A strategy for rapid in situ elimination of interfering substances that are present in extracts of food samples during assay is described in this article. The novel feature of this method is that the sample purification is carried out as a part of the assay, and a separate sample cleanup step is not required. The assay procedure involves the sequential addition of standard or sample, cleaning solutions, and aflatoxin B1-horseradish peroxidase conjugate (AFB1-HRP) over antibody-spotted zones of a membrane, and 3,3'-diaminobenzidine was used as the substrate for visualization. We have determined that trifluoroacetic acid and propionic acids at concentrations of 100 mM are highly effective for cleaning groundnut, wheat, corn, and poultry feed samples and that NaHCO3 (100 mM) is successful in cleaning processed soybean. In all cases, subsequent washing was performed with phosphate-buffered saline solution to facilitate the removal of traces of adhering interfering substances. A batch of 12 samples can be analyzed within 8 min either by visual comparison of the color intensity (inversely related to the analyte concentration) of a sample spot with those of reference standards or, more precisely, by densitometry. The method was tested for the analysis of AFB1 in groundnut, wheat, corn, processed soybean, chili, and poultry feed. The detection limit obtained was 5 microg/kg, except for chili, where it was 10 microg/kg. The average recoveries from different noninfected food samples spiked with AFB1 at concentrations of 5 to 100 microg/kg were between 99 and 105%. The values obtained for infected corn and groundnut samples correlated well with the estimates obtained by high-pressure liquid chromatography. The absence of a sample extraction step reduces the cost and labor involved in the assay. The method may be potentially applicable to the assay of other mycotoxins and environmental pollutants.

A simple analytical device has been developed for performing noninstrumental immunofiltration-based assay on a batch of samples. The device consists of membrane strips, with antibody-immobilized zones, attached to a polyethylene card. A moist filter paper placed between the membrane and the polyethylene card acts as the absorbent body. The device was used to estimate very low concentrations of aflatoxin B1 (AFB1) present in food samples by using an improved catalyzed reporter deposition (Super-CARD) method of signal amplification involving biotinylated tyramine (B-T) and avidin-horseradish peroxidase conjugate. 4-chloro-1-naphthol was used as the substrate for visualization. Semiquantitative results are obtained by visual comparison of the color intensity (inversely related to the analyte concentration) of a sample spot with those of reference standards. Quantitative estimation is possible by densitometric analysis (detection limit 0.25 pg/spot, 0.01 ng mL(-1)). Dilute samples can be assayed by in situ concentration with improved dose-response characteristics. A batch of 12 extracted samples can be analyzed in a single test card within 12 min. Spiked and contaminated samples of groundnut, corn, wheat, cheese, and chilli were analyzed without sample cleanup. The matrix interferences were eliminated by using appropriate dilution of the aqueous methanol extracts. Mean recoveries from different food samples were between 91 and 104%. The values obtained for infected corn and groundnut samples correlated well (R2=0.99) with the estimates by HPLC. The method is well-suited for visual screening of agricultural and food samples for AFB1 under field conditions.

An improved analytical device capable of performing simultaneous immunofiltration-based immunoassay on 30 samples in the presence of reference standards has been developed. The device consists of a rectangular membrane with 36 antibody spotted zones, one end of which was attached to a semirigid polyethylene card. A piece of wetted filter paper between the membrane and the polyethylene card absorbs the added reagent. The assay is a competitive one using T-2 toxin-horseradish peroxidase (T-2 toxin-HRP) as the labeled analyte and 4-chloro-1 naphthol (4CN) as the substrate. Signal amplification was done by the Super-CARD signal amplification method. Semiquantitative results were obtained by visual comparison of the color intensity of a sample spot with those of reference standards. Densitometric analysis was used for quantitation. The method allows rapid and easy determination of T-2 toxin in wheat and poultry feed with detection limits of 12.5 and 25 microg x kg(-)(1), respectively, with accuracy and precision. Matrix interference was eliminated by appropriate dilution of sample extracts with assay buffer. The detection sensitivity in ELISA was 10-fold higher than that in the membrane-based method. Noninfected samples were spiked with T-2 toxin at several concentrations and analyzed by the present method and rapid ELISA. Mean recoveries by both methods were between 80 and 108%. The correlation between the two methods was excellent (R(2) = 0.99).

A novel strategy to improve significantly antigen detection sensitivity of Dot-ELISA by catalyzed reporter deposition (CARD) method of signal amplification has been developed. The method, termed Super-CARD, utilizes synthesized electron rich proteins having multiple binding sites as blocking agents. After completion of conventional Dot-ELISA, the solid phase bound horseradish peroxidase (HRP) oxidises the added labeled substrate, which deposits onto the solid phase. This deposition is markedly increased in the presence of immobilized electron rich proteins, which not only amplifies the signal but also increases the sensitivity. The high specificity of the amplification reaction avoids the generation of any false positive signal. The extremely high sensitivity of Super-CARD technology permits visual detection of as few as 800 rabbit IgG molecules (1.33 x 10(-21) mol). The method is approximately 10(5)-fold more sensitive than conventional Dot-ELISA. Direct comparison with existing CARD methods demonstrates approximately 1.6 x 10(4)-fold enhancement in detection sensitivity which is much higher than that of any other existing methods. The Super-CARD technology is specific, flexible and may be applied to clinical diagnostics.

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.

A new and simple method for enzyme immunoassay of folic acid (FA) has been developed, which does not require extraction or heat denaturation of serum. FA-free serum for standards was prepared by a new immunosorbent technique as conventional methods were unsuccessful. The detection limit of the assay is 0.05 ng/ml. Intra- and interassay variabilities ranged between 5-13.3%. Analytical recoveries obtained after spiking with different amounts of FA ranged between 93-110%. We eliminated the interference of endogenous folate binding protein - a major problem in direct FA assay by incubating serum samples (or standards) with FA-HRP conjugate in antibody coated plates at 50 degrees C. Comparison of our data with results obtained by microbiological assay and also by heating samples in alkaline buffer showed good correlation.

We have developed an improved method, using 96-well microtiter plates, for the microbiological assay of folic acid. With this method, the tedium of conventional microbiological analysis is substantially decreased. Culture volumes have been reduced 33-fold, and pipetting procedures have been simplified. Assay time has been reduced to 14 h, and sensitivity has increased 10-fold (0.1 ng/mL). Analytical recoveries range from 98 to 104%. Intra-assay and interassay variabilities are less than 11%. The assay does not require extensive manipulation of inoculum. Day-to-day variability has been minimized by using saline aliquots of the bacterial suspension stored at 4 degrees C. The procedure is accurate, selective, and useful for direct measurement of folic acid in multivitamin formulations.

For raising high titre and specific antibody to haptens or drugs, epsilon-aminocaproic acid modified bovine serum albumin (epsilon-ACA-BSA) was prepared for use as a carrier protein. Folic acid (FA) was coupled to epsilon-ACA-BSA, Imj.BSA and BSA for raising antibodies in rabbits. Enhancement of FA immunogenicity with FA-ACA-BSA was observed. Apart from determination of titre by indirect ELISA, dose-response behaviour and specificity of these antisera were also compared. FA-ACA-BSA antibody showed high sensitivity and specificity. Using this antibody, an ELISA method for the determination of FA was developed. The study provides a simple approach to raise highly specific and high titre antibody against small molecules.

The production of phaseolinone, a phytotoxic metabolite of Macrophomina phaseolina in infected Phaseolus mungo seeds grown on soil, was estimated by enzyme‐linked immunosorbent assay and HPLC. The degree of inhibition of seed germination correlated well with the amount of toxin produced; 50% inhibition was observed at a toxin level of 2.1 μg g‐1 of wet tissue. A comparison of the toxin‐producing ability of nine isolates of the fungus obtained from different hosts and localities showed that the strain MPK'83 produced a significantly larger amount of the toxin, both in liquid culture and in infected seeds. The virulence of the isolates was related to their ability to produce phaseolinone.

The effect of dimer heterology in the sandwich immunoassay of testosterone was studied using symmetrical and asymmetrical dimers prepared from testosterone-3-(O-carboxymethyl)oxime and 4-(carboxymethyl-mercapto)testosterone. The effect of antibody heterology was studied using antibodies against 3-carboxymethyl oxime and 17-hemisuccinate derivatives of the steroid and an asymmetrical dimer prepared from the same two derivatives. Using antibody against the 3-carboxymethyl oxime and the dimer of 4-(carboxymethylmercapto)testosterone, the sensitivity of direct enzyme immunoassay of testosterone in serum by this method was 0.5 pg/well and the cross-reactivity of 5-alpha-dihydrotestosterone was 5%.

A method for enzyme immunoassay of testosterone in serum has been developed which does not require extraction of the serum with organic solvents. The release of testosterone from the binding proteins is achieved by heating the serum at 70-degrees-C for 30 min in an alkaline buffer. The results correlated well (r = 0.96) with those of a radioimmunoassay using I-125-labelled testosterone and with enzyme immunoassay with prior extraction of samples (r = 0.98). The detection limit of the assay is 1 pg per well and the turn around time for 36 samples is 3.5 h. The procedure is simple and well suited for routine analysis.

A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue.

A homogeneous enzyme immunoassay for estriol has been described using estriol-glucose-6-phosphate dehydrogenase (E3G6PD) as enzyme conjugate. Antisera for estriol were raised by immunising rabbits with two different immunogens, estriol-6-(O-carboxymethyl oxime) bovine serum albumin (E36CMOBSA) and estriol-3-carboxymethyl ether bovine serum albumin (E33CMEBSA). A new method for preparation of estriol 3-O-carboxy methyl ether in high yield (90%) has also been described. Addition of anti-estriol antibodies to E3-G6PD conjugate resulted in inhibition of enzyme activity. In the presence of free estriol, the antibody induced inhibition of enzyme activity was reduced in a concentration dependent manner. The use of the heterologous combination between immunogen and enzyme conjugate i.e. using anti-E33CMEBSA and E36CMOG6PD improves the sensitivity at the expense of specificity, the cross-reaction with other estrogenic hormone being 10–15%.

Structure of phaseolinone, a phytotoxin isolated from the fungus Macrophima phaseolina (Tassi) Gold, has been established as 1.