Spectra analysis of coating antigen: A possible explanation for difference in anti-AFB1 polyclonal antibody sensitivity

Abstract

For the detection of small hapten molecules, indirect competitive enzyme-linked immunosorbent assay (icELISA) is a preferred method. However, diverse coating antigen might bring different antiserum titer and sensitivity for the identical antiserum. In the present study, four AFB1-protein (aflatoxin B1-carrier protein) conjugates were prepared by activated ester method (AFB1O-BSA/AFB1O-OVA) and mannich method (AFB1-cBSA/AFB1-cOVA), and then applied as coating antigen for titer and sensitivity detection of the identical antiserum obtained from rabbit immunized by AFB1-KLH. Afterwards, the ultraviolet-visible, fluorescence and far-ultraviolet circular dichroism (far-UV CD) spectra were recorded for understanding the difference in titer and sensitivity obtained. Results revealed that AFB1O-BSA/AFB1O-OVA showed a strong intrinsic fluorescence band centered at 450 nm that originated from the emission of AFB1, which differed from AFB1-cBSA/AFB1-cOVA, while the decrease of α-helical and increase of β-sheet in AFB1-cBSA was the most remarkable. This indicated that the better sensitivity obtained by using AFB1O-BSA as coating antigen might be caused by its extended structure, because such structure affect the binding between AFB1 and antibody. The study might offer structural information for understanding the titer and sensitivity difference caused by coating antigen.

Dual-Modal Split-Type Immunosensor for Sensitive Detection of Microcystin-LR: Enzyme-Induced Photoelectrochemistry and Colorimetry

“…In Figure D, there is a positive peak at about 275 nm for S 0 (Figure D, curve a), the characteristic absorption of single-strand DNA. In Figure D, curve b, for the spectrum of Ab 2 , two negative peaks at about 209 and 223 nm appeared, demonstrating that the structure of the protein was mainly α-helical secondary structure . As for Ab 2 –MSNs–S 0 conjugate (Figure D, curve c), the absorption peaks of Ab 2 and S 0 were both detected, proving that biomolecules were successfully modified on MSNs without changing their secondary structure.…”

“…In Figure 2D, curve b, for the spectrum of Ab 2 , two negative peaks at about 209 and 223 nm appeared, demonstrating that the structure of the protein was mainly α-helical secondary structure. 36 As for Ab 2 −MSNs−S 0 conjugate (Figure 2D, curve c), the absorption peaks of Ab 2 and S 0 were both detected, proving that biomolecules were successfully modified on MSNs without changing their secondary structure. To prove the formation of dsDNA after HCR, the gel electrophoresis was investigated.…”

Conjugation of Different Aflatoxin Derivatives to Proteins and Their Use as Heterologous Antigens in Immunoassay Development

“…So, we propose that a change at secondary structure which cannot be resolved under reducing conditions of SDS PAGE occurred upon conjugation reaction. The structural changes upon hapten conjugation is a known phenomenon which was discussed by Ye, …”

Camelization of a murine single-domain antibody against aflatoxin B1 and its antigen-binding analysis

“…HisPur Ni–NTA resin, B-PER extraction reagent, and Nunc MaxiSorp flat-bottom 96-well plates were obtained from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Coating antigens AFB 1 -OVA were formed by AFB 1 -oxime derivative coupling with carrier proteins in our laboratory (Ye et al 2016 ).…”