining is desired, the plate should be placed in normal saline for 24 to 48 h to leach out soluble, non-precipitated proteins. A brief soak in distilled or deionized water will remove excess salts prior to staining. Place the slide in the amido black stain for 10 to 15 min and remove excess stain with a brief rinse with distilled water followed by destaining in acetic acid, ethanol (methanol), water (10, 45, 45 by volume). After destaining the gel can be dried and mounted for a permanent record. Interpretation of the resulting immunoprecipitates can be aided by the use of known, speciﬁc antisera. A monospeciﬁc antiserum results in the generation of a single precipitin line while antiserum containing multiple antigenic speciﬁcities will produce multiple arcs.

Serum sample purification

Post production of Polyclonal antiserum needs purification as it contains antibodies that will bind many different antigens. Depending on the requirement purification strategy has to be decided. In some assay systems, impurity is not a problem because the speciﬁc antibody titer is high enough that simple dilution eliminates the interference. Or else the antigens recognized by the nonspeciﬁc antibodies are not present in the assay system. When this is not the case, there are approaches for the generation of a monospeciﬁc polyclonal antiserum:

afﬁnity puriﬁcation of the speciﬁc antibodies,
afﬁnity depletion of the nonspeciﬁc antibodies, leaving behind the speciﬁc antibodies.

The basic method involves the immobilization of the speciﬁc antigen on a solid phase (usually activated Sepharose-like beads) for afﬁnity puriﬁcation or immobilization of the nonspeciﬁc antigen(s) on a solid phase for the afﬁnity depletion method. Passing the antiserum over the immobilized antigen(s) results in binding of the appropriate antibodies, which can be recovered following elution (afﬁnity puriﬁed antibody) or discarded following elution (depletion methods). These procedures allow the generation of highly speciﬁc polyclonal antisera. There are many well-documented approaches to purifying antibody from a variety of source. The method of choice depends upon the antibody source, time available, cost considerations, and ﬁnal use of the antibody. One should begin by determining the use and ﬁnal level of purity required for the antibody generated in immunization process. Some form of puriﬁcation will be required in instances were the antibody will be used for immunoassay, immunotags, and as immunoafﬁnity reagents. Puriﬁcation is also required when background or nonspeciﬁc binding is observed in assay negative controls. Consider the degree of purity needed and how the method of choice will affect the ﬁnal yield, purity, and especially antibody activity. Purifying polyclonal antibody from ascites (1 to 10 mg/ml), sera (20 to 30 mg/ml), cell culture supernatant [static (50 µg/ml or bioreactor (0.1 to 10 mg/ml)] and what is the host species and isotype of the antibody, are important questions to be considered. The economical two-step methods are more commonly employed for purifying polyclonal antibody ﬂuids. Factors such as the effect the puriﬁcation method has on the antigen (Ag) binding afﬁnity and/or Fc region and the time and cost may also inﬂuence the method selected.

Clarification of sample

After collection of fluids (serum, ascites ﬂuid, and culture ﬂuid) containing antibody will be collected, pooled, and frozen for later puriﬁcation. Contaminating cells and cellular debris should be removed by centrifugation (1500 × g for 15 min) before the ﬂuids are pooled and frozen. In the case of ascites ﬂuid, it may also be necessary to remove lipids by ﬁltering the ascites through glass wool prior to puriﬁcation. Not only while storing it is very important to clarify sample upon thawing by centrifugation at 20,000 × g for 30 min before proceeding with any puriﬁcation procedure. Centrifugation will remove any ﬁnal cellular debris and protein/antibody aggregates that may have formed during the freezing and thawing process. A step ﬁltration through a 0.45-µm ﬁlter and then a 0.22-µm sterile syringe ﬁlter prior to puriﬁcation is also recommended.

Characterization

Quantitation of the total protein in the sample is an important ﬁrst step and can be accomplished using a protein assay (Bradford, Lowry, BCA) or by measuring the absorbance at 280 nm using a UV spectrophotometer (A280 × 0.8 = mg/ml). Next important thing is to check antibody activity or antibody titer in the unpuriﬁed sample as a point of reference for later evaluating the puriﬁed product. Finally, using capture ELISA kits can be used to quantitate immunoglobulin in a sample. The total starting protein concentration and the level of antibody reactivity at a given dilution or titer will provide a reference point for determining the yield and the potential for loss of antibody activity when puriﬁcation is complete.

Purification of IgG

One-step method using affinity chromatography

Protein G, A and L affinity matrix are used in this purification. They have affinity for binding to either constant region or kappa light chain. Protein G (30 to 35 kDa), isolated from β-hemolytic streptococci (group G) has a natural afﬁnity for the constant region of the antibody heavy chain. Recombinant forms of Protein G are also available and binding site for albumin is deleted which is originally present in native form. However Protein A afﬁnity matrix does not have a comparable binding afﬁnity or diversity in subclass binding that can be found with Protein G. Protein L, originally isolated from Peptostreptococcus magnus. This the most versatile of the three proteins, it has binding for the kappa light chain and thus binds to all antibody classes, including IgM. Protein L is especially useful for purifying Fab and single-chain variable fragments (ScFV). Protein L has also been used in purifying monoclonal antibodies from hybridoma cell culture medium containing bovine serum as it does not bind bovine antibodies. Protein L provides a better afﬁnity for purifying mouse IgG3 when compared to Protein G. Protein L is useful for purifying chicken IgG and IgY and porcine. This method of puriﬁcation involves a higher initial expense, with chromatography gels costing over $200 for a 2-ml volume, but the columns can be reused multiple times, reducing the long-term costs of puriﬁcation and is recommended for both polyclonal and monoclonal antibody puriﬁcation.

Antigen Antibody

In this method puriﬁed antigen is bound to a support matrix to be used in column chromatography for antibody puriﬁcation. This method requires milligram quantities of the puriﬁed antigen. This method increases the speciﬁcity of the puriﬁed product as it eliminates any other immunoglobulins that do not recognize the antigen. This is particularly useful for improving the speciﬁcity of polyclonal antisera or ascites ﬂuid. Because the antibody is eluted by changing the pH of the elution buffer this method is considered harsh and may lead to loss of antibody activity. Additionally, the antigen must be able to withstand the harsh elution conditions and have the ability to renature after equilibration so that immunoafﬁnity matrix can be reused. This method can be used for monoclonal antibody puriﬁcation, but antibodies with low afﬁnity to the antigen may wash off with the rest of the unbound proteins before the elution step.

Thiophilic Chromatography

Thiophilic absorbent is a modiﬁed sulfur containing silica bead that has a high afﬁnity to all IgG subclasses in a high-salt environment (e.g., 0.5 M K₂SO₄). This method provides a fast enrichment of both monoclonal (IgG) and polyclonal antibodies (IgG and IgY — chicken), but has poor IgM binding capacity. Puriﬁcation can be completed in one step and with a high yield and good purity (20 mg goat, pig, cow IgG/ml resin, 10 mg mouse IgG/ml resin). Antibody binding occurs through sulfone and bisthioether groups on the column with phenylalanine-phenylalanine and tryptophan/tryptophan groups on the protein. The antibody is eluted in a concentrated form in low salt without the adverse effects of low pH on antibody activity that sometimes occurs during immunoafﬁnity chromatography elution. This method is inexpensive. This column resin can also withstand pressures necessary to make it useful in HPLC columns, and the rapid binding kinetics allow for faster ﬂow rates.

Two -step methods

Caprylic Acid

Caprylic or octanoic acid, is a weak acidic buffer, and with the addition of short-chain fatty acids will precipitate most serum proteins except IgG. This method is inexpensive and a crude ﬁrst step for IgG puriﬁcation from serum or ascites. It is useful as an initial step in purifying IgG1, 2a, and 2b from mouse ascites, and not for IgM, Ig3 and IgA. Additional techniques such as ammonium sulfate precipitation or anion exchange chromatography are needed to further purify the antibody fraction.

Ammonium Sulfate Precipitation

This method is useful as a second step following caprylic acid precipitation negative selection method. Sometimes it is ﬁrst step prior to anion exchange chromatography for purifying antibody from serum and ascites ﬂuid. This method principally works on the basis that ammonium sulpfate dehydrates protein molecules. The high-salt concentration (i.e., small and highly ionic molecules) used in this method removes water molecules that interact with antibodies, thus decreasing the solubility of the proteins and leading to protein precipitation. Various concentrations of ammonium salt precipitate out different proteins depending upon their size and charge (as well as species of origin, ﬂuid type, pH, temperature, and the number and position of polar groups on the protein). Typically the clariﬁed antibody solution is mixed with a saturated ammonium sulfate solution to a 45% volume/volume ratio. Precipitation occurs for over a period of 2 h in the cold. The precipitate is resuspended in PBS and desalted via gel ﬁltration, dialysis against PBS, or ﬁlter centrifugation. The antibody solution is then concentrated. This is an inexpensive method to perform.

Anion Exchange Chromatography

This is a second step of purification and separates protein based on charge. Proteins electrostatically bind onto a matrix bearing the opposite charge. Typically, these bound proteins are eluted by a change in the pH or an increase the concentration of the buffered salt. Anion exchange resin [e.g., DEAE (dimethylaminoethyl) cellulose/Sepharose®)] can be used for positive (column chromatography) or negative (batch chromatography) selection of antibody. Antibodies have basic isoelectric points when compared to most of the proteins in serum. During the batch method, lowering the pH will prevent the antibodies from binding the resin, providing a negative selection for antibodies in batch quantities. An increase of the pH of the antibody solution will facilitate antibody binding to the column (positive column selection method). Initially, the antibody is puriﬁed using caprylic acid precipitation or ammonium sulfate precipitation. This method is inexpensive and approximately 2 ml of resin is required for 1 ml of serum. Conditions for negative selection are 70 mM sodium phosphate buffer (pH 6.3) for rabbit, 20 mM sodium phosphate buffer (pH 8) for human, and 20 mM sodium phosphate buffer (pH 7.5) for goat. Positive selection conditions for mouse and rat IgG binding occur at pH 8 and are then eluted in a linear gradient of 0 to 300 mM NaCl.

Batch method: A batch method for negative selection of antibodies is outlined by Harlow and Lane. In brief, the anion exchanger is equilibrated to pH 6.3 (for rabbit serum puriﬁcation) and pH 7.5 (for serum puriﬁcation) in sodium phosphate buffer and mixed with antibody solution (dialyzed to an equivalent pH). The mixture incubates on a rocker for 1 h and the slurry is ﬁltered, collecting the antibody in the ﬂow through. This method has a lower yield but a fast processing time.

Column method (DEAE anion exchange): Used in combination with ammonium sulfate precipitation, this method yields a relatively pure antibody preparation at a moderate cost. The antibody solution is adjusted to pH 9 and loaded onto a DEAE column. The antibody is eluted with a high salt (less than 0.5 M NaCl) and the fractions are collected and monitored for protein measuring the absorbance at 280 nm (OD = 1 is roughly 0.8 mg/ml). This method is useful for eliminating albumins from the antibody preparation.

Purification of IgM

One-step method using affinity chromatography

Protein L or Mannan Binding

Protein L binds equally well to the light chain of IgM and can be useful as a one-step method for purifying this mouse isotype. Mannan-binding protein (MBP) is a lectin speciﬁc to mannose and N-acetylglucosamine in mammalian sera. These binding characteristics have made it useful as a ligand in chromatography for mouse IgM puriﬁcation and purities of at least 95% have been observed following this method. The immobilized MBP is also available in the market. The cost of this resin may make this method impractical, however the method provides a fast one-step method with excellent ﬁnal purity and yield.

Anti-Antibody

Anti-IgM (mouse) covalently linked to agarose is available commercially for use in afﬁnity chromatography of mouse IgM. Typically, an immunoafﬁnity column of this type will have 5 to 10 mg of IgG covalently attached to 1 ml of resin. The IgM binding capacity is then approximately 10% or 0.5 to 1 mg/ml of resin.

Two step methods

Ammonium Sulfate Precipitation

This method is a common ﬁrst step in IgM puriﬁcation. Yield (>80%) and purity (<50%) are similar to IgG puriﬁcation.

Gel Exclusion Chromatography

Gel exclusion chromatography separates proteins on the basis of molecular size using porous beads of cross-linked dextran or agarose. A molecular sieve of a deﬁned pore size sifts for the molecular weight range of interest. This method may be considered as a secondary step in a two-step puriﬁcation of IgM with ammonium sulfate precipitation or ultracentrifugation as the primary ﬁrst step. Because a neutral wash buffer is used to elute the antibody, there are no deleterious effects on the antibody activity. This method is inexpensive, but time consuming.

Hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is similar to the approach of reverse phase chromatography utilizing aqueous rather than organic solvents. The antibody solution is placed in a high-salt buffer (20% saturated ammonium sulfate) to facilitate binding of hydrophobic domains in the protein structure to the column matrix of derivatized hydrocarbons. The salt concentration is reduced with a gradient to elute the antibody. An advantage to this method is minimal disruption of the antibody structure and function. This method removes endotoxins, viral particles, and nucleic acids and is invaluable in antibody production. However, since HIC does not separate albumin from immunoglobulins a preliminary ammonium sulfate precipitation may be needed. HIC’s disadvantage is the variable interaction between monoclonal antibodies and the matrices. If the antibody binding to the column is too strong, the elution conditions may cause a loss of antibody activity. Thus, the optimal column derivatization for the isolation of a speciﬁc monoclonal antibody must be determined empirically. If albumin is present, this method does not work well to separate albumin from the immunoglobulin fraction. Ammonium sulfate precipitation works well as a preliminary step prior to using the method. This method is invaluable from a production standpoint for antibody use as bioagents because of the removal of endotoxins, virus particles, and nucleic acids.

Liquid polymer precipitation

These methods have been documented in the literature which demonstrate polymer speciﬁc precipitation of antibody fractions from a variety of samples. For example, PEG 6000, at a volume/volume percent of 4 to 6% can be used to precipitate murine IgM from delipidized ascites ﬂuid (>90% pure, >80% yield). This method requires a second step of puriﬁcation (e.g., ion exchange) if antibodies have to be used for ELISA to remove interfering polymers. There also tends to be variability in the purity and yield documented with this method.

Hydroxylapatite chromatography

This method has been used to process bulk amounts of antibody into a concentrated form in buffers that do not require post-puriﬁcation dialysis. Antibodies are eluted with increasing concentrations of salt in wash buffer (120 to 300 mM PBS, pH 6.8). This method has been shown to provide a good yield and purity from ascites and sera, but has not been useful for tissue culture because of the contaminating sera immunoglobulin components. As with the other column chromatography methods, this procedure is time consuming but inexpensive. Typically, puriﬁcation of 5.0 ml of sera will require the use of 100 ml of hydroxyapatite.

Continuous flow centrifugation and tangential flow filtration

This method is utilized for very large-scale production of antibodies from cell culture media (>100 L/h capacity) to remove cells and debris. A secondary step involves the use of cross ﬂow or tangential ﬂow ﬁltration devices to concentrate large volume of cell culture product with reduced fouling of the membrane ﬁlter surface.

Final product

Yield and purity

The use of gel electrophoresis will allow determination of non-immunoglobulin contamination of the ﬁnal product. However, polyclonal sera and mouse ascites ﬂuid also include nonspeciﬁc immunoglobulins unless antigen speciﬁc afﬁnity puriﬁcation is performed. In these cases, these can represent anywhere from 10 to 50% of the puriﬁed fraction. Also, tissue culture supernatant and bioreactor products can be contaminated with serum supplement immunoglobulins unless serum-free media are utilized.

SDS-PAGE

This gel electrophoresis method is useful for determining the purity of the antibody product with IgG providing 2 bands (25 and 55 kDa) and IgM (25 and 78 kDa).

Quantitative ELISA (Mouse IgG) – Specific activity (ELISA assay/titration)

Antibody titer is deﬁned as the highest dilution that still allows observation of an antigen detection signal in the assay. A titration assay using ELISA with puriﬁed antigen as the coating layer is a common method for determining the level of enrichment in the antibody solution after puriﬁcation. Samples of pre- and post puriﬁcation antibody are used in a serial dilution on replicate wells coated with antigen to determine the antibody titer before and after puriﬁcation. Alternatively, antibody titer can be evaluated using a dot-blot analysis, immunoﬂuorescence assay (IFA), antibody neutralization of virus, or other methods that provide relevant information on the antibody’s ability to bind antigen.

Storage

For long-term storage of puriﬁed antibody, the best scenario is in the freezer at –20°C or at –70°C (further reduces protease activity). Antibody storage should be at a neutral pH and a concentration of 1 to 10 mg/ml. Further, a rapid dry-ice methanol bath to freeze, reduces the formation of antibody precipitation. Freeze-drying can cause aggregate formation and should be avoided. Avoid repeated freezing and thawing, as the antibody molecule, especially IgM, will denature and/or aggregate. Sodium azide of 0.05 to 0.1% concentration can be used as a bactericidal agent and stored at 4°C for long periods, assuming no protease activity exists. But Sodium azide can have detrimental effects on alkaline phosphatase activity. Thimerosal can be used in place of azide as a bactericide. The additional precaution of ﬁltering through a 0.2-µm membrane will also reduce microorganism growth. IgM antibodies may precipitate out in solution and can be re-dissolved with the addition of more salt to the diluent (i.e., NaCl, if PBS is used). Antibodies that have been conjugated can be stored at –20°C in a 50% glycerol solution.

Recombinant renewable polyclonal antibodies (MAbs. 2015;7(1):32-41 2015)

In a recent research by Andrew et al, generation of high-quality recombinant polyclonals has been observed in vitro by combining phage and yeast display, in which hundreds of different antibodies are all directed toward a target of interest. The variable quality of most commercial polyclonal antibodies may be due to polyclonal antibody generated following traditional methods contain barely 0.5–5% of antibodies that will recognize the target. Frequent results of undesirable polyreactivity are because only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest. Although this problem can be overcome by afﬁnity puriﬁcation but, the ﬁnite and non-renewable nature of polyclonal antibodies limits the amount of speciﬁc antibody that can be isolated. They also showed that unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be ampliﬁed over one hundred million-fold without losing representation or functionality. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly speciﬁc recombinant renewable antibodies against all human gene products. An advantage of producing antibodies recombinantly rather than by immunization is that the selection conditions can be modiﬁed to generate antibodies against speciﬁc target forms. In vitro display methods (e.g., phage/yeast display) yield recombinant antibodies that are usually deconvoluted to single clones for further testing and can be a valid alternative to the non-renewable polyclonal antibody products available at present.

Adjuvants

Adjuvants are the type of compounds that are known to supplement or enhance the immune response that is elicited against an immunogen. Most of the purified proteins are non-immunogenic in nature thereby rarely are able to induce a vigorous immune reaction in absence of an adjuvant. For an antigen to successfully generate an immune response it is mandatory that it produces an inﬂammatory response as it results into the involvement of inﬂammatory cells alongwith instigating the dendritic cells and macrophages which together forms an indispensable element towards developing a successful immune response. The event of inﬂammation is marked by the variable amount of destruction of tissues involved together with pain and the area effected depends on the physiognomies and degree of the inﬂammatory response. Out of the other important elements, one of the essential decision during the production of polyclonal antibodies is the type of the adjuvant that is to be implemented as is one of the most significant and often the most debated decision. In choosing an adjuvant the overall goal is a critical component. Therapeutic vaccines are supposed to provide protection against pathogens with the expression of least side-effects. The anticipated reaction will provide protection to the receiver from harbouring a grave disease followed by experiencing contact with the antigen and will necessitate a humoral response. Generally, antibody production is principally focused in the effective and cost-effective production of antibody in a high-titer having higher affinity to a specific immunogen. The technique by which an adjuvant stimulates an immune response is well understood slowly with the development in the studies dealing with the features of an antigen recognition pathway and subsequent immune response. Adjuvants are known to manifest five different mechanisms with specificity in action that includes an antigen presentation, antigen distribution effect, “depot” effect together with an immunomodulatory effect. In addition, they also feature a cytotoxic induction of lymphocyte effect. Among these, the important ones that have a significant impact on inducing a humoral response to a specific antigen are the depot activity and the immune modulation properties. The “depot” effect of an adjuvant defends the immunogen against dilution and degradation in presence of a host. This is done by ensuring the localised sustained release of the immunogen for a longer period of time so that it is available for interaction with the immune system for a considerable amount of time. This fact has been experimentally proved by researchers in various studies. The most frequently applied adjuvants in the production of polyclonal antibodies with a “depot” effect are the emulsions which are either water-in-oil or oil-in-water emulsions though many complicated forms of emulsions are also present. Non-biodegradable mineral oils or biodegradable oils are used along with emulsifiers. Aluminium, liposomes, microparticles, nitrocellulose are some of the other elements which can potentially act as “depot” agents for adjuvant. It is essential that the depot substance implemented to conserve the original nature of the immunogen alongside shielding and slowly discharging the immunogen. This is essential so that the immunogen is presented in a correct form for the development of antibody response. For maintaining the immunogen in the aqueous phase the Water in-oil emulsions are considered while for maintaining the immunogen in the oil phase the oil-in-water are preferred. For hydrophobic immunogens, oil-in-water emulsions are suited while for hydrophilic immunogens water-in-oil emulsions work better. In case of adjuvants like aluminium salt, it is possible to utilise the electrostatic forces for binding immunogens that depends on the suitable pH and the buffer applied and also it varies depending on the aluminium salt type and the ratio of the immunogen to the salt. It is necessary for an adjuvant to also make sure that the immunogen is processed correctly and circulated extensively all over the immune system, in turn, maximizing the immune response. Adjuvants are known to form massive aggregates alongwith the immunogen that enables its uptake and processing by macrophages and APCs (dendritic cells). In certain cases, the adjuvants may integrate carbohydrate species or conformations in molecular structure that is specific for macrophage or dendritic cell recognition and uptake followed by processing. The adjuvant is also responsible for the processing and transportation of the immunogen to the local lymphoid tissues in a short time and even the removal of the original injection sites by surgery does not affect the immune response in the animal although the antigen is still existing in the excised site of injection. The adjuvant should have low toxicity to the immunised animal subject as well as to the person responsible for carrying out immunisation. There are numerous adjuvants that are known scientifically, but the majority of them do not qualify for routine usage in the production of pAbs mainly attributed to the expense involved or the struggle involved in the injection formulation preparation. The different classes of adjuvant that can be applied for pAb production are oil emulsions that are immunostimulatory, mineral salts containing aluminium, microbial products, saponins, a combination of above adjuvant formulations. The immunization blend containing the antigen and the adjuvant should be prepared by maintaining proper hygiene and aseptic conditions so as to lessen the risks of a likely event of contamination.

Water-In-Oil Emulsion Adjuvants Freund’s Adjuvants

Freund’s complete adjuvant (FCA) has occupied the position of most extensively used adjuvants since the time of inception of the concept of adjuvants for polyclonal antibody production. The capability of FCA to effectively stimulate the immune system is considered to be a “benchmark” for comparison of freshly formulated adjuvants. However the FCA is known to be the source of a number of types of lesions which includes granulomas of the confined site of injection and also granulomas are also seen in different of organs that are situated quite far from the site of injection, dermatitis that is necrotizing, granuloma development that causes damage to the spinal cord. FCA’s use has been limited due to the severe lesions created by adjuvant in both human/animals and have resulted in the formation of many controlling guidelines for their use in vaccinations and approvals concerning their usage in polyclonal antibody production. FCA is made up of light mineral oil, a surfactant called mannidemonooleate and that are in dried form. FIA( Freund’s incomplete adjuvant) on the contrary is devoid of the heat-killed mycobacterium keeping all other components same. The presently available FCA and FIA adjuvants are much more purified and lower in toxicity compared to the originally developed formulation. More recently new formulations like Specol are supposed to be alternatives which are composed of purified oil and emulsifiers and they exhibit profound immunomodulation effects alongwith less toxicity. They constituents are FDA approved for use in animals and has the flexibility to be acquired independently. Studies have been conducted which equate the responses of the antibody exposed to different immunogens and Specol/ FCA/FIA combinations and have mostly conveyed comparable outcomes in rabbits. The Montanide adjuvants that are also developed as an alternative are founded on the basis of a purified mineral oil a (water-in-oil emulsion) quite alike to FIA. Studies have been carried out in comparing these adjuvants to FCA/FIA and the results have corroborated to those of FCA/FIA with rather same or less amount of inﬂammatory responses.

Other water-in-oil emulsion adjuvants like TiterMax and TiterMax Gold contains oil named squalene that can be metabolized, an emulsifier with a block copolymer, and silica that is microparticulate in nature. Non-ionic block copolymers having a high-molecular-weight due to the presence of linear chains of polyoxyethylene that is hydrophilic and the linear chains of polyoxypropylene that is hydrophobic are found to be efficient in developing a better effect via heightened antigen presentation, macrophage, activation of complement followed by chemical taxis. These properties have tagged the block copolymers as efficient immunomodulators in the expansion of vaccination and formulation. Additionally, these adjuvants (TiterMax, TiterMax Gold) can also be implemented as alternative approaches to FCA/FIA in the making of a polyclonal antibody with reduced tissue toxicity while maintaining the same effectiveness in its role as an adjuvant.

The oil-in-water adjuvants have differences with respect to water-in-oil adjuvants in terms of the fact that they are characteristically formulated in presence of a less amount of oil distributed in an aqueous phase where as in water-in-oil emulsions it contains normally 50% of oil. Oil-in-water emulsions are not reported to induce localized depot effect like in case of most of the water-in-oil adjuvants. Nevertheless, they are promptly carried away from the site of injection to the lymphatic system thereby dropping the possible chances of long-lasting localized inﬂammation at the site where the injection is done. This property has helped the adjuvants like oil-in-water emulsion become more prospective contenders in comparison to water-in-oil emulsion adjuvants in development of vaccines.

RIBI Adjuvant System

The adjuvant system known as (The RIBI Adjuvant System) is one of the initially developed commercial adjuvant systems for investigational antibody production. The resultant emulsion involves a minor amount of squalene oil and surfactant Tween80 and delivers considerable less viscosity than classical water-in-oil emulsions which renders them to be successfully sterilized via filtration and also promotes easy administration. These are compounds that are supposed to be present in the emulsion as an auxiliary constituent when the emulsion is having poor adjuvant characteristics. Trehalose 6,6-dimycolate, Monophosphoryl lipid A (MPL), cell wall skeleton (CWS) are the various available adjuvants that can enhance the immune response by supplementing both the humoral and the cellular immune responses although it is apparently found to be less effective in rabbit compared to mice, the MPL is known to act as agonist maintaining the immune stimulation properties but lowering the toxicity. It is recommended for use in mice in presence of strong antigen and is found in formulations recognised for application in rabbits, goats as well as primates. The activity of immune stimulation found in the CWS is mainly due to the muramyl dipeptide (MDP) accountable for the considerable amount of the adjuvant activity. The investigations that have compared the pathological lesions produced by the above mentioned adjuvants have produced variable outcomes analogous or minimised compared to those realized with FCA/FIA

Syntex Adjuvant Formulation

The Syntex Adjuvant Formulation developed by the Chiron Corporation is a microﬂuidized oil-in-water emulsion that contains a 2.5% pluronic, 5% squalane, 0.2% polysorbate 80 surfactant and PBS together with threonyl modified muramyl dipeptide (t-MDP). The Pluronic that is a synthetic block copolymer and t-MDP attends as the immune stimulating agents. This particular adjuvant formulation has been developed as an adjuvant for vaccination and evaluation has been carried out in humans alongwith other species to understand its effectiveness and safety. As per a trial conducted in human cancer immunotherapy complications such as local erythema, edema, arthralgia, myalgia, and pyrexia has been repoted. Compared to other contemporary adjuvants, the efficacy of this adjuvant in antibody production have yielded varied outcomes.

Particulate Adjuvants

In this category of adjuvant absorption of immunogens is carried out onto aluminium phosphate/aluminium hydroxide and is the utmost prevalent technique implemented in producing vaccines applicable for both human and animal purposes. The aluminium salts are known to bind the immunogens by electrostatic interaction and via exchange of ligand that provides a comparatively shorter depot effect even with maximum antibody titers post injection. Aluminium adjuvants display some of the characteristic immunomodulation signatures like properties induction of secretion of CCL3, CCL4, CXCL8 from the macrophages and human monocytes and the CCL2, CXCL1, CCL11 and IL-1β in case of mice. They also have the ability to stimulate an approximately selective immune response of Th2 type with very less side effects with an established safety record. They are also generally not found to participate in localized erythema or lesion/granuloma development at the site of injection and because of this is generally favoured with administration via the subcutaneous route.

Immune-stimulating complexes (ISCOMs)

They appear as ear 40–100-nm honeycomb shaped complexes consisting of immunogen, phospholipids, cholesterol and saponin from soap tree. This saponin is a mixture of glycosides mixed heterogeneously and forms an essential component in the development of the ISCOM serving as a powerful adjuvant acting via apparently enhancing the uptake of the said ISCOMs by the dendritic cells. Numerous forms of half purified or purified saponin formulations are available thatb are extensively used in vaccines in veterinary medicine but owing to the haemolytic activity of some of them during administration via parenteral route has prohibited their usage in further applications. They are characteristically designed by the reaction of saponin in presence of cholesterol as well as phospholipids together with an antigen of hydrophobic behaviour in the existence of an appropriate detergent. Some of the hydrophilic immunogens if desired to be used in ISCOMs like in case of small peptides they should be obligatorily modified through the incorporation of hydrophobic residues. Since the making of the matrix of ISCOM is reliant on the ratio of the immunogen to saponin, hydrophobicity content and phospholipid component of the immunogen for producing a proper functioning formulation of immunogen–adjuvant mix, it becomes challenging and estimation of the end product is tedious. Out of these for polyclonal antibody production, most of the challenges associated can be overcome by the implementation of pre-prepared ISCOM-matrix that can be immediately associated with the immunogen of choice. Initially, though the ISCOM-matrix has been primarily developed for use in vaccines it opens up avenues for a ready-to-use adjuvant in the advancement of polyclonal antibodies production.

Existing Guidelines on the Production of Polyclonal Antibodies

A number of guidelines have been framed to oversee the production of pAbs by several nationwide governing authorities, welfare organisations and academic institutions. These guidelines mainly target the unwanted abuse that may take place due to the side-effects of the adjuvants used during the process as well as the injection and sample collection routes. Importantly, it to be noted that majority of the protocols followed for the pAb production relies on conventionally followed procedures which are not based on scientific principles. The ultimate aim of framing these guidelines is to regulate the procedures followed for the production of pAbs ensuring acceptable outcome but not at the cost of discomfort and pain of the participating animals. In 1988, NIH in the USA issued their internal approvals for using FCA in research followed by guidelines on suitable immunological protocols framed by Canadian Council on Animal Care. These guidelines thereafter gave a platform for the launch of guidelines in many other nations. European countries Switzerland, Denmark, The Netherlands, the UK and Sweden have their own set of code of national guidelines issued on the pAbs production. This particularly has helped to achieve welfare of the animal involved along with an optimistic impression on pAb production though there is some difference in opinion in regard to the immunisation protocols followed for the pAbs production. However, in spite of this most of the guidelines cover topics ranging from species choice, preparation of antigen, the route to be followed for injection as well as volume, adjuvants choice and judicious use, injection method including immunisation routine, finally blood sample collection and post-injection monitoring.