Development of anti-immunocomplex antibodies for rapid detection of sex hormones
Bäckström, Ida (2024-05-14)
Development of anti-immunocomplex antibodies for rapid detection of sex hormones
(14.05.2024)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
suljettu
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2024060747120
https://urn.fi/URN:NBN:fi-fe2024060747120
Tiivistelmä
The steroid sex hormones testosterone and estradiol (E2) regulate sexual differentiation and reproductive physiology. Substantial advancements have been made in understanding the relevance of sex hormones in both health and disease, and their broader significance now extends to conditions such as polycystic ovary syndrome, hypogonadism, osteoporosis, cancer, diabetes, and cardiovascular disease.
Even minor hormonal abnormalities can significantly impact health and disease risk. Precise detection and measurement, even at low concentration levels, is therefore essential. For this purpose, immunoassays offer a rapid, straightforward, and high-throughput method, in which the performance is influenced by assay format and antibody selection. While the non competitive sandwich enzyme-linked immunosorbent assay (ELISA) is effective for larger molecules, steroid hormones and other small analytes lack sufficient epitopes for this method. Consequently, the competitive ELISA, although standard, does not offer comparable sensitivity and reproducibility for measuring small analytes. Given the demand for high-performance assays, novel non competitive immunoassay concepts are needed. Anti-immunocomplex antibodies, which bind to the immunocomplex (IC) between a primary antibody and its small analyte, can be used to construct an immunoassay that resembles the sandwich ELISA.
The aim of this thesis was to generate anti-IC antibodies for testosterone and E2 ICs, with the ultimate goal of utilizing them in the development of a lateral flow (LF) assay. A synthetic fragment antigen binding (Fab) library was screened for anti-IC Fabs using phage display panning. After four rounds of enrichment followed by screening, specific clones were found for the testosterone IC and one clone demonstrated superior performance. The EC50 was 570 pM, indicating that the Fab can be used to detect samples within and below the reference ranges for testosterone in serum. The successful development of the anti-IC Fab opens possibilities for novel immunoassay applications for enhanced testosterone detection.
In addition, a primitive LF assay for detection of E2 was constructed using a smaller anti-IC antibody fragment. With optimization, this proof-of-concept assay could demonstrate how anti IC antibodies can be used in a point-of-care application.
Even minor hormonal abnormalities can significantly impact health and disease risk. Precise detection and measurement, even at low concentration levels, is therefore essential. For this purpose, immunoassays offer a rapid, straightforward, and high-throughput method, in which the performance is influenced by assay format and antibody selection. While the non competitive sandwich enzyme-linked immunosorbent assay (ELISA) is effective for larger molecules, steroid hormones and other small analytes lack sufficient epitopes for this method. Consequently, the competitive ELISA, although standard, does not offer comparable sensitivity and reproducibility for measuring small analytes. Given the demand for high-performance assays, novel non competitive immunoassay concepts are needed. Anti-immunocomplex antibodies, which bind to the immunocomplex (IC) between a primary antibody and its small analyte, can be used to construct an immunoassay that resembles the sandwich ELISA.
The aim of this thesis was to generate anti-IC antibodies for testosterone and E2 ICs, with the ultimate goal of utilizing them in the development of a lateral flow (LF) assay. A synthetic fragment antigen binding (Fab) library was screened for anti-IC Fabs using phage display panning. After four rounds of enrichment followed by screening, specific clones were found for the testosterone IC and one clone demonstrated superior performance. The EC50 was 570 pM, indicating that the Fab can be used to detect samples within and below the reference ranges for testosterone in serum. The successful development of the anti-IC Fab opens possibilities for novel immunoassay applications for enhanced testosterone detection.
In addition, a primitive LF assay for detection of E2 was constructed using a smaller anti-IC antibody fragment. With optimization, this proof-of-concept assay could demonstrate how anti IC antibodies can be used in a point-of-care application.