Development of anti-immunocomplex specific antibodies and non-competitive time-resolved fluorescence immunoassay for the detection of estradiol

Abstract

Detection of circulatory estradiol has widespread use in various clinical applications. Particularly, the use of estradiol-specific antibodies in immunoassays is routinely used, mainly due to the cost efficiency and simplicity of the sample handling process. However, the circulatory levels of estradiol can be extremely low in some conditions, and beyond the current detection limit of existing competitive immunoassays. We describe the generation of anti-immunocomplex specific antibodies derived from synthetic antibody repertoire and the development of high-performance non-competitive immunoassay for the detection of estradiol. Phage display selections were used to isolate new antibodies from synthetic antibody library with the use of existing estradiol specific Fab fragment. The found antibodies were consecutively used to set up a time-resolved fluorescence-based immunoassay (TRFIA), which can be used to detect estradiol with exceptional sensitivity and specificity. The limit of detection and EC50 were shown to be 3.0 pg mL −1 and 32.4 pg mL −1 respectively. Graphical abstract

Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins

“…The phages were repropagated from the cells collected from the output plate as described in Ref. [ 20 ]. A total of three selection rounds were performed whereby the amount of antigen was reduced to half after each round.…”

Site-Specific Linking of an Oligonucleotide to Mono- and Bivalent Recombinant Antibodies with SpyCatcher-SpyTag System for Immuno-PCR

“…The applicability of the system was also studied using another immunocomplex assay system intended for the detection of 17β-estradiol (E2), a steroid hormone essential for the development and sustaining of female reproductive tissues. 17 To this end, the synthetic antibody library-derived scFv C6 18 (termed later as anti-E2 scFv), which binds to the immunocomplex of E2 and antibody S16, was cloned to pLK06SC vector, thus creating scFv-AP-SpyCatcher construct. The expression yield was similar to anti-MC-LR scFv-AP-SpyCatcher ( Figure 2 ).…”

Application progress of magnetic molecularly imprinted polymers chemical sensors in the detection of biomarkers

“…Immunoassay mainly includes polymerase chain reaction (PCR), 2 enzyme-linked immunosorbent assay (ELISA), 3 radioimmunoassay (RIA) 4 and fluorescence immunoassay (FIA). 5 The above methods have good selectivity and accuracy, and have been gradually applied to formal medical tests. However, natural recognition molecules such as enzymes and antibodies have poor stability, environmental sensitivity, high prices, and limited sources, 6 which hinder the further application of immunoassays in biomarker detection.…”