Development and validation of carbodiimide-activated ELISA plate for quantifying IgG levels against Streptococcus pneumoniae capsular polysaccharides

Abstract

Background: Conventional microtiter plates lack the surface strength needed for effective binding of pneumococcal polysaccharide antigens. This study tackles the limitation by altering the surface of polystyrene plates through carbodiimide activation under acidic pH conditions. Method: The microtiter plates were activated with carbodiimide coupling agents, N,N′-Dicyclohexylcarbodiimide (DCC) and N-Hydroxysuccinimide (NHS). They were subsequently coated with 13 pneumococcal antigens at a concentration of 5 μg/ml with a pH of 3.5. The IgG antibody titer was assessed utilizing the World Health Organization (WHO) ELISA protocol for 30 human serum samples. In addition, validation experiments were conducted to evaluate specificity and precision. Results: The modified plates exhibited two-times higher antibody titers compared to conventional plates across all 13 serotypes. Observations revealed elevated antibody levels, with geometric concentrations ranging between 0.96 μg/ml and 4.24 μg/ml. Conclusion: Carbodiimide activation and acidic pH modification of microtiter plates enhance sensitivity and specificity in detecting pneumococcal antibodies, critical for vaccination planning and immunity assessment.

Article highlights

• Accurate detection of pneumococcal antibodies is vital for evaluating individual immunity and designing vaccination strategies, highlighting the need for a dependable method to assess specific immunity to pneumococcal polysaccharide antigens.

• Despite World Health Organization guidelines, it is essential to adapt methods to local settings and resources when measuring serotype-specific pneumococcal antibodies.

• The WHO's commonly recommended polystyrene microtiter plates are expensive, prompting the exploration of alternative methods.

• A simple, sensitive method involves covalently attaching polysaccharide antigens to polystyrene surfaces at pH 3.5, ensuring robust binding for precise assessment.

• Modified ELISA plates show two- to four-times higher antibody titers and outperform conventional plates, enhancing pneumococcal antibody detection reliability.

Keywords: :

• carbodiimide activation
• IgG antibodies
• microtiter plate
• polysaccharide antigens
• Streptococcus pneumoniae
• validation

Supplemental material

Supplementary data for this article can be accessed at https://doi.org/10.1080/17576180.2024.2349453

Acknowledgments

Rijo Joseph John (Instruments Manager), G K Netravathy (Accounts Manager), and K N Ravishankar (Accounts Manager) of the Central Research Laboratory, Kempe Gowda Institute of Medical Sciences, Bengaluru, India.

Author contributions

Shincy MR and Akhila MM collaborated on the study, jointly conducted the research, analyzed the data, and drafted the manuscript. Both authors made equal contributions to the study. V Govindan assisted in conducting the assays. G Nagaraj helped in analyzing the data. KL Ravikumar provided revisions to the manuscript. All authors reviewed and approved the final version of the manuscript.

Financial disclosure

This work was supported with funding from the Biotechnology Industry Research Assistance Council (BIRAC), Govt of India under Award No. BT/NBM/0097/02/18 with a project entitled ‘‘Establishment of Pneumococcal Vaccine Immunogenicity Evaluation Center at CRL-Kempegowda Institute of Medical Sciences, Bangalore” under Industry-Academia Collaborative Mission for Accelerating Discovery Research to Early Development for Biopharmaceuticals. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The study was conducted in compliance with KIMS-IRB guidelines, under an approved protocol (KIMSIEC/S04-2020).Written consent was obtained from all participants prior to their involvement in the study. Participants were provided with a detailed consent form outlining the purpose of the research, potential risks and benefits, confidentiality measures, and their rights as participants. They were given ample time to review the information and ask any questions before providing their written consent.

Additional information

Funding

This work was supported with funding from the Biotechnology Industry Research Assistance Council (BIRAC), Govt of India under Award No. BT/NBM/0097/02/18 with a project entitled ‘‘Establishment of Pneumococcal Vaccine Immunogenicity Evaluation Center at CRL-Kempegowda Institute of Medical Sciences, Bangalore” under Industry-Academia Collaborative Mission for Accelerating Discovery Research to Early Development for Biopharmaceuticals.