Enzyme-Linked Immunosorbent Assay

Enzyme-Linked Immunosorbent Assay (ELISA)

Widely utilized throughout research and medicine, the ELISA can measure the amount and specificity of an antibody through its direct binding to antigen. The ELISA measures the direct binfing of the antibody to its antigen, and is therefore referred to an assay that is based in primary interactions. In order to perform an ELISA, a pure preparations of a knwon antibody or antigen, or both is required. With the ELISA, both competitive and noncompetitive procedures can be utilized to quantify antigens (antibodies). However, we used a noncompetitive ELISA assay, the "sandwich" ELISA (2).

Sandwich Enzyme-Linked Immunosorbent Assay

The "sandwich" ELISA involves the use of a constant amount of immobilized antibody, with an excess of enzyme-linked antibody possessing the same or similar specificities as the immobilized antibody. The antigen (or antibody) is incubated with the immobilized antibody, followed by washings. After, the enzyme-linked antibody is added and allowed to incubate, with further washings, the enzyme becomes associated with the solid phase and its activity measured with the addition (excess) of a chromogenic substrate, which will be cleaved from colorless to a color that will be measured. The enzyme activity is correlated, proportionally, to the amount of antigen present in the test solutions (1,2,3).

Enzyme-Linked Immunosorbent Assay Protocol

This procedure can be used to determine if IgM is present in the supernatants, whether IgM was secreted. Goat anti-IgM antibodies are allowed to bind to the wells of a microtitre plate, followed by the addition of the culture supernatants, with further incubation. Moreover, the goat anti-mouse IgM antibodies used were polyclonal. Monoclonal antibodies are usually not utilized for experiments involving mutations, because if a B cell hybridoma had a mutation that altered this one epitope on its' IgMs, there would be no binding and no detection of IgM, even though there would be IgM within the supernatants of the samples. During this incubation, any secreted IgM present in the supernatants will bind to the anti-IgM. If there is specific binding, the IgM will be non-covalently bound to the antibodies on the wells, and not be removed by the subsequent washing steps. The "sandwich" was completed with the addition of the anti-IgM linked to an enzyme, that will bind to any bound IgM. If IgM initially interacted and bound to the well-coated anti-IgM, then the enzyme conjugated anti-IgM will bind, and the addition of a chromogenic form of the enzyme substrate will cause the color change that will be measured spectrophotometrically. The chromogenic substrate utilized in our investigation was phosphatase substrate, turned the solution yellow, when cleaved by the phosphatase, which was conjugated with the goat anti-mouse IgMs. The color change was measured at an absorbance of 405 nm (1,3).

Enzyme-Linked Immunosorbent Assay Troubleshooting

Poor washings and failure to dispense the entire volume from the pipette, supernatant, could drastically affect the absorbances, as very small quantities were pipetted, utilized and measured. Nonspecific adsorption could have been reduced by including the medium during incubation and washing steps. Repetition of samples would have improved the results and made them more reproducible and accurate. Moreover, antibody quantification using the "sandwich" ELISA produces suitable results, in both sensitivity and reproducibility. However, because antibodies possess different affinities for a given antigen, unlike antigen measurements, measurements of antibody is not strictly possible. Any values obtained display the combination of both the quantity and the affinity of the antibody and will have to be viewed in terms of titration, in contrast to quantitation. (3).

Enzyme-Linked Immunosorbent Assay Quantification

Quantification using ELISA, could be done in both competitive and noncompetitive procedures. Incubation of a constant amount of the same antigen, that is also to be measured, is labeled with anenzyme, with a constant amount of immobilized antibody. After washings, the enzyme activity associated with the solid phase is further measured, and due to the antibody-combining sites being less than the number of epitopes present on the unlabeled and labeled antigen, competition is formed between them. Because the only variable is the level of unlabeled antigen, enzyme retained by the immobilized antibody is inversely proportional to the amount of antigen to be determined. Moreover, the immunometric procedure can also be used, which depends on the use of a constant and limited amount of enzyme bound to solid phase decreases with increasing concentrations of the antigen to be measured. The "sandwich" ELISA is the only noncompetitive procedure, which involves the use of a constant amount of immobilized antibody and an excess of enzyme-labeled antibody with same specificities as first. Incubate the antigen with the immobilized antibody, followed with washings and the enzyme-labeled antibody is then added, incubated, and after washings, the enzyme associated with the solid phase is measured. Therefore, the enzyme activity is proportional to the amount of antigen added. Furthermore, calibration curves are created with varying amounts of antigen, and unknwon concentrations of antigen is determined by reference to these standard curves (3,4).

Enzyme-Linked Immunosorbent Assay Controls

Controls for ELISA are essential. A negative control would be the medium alone, while a positive control could be the supernatant from a normal B cell wild-type or other normal non-altered cell line. This would allow for an indication as to the absorbance derived from a normal concentration of IgM (in addition, IgG could be used, to depict any binding of non-IgM that could have been detected as IgM, due to Fc binding antibodies, binding other Ig's). The use of the consistent microtitre plastic surface is essential, as the adsorbing capacity varies from one set to another. Moreover, nonspecific adsorption can be further reduced with the use of includingin the medium, through washings and incubation, a nonionic detergent, such as Tween-20, either alone of supplemented with a protein, as bovine serum albumin. In addition, if using large numbered samples, it is better to use enzymes for which stable substrates are available and therefore allow for reactions of longer duration, stability. Moreover, the limiting factor is the binding affinity of the antibody, which ultimately determines the sensitivity of the heterogeneous enzyme immunoassays (3,4).

References:

1. Janeway C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA pages 1-40, 2.5-2.22, 3.1-3.12

2. Delves P., and Roitt I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA

3. 1994 Current Protocols in Molecular Biology. Volume 2. John Wiley & Sons Inc., USA

4. Cruse J., and Lewis R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA pages 1960-1965

5. Chen F.H., and Painter R.H. 1997. Domain switched mouse IgM. IgG2b hybrids indicate individual roles for C domains in the regulation of the interactions of IgM complement C1q. J. Immunol. 159, 3354-3363

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