Skip to main content
SpringerLink
Log in

Development and Characterization of Guinea Pig Anti-Insulin Polyclonal Antibody

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Development and characterization of guinea pig anti-insulin polyclonal antibody against a target-specific insulin antigen. In India, an insulin immunogenicity kit for detecting insulin antibodies (neutralizing Nab) is an unmet medical need for diabetic patient’s routine diagnosis. Type 1 diabetics rely on insulin injections daily basis for survival; if the body develops anti-insulin antibodies and neutralizes the exogenous recombinant insulin, glucose control is lost, and the patient eventually dies. Antibodies are excellent diagnostic reagents due to the specificity and sensitivity they provide in recognizing specific and unique target antigens. The paper describes the use of insulin as a target antigen and the development of target (insulin) specific antibodies in guinea pigs for use as a positive control for immunogenicity kit validation. Anti-insulin polyclonal antibody was raised against insulin in the Dunkin Hartley guinea pigs host. Anti-insulin antibody titer of all bleeds from four animals was tested using an indirect ELISA assay format. All four animals responded to the target-specific antigen but only one animal (#4) responded with a high-affinity antibody titer. The hyperimmune sera were purified using a protein A column. The purified anti-insulin antibody was characterized through SDS Page and western blot. The specificity, reactivity, and antibody binding efficiency were confirmed through immunoassays. Guinea pig anti-insulin polyclonal antibody developed in this study showed good specificity, reactivity, and efficiency in the immunoassays. This paper describes the development and characterization of anti-insulin antibodies for use as a control in developing a user-friendly insulin immunogenicity kit.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

Data Availability

The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Mathieu, C., Gillard, P., & Benhalima, K. (2017). Insulin analogues in type 1 diabetes mellitus: Getting better all the time. Nature Reviews Endocrinology, 13, 385–399.

    Article  CAS  PubMed  Google Scholar 

  2. Sharma, S. K., et al. (2018). Immunogenicity, Safety and efficacy comparison of Wockhardt’s biosimilar insulin glargine—Glaritus® with Reference Product— Lantus®: Study protocol & early data trends. Open Journal of Endocrine and Metabolic Diseases, 08, 157–166.

    Article  CAS  Google Scholar 

  3. Tieu, C., Lucas, E. J., DePaola, M., Rosman, L., & Alexander, G. C. (2018). Efficacy and safety of biosimilar insulins compared to their reference products: A systematic review. PLoS One, 13(4), e0195012.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Home, P., et al. (2018). Anti-insulin antibodies and adverse events with biosimilar insulin lispro compared with humalog insulin lispro in people with diabetes. Diabetes Technology & Therapeutics, 20, 160–170.

    Article  CAS  Google Scholar 

  5. Fineberg, S. E., et al. (2007). Immunological responses to exogenous insulin. Endocrine Reviews, 28, 625–652.

    Article  CAS  PubMed  Google Scholar 

  6. Hu, X., & Chen, F. (2018). Exogenous insulin antibody syndrome (EIAS): A clinical syndrome associated with insulin antibodies induced by exogenous insulin in diabetic patients. Endocrine Connections, 7, R47–R55.

    Article  CAS  PubMed  Google Scholar 

  7. Lablanche, S., et al. (2014). Impact of anti-insulin antibodies on islet transplantation outcome: Data from the GRAGIL network. Transplantation, 98, 475–482.

    Article  CAS  PubMed  Google Scholar 

  8. Wright, P. H., & Malaisse, W. J. (1966). A simple method for the assay of guinea pig anti-insulin serum. Diabetologia, 2, 178–188.

    Article  CAS  PubMed  Google Scholar 

  9. Even, M. S., Sandusky, C. B., Barnard, N. D., Mistry, J., & Sinha, M. K. (2007). Development of a novel ELISA for human insulin using monoclonal antibodies produced in serum-free cell culture medium. Clinical Biochemistry, 40, 98–103.

    Article  CAS  PubMed  Google Scholar 

  10. Boto, R. E. F., Almeida, P., & Queiroz, J. A. (2008). Thiacarbocyanine as ligand in dye-affinity chromatography. Biomedical Chromatography, 288, 278–288.

    Article  Google Scholar 

  11. Fishman, J. B., & Berg, E. A. (2019). Peptide affinity purification of antibodies. Cold Spring Harbor Protocols, 2019(5), pdb.prot099176.

    Article  Google Scholar 

  12. Laemmli, U. K. (1970). Denaturing (SDS) discontinuous gel electrophoresis. Nature, 227, 680–685.

    Article  CAS  PubMed  Google Scholar 

  13. Valedkarimi, Z., et al. (2018). Production and characterization of anti-human IgG F(ab’)2 antibody fragment. Human Antibodies, 26, 171–176.

    Article  CAS  PubMed  Google Scholar 

  14. Nell, L. J., Virta, V. J., & Thomas, J. W. (1985). Application of a rapid enzyme-linked immunosorbent microassay (ELISA) to study human anti-insulin antibody. Diabetes, 34, 60–66.

    Article  CAS  PubMed  Google Scholar 

  15. Ueno, H., Nishiyama, A., Akita, M., et al. (1994). The measurement of insulin antibodies and insulin autoantibodies by enzyme-linked immunosorbent assay using recombinant human insulin antigen and its clinical application. Nippon Naibunpi Gakkai zasshi, 70(6), 585–596. https://doi.org/10.1507/endocrine1927.70.6_585

    Article  CAS  PubMed  Google Scholar 

  16. Casesnoves, A., Mauri’, M., Dorninguez, J. R., Alfayate, R., & Picó, A. M. (1998). Influence of anti-insulin antibodies on insulin immunoassays in the autoimmune insulin syndrome. Ann Clin Biochem, 35(6), 768–774.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Affigenix Biosolutions Pvt Ltdprovided the equipment and laboratory facility for the investigation.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Sathyabama Institute of Science and Technology, Chennai, India

    Sathiya Varadarajan & V. Ramesh Kumar

  2. Affigenix Biosolutions Pvt Ltd, Chennai, India

    Arumugam Muruganandam

Authors
  1. Sathiya Varadarajan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arumugam Muruganandam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. V. Ramesh Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

In this study, Sathiya Varadarajan: methodology, writing (original draft preparation); Dr.Arumugam Muruganandam: formal analysis and investigation, writing (review and editing); supervision: Dr. V Ramesh Kumar. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to V. Ramesh Kumar.

Ethics declarations

Ethics Approval

IAEC approval number ABS-IAEC-032–2021-22

Consent to Participate

This article is done by us together.

Consent for Publication

All authors are willing to publish this work.

Conflict of Interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Varadarajan, S., Muruganandam, A. & Kumar, V.R. Development and Characterization of Guinea Pig Anti-Insulin Polyclonal Antibody. Appl Biochem Biotechnol (2023). https://doi.org/10.1007/s12010-023-04670-z

Download citation

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12010-023-04670-z

Keywords

Search

Navigation