Who invented ELISA?
Two different teams led by Stratis Avrameas and G. B. Pierce in 1960s laid the ground work for enzyme linking process. In the same period, immunosorbent preparation technique was published by Wide and Jerker Porath.
Following which, scientists in Sweden (Peter Perlmann and Eva Engvall at Stockholm University) and Netherland (Anton Schuurs and Bauke van Weemen) generated knowledge that go into making ELISAs.
Before ELISA, radioimmunoassay employing radioactively labelled antigens and antibodies were used. Radioactivity served as the reporter signal indicating specific antigen or antibody. As radioimmunoassay posed significant health risks to researchers, alternatives were sought.
H2: What is the principle behind ELISA?
ELISA works by coupling antibody or antigen to assay enzyme. The assay combines the specificity of antibody and sensitivity of assay enzymes to primarily detect antigens through assay antibody or antibodies through assay antigens.
The sensitivity and precision of the assay is enhanced by coating the plate with high-affinity antibodies.
H2: How does ELISA work?
The conventional ELISA involves usage of chromogenic reporters and substrates to produce color changes to indicate the presence of specific antigen or an analyte. Newer Assay techniques make use of fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals.
The advantage of using advanced reporters help in measuring multiple analytes in a single or cycle of assays (Multiplexing) and higher sensitivities (specificity and sensitivity).
Technically, newer assays use reporters that are not enzymes, nonetheless the underlying principles of the assays are similar. Therefore, these assays are grouped as ELISAs.
To know more about running ELISA assays, Jump to the protocols page.
H2: How does ELISA assay detection work?
The detection of the assay is carried out by evaluating the enzyme activity of the conjugate which is implemented by incubation with a suitable substrate for the enzyme resulting in a generation of a quantifiable product. Generally, the ELISA technique results in a colored end product.
The colored product exhibits absorption at a specific wavelength and can be correlated to the quantity of analyte in question present in the sample. An extremely specific antibody-antigen interaction is the utmost critical component of the entire process.
The ability to wash away non-specific unbound reactants makes the ELISA technique an influential and reliable means for gauging precise information on the analytes even when present within a crude and impurified sample.
H2: What are the applications of ELISA?
- Cancer screening
- Pregnancy testing
- Drug testing
- Detection of platelet antibodies
- Detection of food allergens
- Detection of viruses (HIV, West Nile Virus, and New Castle Disease Virus)
ELISA has been implemented in diagnostics as well as for quality-control programmes throughout a number of industries. ELISA tests are rapid and easy to carry out, and because they are premeditated to swiftly carry out high throughput screening and multiplexing, they have made a breakthrough in the widespread assessment of various investigations as well as diagnostic assays.
ELISA has retained its position as widely used detection technique either in their original form or in extended forms with certain amendments i.e. changes focused on allowing for more than two analytes in each well, improved sensitivity and straightforward output. These assays function on common foundation (shared with conventional ELISA) compared other immunoassay techniques available.
H2: What are the different types of ELISA?
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competition/Inhibition ELISA
H2: What is direct ELISA?
Direct ELISA involves coating of antigen directly to wells of microtitre plate, and addition of enzyme labelled primary antibody.
H2: What are the advantages and disadvantages of Direct ELISA?
Direct ELISA is easiest to carry out as a technique among all ELISA formats. Given its simplicity, it is also the fastest (not prone to errors) due to limited number of steps followed.
The disadvantages can arise in the form of specificity (the immobilization of the antigen is not specific due to which background related issues are seen), less flexibility in terms of primary antibody, and absence of signal amplification reduces sensitivity.
H2: What is Indirect ELISA?
The indirect ELISA method exhibits higher sensitivity since it employs enzyme labelled secondary antibody which interacts with a primary antibody. The technique is considered to be comparatively economical than direct ELISA due to the requirement of fewer labelled antibodies.
The higher flexibility of indirect ELISA is notable since it enables enzyme labelled secondary antibody to bind with various primary antibodies.
The secondary antibody is mostly polyclonal in origin with anti-species reactivity. The limitation to be concerned in this technique is the issue of cross reactivity of secondary antibody with a bound antigen that may create more background noise. There is a necessity of an extra step where incubation of secondary antibody is performed that leads to more time consumption.
The indirect ELISA helps in quantifying the total concentration of antibody in samples.
H2: What are the advantages and disadvantages of Indirect ELISA?
Some of the advantages of using indirect Elisa:
- Offers high sensitivity and flexibility as the number of secondary antibodies can bind to a primary antibody and one type of secondary antibody can label different primary antibodies
- It is cheaper as there is a requirement of fewer labeled antibodies.
Disadvantages:
- Higher signal to noise ratio.
- Time consuming and extra labor required.
H2: What is sandwich ELISA?
Monoclonal (or polyclonal) antibodies are used in pairs as capture antibody and detection antibody in sandwich ELISA. Each antibody is highly specific towards epitope of an antigen and the assay is found to be more suitable for antigens possessing two epitopes.
The specificity of matched antibody pairs is very important to confirm their binding to different epitopes in obtaining precise results. As its name indicates, the capture antibody interacts with an antigen which can then be detected via both direct and indirect ELISA techniques.
Since the antigen quantification is taking place between upper and lower layer of antibodies, the entire method is termed as sandwich ELISA.
H2: How is Sandwich ELISA carried out?
A sandwich ELISA applied for investigation more often demands validation as it falls under the risks of delivering false positive results. Sometimes the development of the sandwich ELISA assay takes considerable time owing to the procurement of matched pairs of antibodies that is required.
The primary step in sandwich ELISA involves coating of ELISA plate with a capture antibody. To this antibody coated plate, a sample containing antigen is added, followed by antibody detection.
The detection antibody can be either enzyme labelled or enzyme unlabelled, depending on which it will be direct sandwich ELISA or indirect sandwich ELISA respectively.
H2: How is direct sandwich ELISA different from Indirect Sandwich ELISA?
In indirect sandwich ELISA, a secondary enzyme-labelled antibody based detection is introduced that binds to the primary unlabelled detection antibody. Sandwich ELISA is found to be 2-5 times more sensitive when compared to direct and indirect ELISA techniques.
The higher specificity of sandwich ELISA is attributed to the usage of two antibodies which detect the antigen of interest. Since the implementation of both direct and indirect methods is possible, the technique promises more flexibility in detection procedure.
H2: When is Sandwich ELISA used?
The analysis of a complex sample with high specificity and sensitivity is possible through this technique as it requires no prior purification of antigen. The technique however brings few disadvantages which are to be considered.
It is important to have an optimized and tested ELISA kit for specific reaction and detection. The unavailability of standardised kit would demand the optimization of antibody to avoid cross reactivity of capture antibodies with detection antibodies which is time consuming.
H2: What is Inhibition ELISA?
The competition/inhibition ELISA which is otherwise termed as blocking ELISA is known to be a plate/surface-based assay.
It is one of the most complex assays performed above all the remaining ELISA techniques. However, it is to be noted that the other ELISA types can also be modified to fit into the competitive format.
The working mechanism of this technique predominantly measures and quantifies the antigen or antibody concentration in a sample based on the supposed signal output by resultant interference.
Basically, it exhibits a competitive interaction of sample antigen or antibody to labelled antibody or antigen which is in limited concentration respectively. The obtained signal output is inversely proportional to the concentration of antigen in a sample where there is an output of a weaker signal at higher concentration of antigen.
H2: What differentiates Inhibition/competitive ELISA from other ELISA formats?
The major step in competitive ELISA or inhibition ELISA is the process of competitive reaction between the target antigen present in the sample and antigen that is bound multiwall plates with the available primary antibody.
The higher antigen concentration in the sample is denoted by a significantly lowered signal output. In contrast, antigen in the sample at a very low quantity will give rise to higher signal output. It is best applicable when the availability of antibody is reduced to one for the target antigen.
It is also apt for distinguishing minor amounts of antigens that cannot be detected even by two distinct antibodies such as in the sandwich ELISA technique. In most of the cases, inhibition assays provide the sample to be tested the opportunity for preferential binding over other interfering components as it is added before them.
In case of competitive assays, the target sample is first incubated with the other component before the reaction proceeds further.
H2: What are the advantages and disadvantages of Inhibition ELISA?
Advantages
- Negligible sample processing is required and can be applicable to crude samples
- Less sensitive to experimental errors.
- Good reproducibility and flexibility.