Development of a highly sensitive icELISA to detect semicarbazide based on a monoclonal antibody

Abstract

A highly sensitive and specific monoclonal antibody was prepared to detect semicarbazide (SEM). Hapten 4-{[(aminocarbonyl)hydrazono]methyl}benzoic acid (4-CPSEM) was synthesised through the condensation reactions of SEM and 4-carboxybenzaldehyde (4-CBA). The active ester method was employed to couple the hapten to bovine serum albumin, keyhole limpet hemocyanin and ovalbumin (OVA) to obtain conjugates of immunogens and coating antigens. A novel hapten 2-[(aminocarbonyl)hydrazono] acetic acid (SEM-A) was also synthesised from SEM and oxoacetic acid to prepare a heterologous coating antigen (SEM-A-OVA). The linear ranges of inhibition curves in the optimised indirect competitive enzyme-linked immunosorbent assay (icELISA) method were in the range of 0.0071-0.056 ng mL −1 and 0.018-0.209 ng mL −1 for coating antigens SEM-A-OVA and 4-CPSEM-OVA, respectively. The IC 50 was 0.019 ng mL −1 for SEM (SEM in the form of 4-nitrobenzaldehyde semicarbazone) and 0.13 ng mL −1 for 2-nitrobenzaldehyde semicarbazone, both of which were well below the minimum required performance limit of 1 µg•kg −1 set by the European Commission.

Establishment of a monoclonal antibody-based indirect enzyme-linked immunosorbent assay for the detection of trimethoprim residues in milk, honey, and fish samples

“…The immunization schedule was performed based on standard protocol (Yin, Liu, Song, Kuang, & Xu, 2015). Briefly, five BALB/c female mice were subcutaneously injected at multiple points with 100 µg of immunogen emulsified with Freund's complete adjuvant.…”

Development of an ELISA and Immunochromatographic Assay for Tetracycline, Oxytetracycline, and Chlortetracycline Residues in Milk and Honey Based on the Class-Specific Monoclonal Antibody

“…On the other hand, immunoassays, based on specific interactions between antibodies and antigens, are rapid and cost-effective, with adequate sensitivity, efficient selectivity, and simple sample pretreatments. I m m u n o a s s a y s , i n c l u d i n g e l e c t r o c h e m i c a l immunosensor methods (Tang et al 2010), chemiluminescent immunoassays (Magliulo et al 2007), enzyme-linked immunosorbent assay (ELISA) (Yin et al 2014), and immunochromatographic lateral flow tests , are popular diagnostic tools for detecting large analytes, e.g., microorganisms (Feng et al 2013), proteins , and viruses (Sithigorngul et al 2006). For smaller analytes such as antibiotics , pesticides (Liu et al 2014a), heavy metals , and hormones (Kong et al 2014), ELISA and immunochromatographic lateral flow tests can be used.…”

Development of immunocolloidal strip for rapid detection of pyrimethanil

“…After each booster, the serum was collected from the tail vein of each mouse to assess the antibody specificity by ic-ELISA. The mouse with the highest titre and the best specificity to PYR was selected, and intraperitoneal immunization with 20μg immunogen, which was dissolved in 200μL normal saline was done (Yin, Liu, Song, Hua, & Xu, 2015). All animal studies in this work were performed according to institutional ethical guidelines and were approved by the Committee on Animal Welfare of Jiangnan University.…”