A Practical ELISA for Azaspiracids in Shellfish via Development of a New Plate-Coating Antigen

Abstract A simple procedure was developed for in vitro synthesis and characterization of aflatoxin B1-lysine adduct using aflatoxin B1, N-α-acetyl lysine and m-chloroperbenzoic acid (MCPBA). At a molar ratio of 1:16 (aflatoxin B1:N-α-cetyl lysine), the recovery of adduct was 62%. Analysis of the adduct by thinlayer chromatography showed a single spot (Rf= 0). Absorption spectra of the adduct showed 2 peaks at 275 and 335 nm. Liquid chromatographic (LC) analysis of th AFB1-lysine adduct showed a relative retention time of 2.1 min. Using the same epoxidation procedure, BSA-AFB1 adduct and ovalbumin-AFB1 adduct were synthesized for production of antibodies and as coating antigen, respectively. Control rat serum, spiked with AFB1-lysine adduct and subjected to LC analysis showed a retention time of 2.1 min, which is similar to that of AFB1-lysine reference standard, synthesized. Further, enzymatically hydrolyzed, control rat serum spiked with BSA-AFB1 adduct showed 2 peaks with retention times of 2.1 and 2.7 min. Based on the LC analysis, recovery of BSA-AFB1 in terms of AFB1-lysine adducts was 67 ± 5%. The major peak (2.1 min) accounted for 72% of the adduct; the second minor peak (2.7 min) accounted for 28% of the total AFB1-lysine adducts formed. Stability studies on the AFB1-lysine adduct synthesized, indicated that it was stable for 1 month. Antibody capture assay showed an absorbance of 0.9 to 1.0 at a dilution of 1:50 000 when ovalbumin-AFB1 was used as a coating antigen. Indirect competitive ELISA showed 50% displacement (IC50) of the antibodies at a concentration of 13 ng AFB1-lysine, whereas the IC50 for AFB1 was 7 ng. The recovery of AFB1-lysine adduct spiked to control rat serum followed by enzymatic hydrolysis and immunoanalysis (indirect ELISA) was 93 ± 6%. The enzyme immunoassay was validated by a rodent model, in which the animals were exposed to aflatoxin B1 (20 μg AFB1/kg body mass/day). The level of AFB1-lysine adduct in the rat serum was 27.3 ± 4.37 μg/mg albumin.

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An enzyme-linked immunosorbent assay for the detection of antibody to avian reovirus by using protein σ B as the coating antigen

Research in Veterinary Science ◽

10.1053/rvsc.2000.0414 ◽

2000 ◽

Vol 69 (2) ◽

pp. 107-112 ◽

Cited By ~ 17

Author(s):

J.H SHIEN ◽

H.S YIN ◽

L.H LEE

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Avian Reovirus ◽

Coating Antigen

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An effective strategy for preparation of a polyclonal antibody with an addition of carbon chain of ciprofloxacin

European Journal of Inflammation ◽

10.1177/2058739218805645 ◽

2018 ◽

Vol 16 ◽

pp. 205873921880564

Author(s):

Dong Wei ◽

Guozhen Fang ◽

Shuo Wang

Keyword(s):

Polyclonal Antibody ◽

Limit Of Detection ◽

High Titer ◽

Carbon Chain ◽

Cross Reactivity ◽

Carrier Protein ◽

Effective Strategy ◽

Coating Antigen ◽

Spacer Arm ◽

Proper Modification

In this study, we synthesized amino propyl ciprofloxacin (CPLX-NH2) as a ciprofloxacin (CPLX) derivative. Moreover, the immune antigen CPLX-NH2-BSA and coating antigen CPLX-NH2-OVA were prepared via CPLX-NH2 coupling with bovine serum albumin (BSA) and ovalbumin (OVA), respectively. Subsequently, the Kunming mice were immunized with immune antigen to obtain the polyclonal antibody with high titer. The regression equation of CPLX-NH2 antibody was y = –17.395x + 89.331 (R2 = 0.9961); IC50 and limit of detection (LOD) were 182.39 and 20.09 ng/mL, respectively. These results were superior to that of CPLX antibody. Meanwhile, the CPLX-NH2 antibody showed cross-reactivity to fluoroquinolones (FQNs) residues. The results of the study indicated that the proper modification of the drug, namely, the addition of a suitable spacer arm between the drug and the carrier protein will improve the efficacy of the antibody, which is a favorable concept for preparation of antibody.

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Heterologous structure of coating antigen on sensitivity of ELISA for sulfamethazine: evidence from molecular similarity analysis

Food and Agricultural Immunology ◽

10.1080/09540105.2010.533752 ◽

2011 ◽

Vol 22 (2) ◽

pp. 115-124 ◽

Cited By ~ 8

Author(s):

Zhanhui Wang ◽

Jing Zhang ◽

Suxia Zhang ◽

Jianzhong Shen

Keyword(s):

Molecular Similarity ◽

Similarity Analysis ◽

Coating Antigen

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Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

Toxins ◽

10.3390/toxins12040273 ◽

2020 ◽

Vol 12 (4) ◽

pp. 273

Author(s):

Caixia Zhang ◽

Weiqi Zhang ◽

Xiaoqian Tang ◽

Qi Zhang ◽

Wen Zhang ◽

...

Keyword(s):

Amino Acid ◽

Ochratoxin A ◽

Limit Of Detection ◽

Basic Knowledge ◽

Affinity Constant ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Assay Sensitivity ◽

Coating Antigen ◽

Relative Standard

Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol−1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

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