Papers by Keyword: Indirect Competitive ELISA

Abstract: This paper reports an indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody (pAb) for estradiol (E2) residues. After derivation, E2 haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method, and New Zealand white rabbits were immunized to produce anti-E2 pAb. The conjugation ratio of E2-BSA was proved to be 18.6:1 by an UV absorbance method. Based on the square matrix titration, an icELISA standard curve was developed. The dynamic range was from 0.16 to 128 ng/mL, with LOD and IC50 value of 0.08 ng/mL and 3.76 ng/mL, respectively. Except for a little cross-reactivity (16.2%) to estrone, this assay showed negligible cross-reactivity to other analogues tested. The results suggest that the produced anti-E2 pAb could be used to develop an icELISA method for the determination of E2 residues in animal-originally products.

Abstract: This study aimed to develop a monoclonal antibody based icELISA method for Ractopamine (Rac) residue. For this purpose, mixed anhydride method was employed to synthesize the immunogen of Rac-BSA and 1, 4-butanediol diglycidyl ether was used to prepare the coating antigen of Rac-OVA, thus pursue the heterologous sensitivity. Through cell fusion technology, four Hybridoma named R1-B5, R2-B3, R2-C6, and R4-C8 were screened out, and the Kas of all mAbs were between 2.7 and 4.8×109 L/mol. Based on the R1-B5 mAb, a heterologous icELISA standard curve was developed. The working range was from 0.013 to 33.7 ng/mL, with LOD and IC50 value of 0.007 ng/mL and 0.67 ng/mL, respectively. Therefore, this icELISA can be used for detecting Rac residue in animal products.

Abstract: A rapid indirect competitive ELISA (icELISA) for the determination of enrofloxacin (ENR) residue has been developed. EDC method was employed to synthesize the artificial antigen of ENR-BSA, and anti-serum produced from rabbits was selected. Based on the square matrix titration, an icELISA method was developed with the polyclonal antibody. The Linear range was from 0.006 to 31.5 ng/mL, with LOD and IC50 values of 0.003 ng/mL and 0.45 ng/mL, respectively. Except for a high cross-reactivity (105.2%) to ciprofloxacin, negligible cross-reactivity to other compounds was observed. The recoveries of ENR were in the range of 98.3-127.5%, 85.7-112.5% and 97.4-103.8% for chicken muscle, liver and kidney, respectively. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in food.

Abstract: This article aimed to optimize a polyclonal antibody based indirect competitive ELISA (icELISA) and compare between the icELSIA method and the LC-MS technology for the determination of enrofloxacin (ENR) residue. Based on the square matrix titration, linear range of the icELISA was from 0.006 to 31.5 ng/mL, with LOD and IC50 values of 0.003 ng/mL and 0.45 ng/mL, respectively. After optimization, 0.03 mol/L of HCl was used in the assay buffer and this ELISA system can tolerate acetonitrile not higher than 10%. The recoveries of ENR were in the range of 98.3-127.5%, 85.7-112.5% and 97.4-103.8% for chicken muscle, liver and kidney. The correlation coefficients (R2) of the ELISA and LC-MS data were 0.9472 in muscle, 0.9843 in liver, and 0.9382 in kidney, respectively.

Abstract: A multiresidue immunoassay method for determination of Fluoroquinolones (FQs) residues has been developed. For this purpose, NHS ester technology was employed to synthesize the immunogen and coating antigen of Norfloxacin (NFLX). SDS-PAGE, UV-visible spectra and Infrared spectra identification showed that the artificial antigen was conjugated successfully. Based on the square matrix titration, an icELISA method was established. The dynamic range in assay buffer was from 0.038 to 112.8 ng/mL, with LOD and IC50 value of 0.02 ng/mL and 1.2 ng/mL, respectively. This assay showed a high cross-reactivity to Ciprofloxacin (86%), Enrofloxacin (75%), Difloxacin (63%), Sarafloxacin (57%) and Pefloxacin (33.8%). The chemical effects on assay performance showed that the physiological pH (7.4) in assay buffer pursued the maximum absorbance (A_max) and the most sensitive IC50 values. The results suggest the artificial antigen was synthesized successfully, and the established immunoassay could be used for simultaneous detecting of Norfloxacin, Ciprofloxacin, Enrofloxacin, Difloxacin, Sarafloxacin and Pefloxacin residues in animal-original food samples.

Abstract: A rapid indirect competitive ELISA (icELISA) format has been developed for the determination of enrofloxacin (ENR) residues in chicken. For this purpose, carbodiimide active ester method was employed to synthesize the artificial antigen of ENR-BSA, and anti- serum produced from the immunized rabbits was tested by indirect ELISA and icELISA. By the square matrix titration, the icELISA method was developed for the quantitative detection of ENR, based on the pAb. The Linear range was from 0.006 to 31.5 ng/mL, with LOD and IC50 value of 0.003 ng/mL and 0.45 ng/mL, respectively. Except for a high cross-reactivity (105.2%) to Ciprofloxacin, negligible cross-reactivity to the other compounds was observed. The recoveries of ENR were in the range of 98.3-127.5%, 85.7-112.5% and 97.4-103.8% for chicken muscle, liver and kidney, respectively. After optimization, 0.03 mol/L of HCl was used in the assay buffer and this ELISA system can tolerate acetonitrile not higher than 10%. The correlation coefficients (R2) of the ELISA and LC-MS data were 0.9472 in muscle, 0.9843 in liver, and 0.9382 in kidney. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in food.

Abstract: This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against enrofloxacin (ENR) through cell fusion procedures, and the development of a mAb-based indirect competitive ELISA (icELISA) method to detect ENR residue using one of these Hybridomas (clone 4B5-D6). Under the optimal experimental conditions, this assay exhibited a working range of 0.004-38 ng/mL with IC50 and LOD values of 0.4 and 0.002 ng/mL, respectively. Except for a high cross-reactivity (105.2%) to Ciprofloxacin, negligible cross-reactivity to the other compounds was observed. After optimization, 10% of methanol was used in the assay buffer and this ELISA system can tolerate acetonitrile not higher than 10%. Recovery studies indicate that an excellent correlation between concentration spiked and concentration determined was found, and the results also suggest this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in poultry tissues.