Tarun K. Dhar

A novel signal amplification technology for ELISA based on catalyzed reporter deposition. Demonstration of its applicability for measuring aflatoxin B1

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.

Homogeneous enzyme immunoassay of estradiol using estradiol-3-0-carboxymethyl ether as hapten

A homogeneous enzyme immunoassay for estradiol estimation has been developed, which can be extended to other steroids. A new procedure for the preparation of estradiol -3-0- carboxymethyl ether by a simple one step reaction in high yield (90%) has been described. This hapten has been used for raising highly specific anti-estradiol antibody in rabbits and for preparation of enzyme conjugates. Two different enzymes, lysozyme and glucose -6- phosphate dehydrogenase have been studied for their suitability as enzyme labels. Our results indicate that lysozyme-conjugate meets the essential requirement for a practical enzyme immunoassay. The advantage of the present nonradioactive procedure is the overall simplicity, low cost and high stability of the reagents.

Structure of phaseolinone, a novel phytotoxin from macrophomina phaseolina

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

Antibody to folic acid: increased specificity and sensitivity in ELISA by using ε-aminocaproic acid modified BSA as the carrier protein

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.

A novel signal amplification technology based on catalyzed reporter deposition and its application in a Dot-ELISA with ultra high sensitivity.

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.

Homogeneous enzyme immunoassay of estriol in picogram amounts

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-carboxymethyl 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%.

Direct microtitre plate enzyme immunoassay of folic acid without heat denaturation of serum.

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.

Sandwich immunoassay of small molecules. II: Effects of heterology and development of a highly sensitive and specific enzyme immunoassay for testosterone

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 filtration method for rapid preparation of conjugates for immunoassay

Modification of protein and other biopolymers by labeling them with small or macromolecules has become a very powerful research tool in biochemistry, molecular biology, diagnostics, and therapeutics. However, current methodologies available for their preparations are not straightforward and take several hours of incubation time. In this paper, we describe a new filtration-assisted technique for covalent conjugation between the reactive functional groups of two different molecules (small or macromolecules). Compared to the current method, this new approach significantly reduces the total reaction time from several hours to just a few minutes. The technique has been used for the preparation of conjugates of a small molecule to a protein such as biotin-BSA conjugate or small molecules to a small molecule such as biotin-tyramine conjugate or protein-protein conjugation such as antibody-horseradish peroxidase conjugate. The procedure consists of filtering the reaction mixture multiple times through membrane micropores with the help of two syringes, which make the cross filtration process less laborious. The method saves time, allows conjugation of less than 1mg protein and produces conjugates better than those obtained by the current methods. Although the present technique has been applied on some common conjugation methods, it provides a potentially general method, and may further be expanded for the synthesis of several other macromolecular conjugates.

Filtration-based staining of proteins on membranes

A filtration-based staining method has been developed for sensitive estimation of proteins in a batch of samples. It is based on focused absorption of an applied protein standard (or sample) and dye solution on nitrocellulose membrane through an aqueous network of capillary channels formed between the membrane and a wetted filter paper. The method does not require any equipment for creating vacuum. Compared with the conventional pouring methods, the new approach reduces the consumption of staining solution and lowers the staining and destaining times (from 1.5 h to approximately 10 min) without compromising the sensitivity.