{"id":2057,"date":"2018-06-04T14:59:51","date_gmt":"2018-06-04T14:59:51","guid":{"rendered":"https:\/\/www.mybiosource.com\/learn\/?page_id=2057"},"modified":"2018-08-10T04:31:57","modified_gmt":"2018-08-10T04:31:57","slug":"2057-2","status":"publish","type":"page","link":"https:\/\/www.mybiosource.com\/learn\/polyclonal-antibodies","title":{"rendered":"Polyclonal Antibodies"},"content":{"rendered":"[et_pb_section fb_built=”1″ _builder_version=”3.0.106″][et_pb_row _builder_version=”3.0.106″ background_size=”initial” background_position=”top_left” background_repeat=”repeat”][et_pb_column type=”4_4″ _builder_version=”3.0.47″ parallax=”off” parallax_method=”on”][et_pb_text _builder_version=”3.0.106″]\n[\/et_pb_text][et_pb_text _builder_version=”3.0.106″]\n

Polyclonal Antibodies<\/strong><\/h1>\n

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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<\/sup>-108<\/sup>), as immunoglobulins with different antigen specificities\u00a0 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. <\/span><\/p>\n

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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.<\/span><\/p>\n

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Polyclonal Antibodies Versus Monoclonal Antibodies<\/strong><\/h2>\n

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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 K\u00f6hler & 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.\u00a0 On the contrary, due to the reason that polyclonal antibody\u2019s 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\u20138 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.<\/p>\n

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The laboratory animals are immunised for induction of a humoral immune response as a general procedure for the production of antibodies.\u00a0\u00a0 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.<\/p>\n

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Selection of host<\/strong><\/h2>\n

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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\u2019t 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 \ufb01ve are commonly used.<\/p>\n

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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.<\/p>\n

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Types of immunogen\/antigen<\/strong><\/h2>\n

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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.<\/p>\n

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Thymus-Independent Antigens<\/strong><\/h2>\n

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The antigens that are able to induce a humoral antibody response through the direct activation of\u00a0 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, \ufb02agella 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.<\/p>\n

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Thymus-Dependent Antigens<\/strong><\/h2>\n

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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\u00a0 MHC class II molecules which results in the initiation of specific helper T-cells towards the antigen. The\u00a0 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\u00a0\u00a0 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\u00a0 longer time.<\/p>\n

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Dosage of immunogen <\/strong><\/h2>\n

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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 \u03b3-globulin (DNP-\u03b3G) at a range of 0.05\u201350mg blended in Freund\u2019s 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 50\u00b5g or 1mg, there observed a quantitative increment in antibody response at a time period of 2 weeks.\u00a0 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 50\u00b5g. An attempt was made by a group of researchers where they have aimed to carry out enzyme immunoassay by developing antibodies against viomycin.\u00a0 By their study, the optimized initial doses of immunisation for FCA as the adjuvant, Freund\u2019s Incomplete Adjuvant (FIA) and aluminium hydroxide (alum) were 10 \u00b5g, 200\u00b5g and 10\u00b5g 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 \u00b5g. 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\u2013250 \u00b5g of proteins contained in adjuvant, an elevated titre of antibody can be observed in rabbits (Cooper and Paterson, 2009; Harlow, 1988).<\/p>\n

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Route of immunization<\/strong><\/h2>\n

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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.<\/p>\n

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Schedule of immunization <\/strong><\/h2>\n

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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\u20133 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.<\/p>\n

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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 in\ufb02uenced 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 nonspeci\ufb01c 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 in\ufb02uence on the immune response after multiple immunizations. Unless the immunogen is highly puri\ufb01ed, 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.<\/p>\n

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Collection of blood<\/strong><\/h2>\n

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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.\u00a0 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\u20134 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.<\/p>\n

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Assessment of Side-effects<\/strong><\/h2>\n

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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.<\/p>\n

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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 -70\u00baC 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.<\/p>\n

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\u00a0<\/strong>Potency of polyclonal protein<\/strong><\/span><\/span><\/h2>\n

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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.<\/p>\n

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\u00a0Enzyme -Linked Immunosorbent Assay<\/strong><\/h2>\n

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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 \u00d7 12 96-well polystyrene plate). Antigen is allowed to bind passively to the plate overnight at 4\u00b0C (100 \u00b5l, 0.1 to 1.0 \u00b5g 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 \u00b5l) 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 \u00b5l 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 \u00b5l per well, dilution depends on speci\ufb01c 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 \u00b5l of 10 mg\/ml OPD in citrate-phosphate buffer containing 0.1% H2<\/sub>O2<\/sub>) is added and incubated for 10 to 30 min, after which the reaction is stopped by the addition of sulfuric acid (50 \u00b5l). 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.<\/p>\n

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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\u2013antigen 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 af\ufb01nity of the antibody for the antigen. Conjugation of the antigen to an inert carrier protein and immobilization via biotin\u2013avidin, as mentioned above, are two remedies for this. Another remedy is to utilize a \u201csandwich\u201d 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 puri\ufb01ed and labeled if the direct method is used.<\/p>\n

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Radial Immunodiffusion<\/strong><\/h2>\n

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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 con\ufb01guration provides a layer 1.0 mm thick. Use of alternative con\ufb01gurations will require a different volume of agarose or a correction factor in the equation. In the protocol agarose is heated to 68\u00b0C, at this temperature it wouldn\u2019t 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 \u00b5l of each antigen solution into separate wells. A minimum of three different concentrations should be used, and \ufb01ve standards seem to give consistently reliable results. The antibody\u2013antigen 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.<\/p>\n

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ELISA and RID methodologies provide measurements of potency. If highly puri\ufb01ed antigens are utilized in those assays, it is possible to get a good estimate of the speci\ufb01c 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 speci\ufb01city of the antibodies.<\/p>\n

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Western blot<\/strong><\/h2>\n

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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 \ufb01nal 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 monospeci\ufb01c antibody relative to the electrophoresed antigen preparation.<\/p>\n

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Immunoelectrophoresis<\/strong><\/h2>\n

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In this process, antibody diffuses into the gel and the antibody\u2013antigen reaction is visualized by staining the resulting immunoprecipitates. It is a two-stage procedure. The \ufb01rst stage separates the antigenic material in biological \ufb02uids by their differential migration in an electric \ufb01eld. The second stage of this technique is the immunological characterization of the separated proteins by the immunodiffusion procedure.\u00a0 This method is commonly utilized to evaluate the speci\ufb01city of antisera raised against serum proteins. The protocol is as followed – Pipet 1 to 2 \u00b5l of bromphenol blue dye into well #1 and allow this to be adsorbed into the gel. Pipet 4 \u00b5l normal serum into well #1, and 4 \u00b5l 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 \u00b5l of antiserum into the appropriate troughs and place the slide in a humidi\ufb01ed 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\u00b0. 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, speci\ufb01c antisera. A monospeci\ufb01c antiserum results in the generation of a single precipitin line while antiserum containing multiple antigenic speci\ufb01cities will produce multiple arcs.<\/p>\n

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Serum sample purification<\/strong><\/h2>\n

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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 speci\ufb01c antibody titer is high enough that simple dilution eliminates the interference. Or else the antigens recognized by the nonspeci\ufb01c antibodies are not present in the assay system. When this is not the case, there are approaches for the generation of a monospeci\ufb01c polyclonal antiserum:<\/p>\n

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