A novel signal amplification technology for ELISA based on catalyzed reporter deposition. Demonstration of its applicability for measuring aflatoxin B(1).
In an earlier communication we have described a novel signal amplification technology termed Super-CARD, which is able to significantly improve antigen detection sensitivity in conventional Dot-ELISA by approximately 10(5)-fold. The method utilizes hitherto unreported synthesized electron rich proteins containing multiple phenolic groups which, when immobilized over a solid phase as blocking agent, markedly increases the signal amplification capability of the existing CARD method (Bhattacharya, R., Bhattacharya, D., Dhar, T.K., 1999. A novel signal amplification technology based on catalyzed reporter deposition and its application in a Dot-ELISA with ultra high sensitivity. J. Immunol. Methods 227, 31.). In this paper we describe the utilization of this Super-CARD amplification technique in ELISA and its applicability for the rapid determination of aflatoxin B(1) (AFB(1)) in infected seeds. Using this method under identical conditions, the increase in absorbance over the CARD method was approximately 400%. The limit of detection of AFB(1) by this method was 0.1 pg/well, the sensitivity enhancement being 5-fold over the optimized CARD ELISA. Furthermore, the total incubation time was reduced to 16 min compared to 50 min for the CARD method. Assay specificity was not adversely affected and the amount of AFB(1) measured in seed extracts correlated well with the values obtained by conventional ELISA.
Bhattacharya
D
D
Indian Institute of Chemical Biology, 4 Raja S C Mullick Road, Jadavpur, Calcutta, India.
Bhattacharya
R
R
Dhar
T K
TK
D016604:Aflatoxin B1 / Q000032:analysis*; D001230:Aspergillus / Q000472:pathogenicity; D002364:Caseins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D012639:Seeds / Q000737:chemistry / Q000382:microbiology; D012680:Sensitivity and Specificity; D014439:Tyramine
D002364:Caseins; D016604:Aflatoxin B1; D014439:Tyramine