Development and Optimization of ELISA for Serum Samples of Bovine Origin

Author: Shweta Yadav, Sushil Kumar, A.K. Mohanty, J.K. Kaushik and Sudarshan Kumar

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Abstract

The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical immunochemistry assay based on the specific bond between the antigen and the antibody. The application of this test has significantly changed the practice of animal laboratories in which it is used for detection and quantification of molecules such as hormones, peptides, antibodies, and proteins. Various technical variants of this test can detect antigen (native or foreign) or antibody, determine the intensity of the protein response whether pathological or not; the type of induced immune response as well as the innate immunity potential; and much more. These capabilities, as well as the high sensitivity and robustness of the test and a small price, make it quick and reliable diagnostic assay. Despite being used for nearly 50 years, a variety of ELISA tests with different technical solutions are still being developed, improving and extending the application of this outstanding test. Some challenges of the study are 1. Optimizing and standardizing enzyme immunoassays for veterinary applications may involve dealing with complex matrices in animal samples, which can introduce interfering substances affecting the assay's accuracy and specificity. 2. Validating the performance of ELISA tests for various veterinary molecules requires addressing issues related to cross-reactivity, sensitivity, and reproducibility, especially when dealing with diverse animal species and varying physiological conditions. 3. Developing and implementing new technical variants of ELISA while ensuring their reliability and comparability to existing methods pose challenges in terms of establishing appropriate reference standards and controls for accurate result interpretation. The purpose of this research article is optimization, standardization and validation of enzyme immunoassays in the veterinary field by overcoming all the challenges.

Keywords

ELISA, Optimization, Validation, Antigen and Antibody

Conclusion

In order to avoid non-specific binding of antigens and antibodies to the microtiter well, bovine serum albumin (BSA) is utilized as a blocking agent (Xiao and Isaacs 2012). Additionally, blocking agents can minimize non-specific interactions and stabilize the biomolecules attached to the well surface (Gibbs and Kennebunk 2001). Among the three blocking reagents, PBST showed higher background means excessive colour development. 1% BSA was used as blocking agent worked better than PBST. Since BSA is also from bovine origin (Majorek et al., 2012), it may affect the binding as well as can show cross reactivity with serum samples of bovines. Thus, to overcome this we used boiled 1% BSA which showed the best result (Fig. 1) among the three blocking reagents used. There are many components in serum samples, such as carbohydrates, proteins, and phospholipids that can interfere with the ability of the antibody pairs to bind to their target. This phenomenon is known as the "Matrix Effect". Due to this OD readings much lower than expected is obtained. Thus, dilution was best way to mitigate matrix effect and also allows the samples to fall under the range of standards. The selection of the diluent is crucial since samples must nearly always be diluted before being used in an ELISA test (Minic and Zivkovic 2020). It can be clearly observed from the results that matrix effect can be overcome by diluting the samples in 1:8 ratios in 1X PBST buffer (pH 7.4) so that one can achieve proper optimization in terms of OD reading for detecting the target samples. It can be clearly observed from the results (Table 1 (B), that matrix effect can be overcome by diluting the samples in proper ratio so that one can achieve proper optimization in terms of OD reading for detecting the target samples. After optimization of ELISA, the standard curve generated has shown R² = 0.9908 (Fig. 2) which would be considered perfect. Thus, it was concluded that the above optimization along with optimization of antibodies concentration has worked best for generation of standard curve based on the OD of samples.

References

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How to cite this article

Shweta Yadav, Sushil Kumar, A.K. Mohanty, J.K. Kaushik and Sudarshan Kumar (2023). Development and Optimization of ELISA for Serum Samples of Bovine Origin. Biological Forum – An International Journal, 15(5a): 217-221.