Kiran P. Kariya

Substitution of carbonate buffer by water for IgG immobilization in enzyme linked immunosorbent assay.

Abstract The first step of enzyme linked immunosorbent assay (ELISA), namely, adsorption of antigen or antibody to the plastic microtiter well plate, was studied as a function of insolubility of IgG in water. Immobilization efficiency was assessed in terms of number of wells coated per milliliter of primary antiserum. We have compared different coating/immobilization protocols, i.e., direct and indirect immobilization of primary antibody to the plastic microtiter well plate using carbonate buffer and phosphate buffer with glutaraldehyde. We have observed efficient coating when the immobilization of primary antibody through an immunobridge technique was performed, where water was used as a coating medium. It gave a higher number of wells coated per milliliter of anti-serum (primary or secondary) than other compared coating protocols and it allowed the use of serum (non-immune) and anti-serum (primary and secondary antibody) dilutions, avoiding the need for γ-globulin purification from normal and immunized serum.

One step enzyme linked immunosorbent assay for direct estimation of serum testosterone.

Abstract One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of testosterone in human serum is described. Testosterone-3-O-carboxymethyl-oxime-bovine serum albumin (testosterone-3-O-CMO-BSA), was used as immunogen and testosterone-3-O-carboxymethyl-oxime-adipic-acid dihydrazide-horseradish peroxidase (testosterone-3-O-CMO-ADH-HRP) was used as tracer. To the testosterone antibody coated microtiter wells, standard or serum samples (100 µL), along with testosterone-3-O-CMO-ADH-HRP conjugate (100 µL) were incubated for 1 h at 37°C. Bound enzyme activity was measured by using tetra methyl benzidine/hydrogen peroxide (TMB/H2O2) as a substrate. In this new strategy, charcoal stripped pooled human serum spiked with non-cross reactive C18, C19, C21, and C27 steroids, used for preparing the standards and blocking the sex hormone binding globulin (SHBG)/and other steroid binding globulins (SBG). The sensitivity of the assay was 0.015 ng/mL. The intra-assay and inter-assay coefficients of variation (CVs) were ranged from 7.8 to 11.8 and 4.8 to 10.4, respectively. The serum testosterone values, obtained by this method, were correlated well with those obtained by radioimmunoassay r = .98 (n = 100).

Use of biotin–streptavidin system for developing a viable, sensitive and specific antigen heterologous assay for hapten

The present study demonstrates improvement in sensitivity and specificity of hapten assay by using antigen heterology in conjunction with low molecular weight biotin label as compared to high molecular weight horseradish peroxidase (HRP) label. For generation of antiserum, cortisol-3-O-carboxylmethyl-oxime-bovine serum albumin (F-3-CMO-BSA) was used as immunogen whereas, for the preparation of primary label, corticosterone-3-carboxymethyl oxime (B-3-CMO) was coupled with biotinylcaproylhydrazide and HRP by employing N-hydroxysuccinimide mediated carbodiimide reaction. The data of the present study revealed that the antigen heterologous assay which employed high molecular weight HRP label showed 100% cross-reaction with corticosterone. On the contrary, when HRP was replaced with low molecular weight biotin label, less than 0.1% cross-reaction was observed with all analogous C(18), C(19), C(21) and C(27) steroids including corticosterone (0.2%). Moreover, the sensitivity of the later assay was 0.09 microg/dL, which is appreciable as compared to previously reported enzyme based assays. The recovery of the exogenously spiked serum pools lies in the range of 90.3-104.2%. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 3.3% to 7.8% and 2.3% to 7.7%, respectively. The serum cortisol values obtained by this method correlated well with those obtained by radioimmunoassay; r=0.9 (n=50). The use of much stable biotin label in place of HRP has made the antigen heterologous enzyme linked immunosorbent assay (ELISA) of cortisol assay highly specific and sensitive.

Preparation of Enzyme Conjugate through Adipic Acid Dihydrazide as Linker and Its Use in Immunoassays

The direct coupling of the carboxylic derivative of steroids to the amino group of enzymes is a well-established method in steroid enzyme immunoassays for making enzyme conjugates (1). Horseradish peroxidase (HRP), containing six lysine residues in the sequence, is a widely used enzyme in enzyme immunoassays; in practice, however, only one or two of these are generally available for reaction (2). This variation in amino group content is caused by changes in the extraction conditions used for the isolation of HRP from the roots of the horseradish plant (2)(3). The low yield of HRP coupled to the IgG by the use of bifunctional reagents, namely glutaraldehyde, carbodiimide, cyanuric chloride, bis-diazotized O -dianisidine, and P - P ′-difluoro- m , m -dinitro-diphenyl sulfone (FNPS), and so forth, prompted Nakane and Kawaoi to investigate another method (periodate method) for the conjugation of HRP to IgG (4). Comparative coupling efficiency studies were carried out with the use of glutaraldehyde, periodate, and N -succinimidyl 3-(2-pyridyldithio) propionate (SPDP) as cross-linking reagents (5)(6) for the preparation of HRP-IgG conjugates. These studies revealed that the most efficient HRP-IgG conjugate was obtained by the periodate method. In practice, the difference in amino group availability in different batches of commercial preparations of HRP makes it difficult to establish standard reaction conditions that could be applicable for more than one batch. Therefore, adipic acid dihydrazide (ADH) has been coupled to HRP with the use of the periodate method reaction to provide the necessary amino group for the preparation of hapten enzyme conjugate with the use of the carbodiimide method. We describe for the first time the use of ADH as a linking reagent between glycoenzyme (HRP) and a steroid carboxylic derivative to prepare enzyme conjugate for ELISA. The conjugation of HRP to cortisol through ADH as …

Development of Rapid and Sensitive One‐Step Direct Enzyme Linked Immunosorbent Assay for 17‐α‐OH‐Progesterone in Serum

Abstract Using a homologous combination of immunogen and enzyme conjugate, a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) was developed to measure 17‐α‐hydroxy‐progesterone (17‐α‐OH‐P) in human serum. The antiserum was raised against 17‐α‐hydroxy‐progesterone‐3‐O‐carboxymethyloxime bovine serum albumin (17‐α‐OH‐P‐3‐O‐CMO‐BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17‐α‐hydroxy‐progesterone‐3‐O‐carboxymethyloxime with horseradish peroxidase (HRP). Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose‐response studies were carried out by incubating 25 µL enzyme conjugate along with 50 µL of standards on the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using Tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100 µL of 0.5 M H2SO4 after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The detection limit of the assay was 180 pg/mL. The assay was more specific as compared to most other reported immunoassays for 17‐α‐OH‐P. Cross reaction with analogous C18, C19, and C21 steroids was less than 0.1% except for progesterone which showed 2.1% cross reaction. The intra‐ and inter‐assay coefficients of variation ranges from 3.7–7.5% and 6.9–11.7%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.9 (n=30).

Development of ELISA for measurement of progesterone employing 17-alpha-OH-P-HRP as enzyme label.

Abstract The present study was aimed to develop a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) to measure progesterone in human serum using a heterologous combination of immunogen and enzyme conjugate. The antiserum was raised against Progesterone-3-O-carboxymethyloxime bovine serum albumin (P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-α-hydroxy-progesterone-3-O-carboxymethyloxime (17-α-OH-P-3-O-CMO) with Horseradish Peroxidase (HRP) to form 17-α-OH-P-3-CMO-HRP. A Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 100µL enzyme conjugate along with 50µL of standards in the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100µL of 0.5 M H2SO4 after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The lowest detection limit of the assay was 0.2 ng/mL. Cross-reaction with analogous steroids pregnenolone and 17-α-OH-P were found to be 6.8 and 6.1%, respectively. For other analogous steroids, it was less than 0.1%. The intra- and inter-assay coefficient of variation ranges from 4.52–7.39% and 4.65–9.55%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.91 (n = 40).

ENZYME LINKED IMMUNOSORBENT ASSAY FOR MILK PROGESTERONE

A direct antigen heterologous enzyme linked immunosorbent assay (ELISA) for milk progesterone has been developed using progesterone-3-O-carboxymethyloxime-bovine serum albumin (P-3-O-CMO-BSA) antiserum and 17-α-hydroxy-progesterone-3-O-carboxymethyloxime-horseradish peroxidase (17-α-OH-P-3-O-CMO-HRP) enzyme conjugate. The data of the present study reveal that the homologous assay, which employed P-3-O-CMO-HRP as the label, showed no displacement. On the contrary, replacement of P-3-O-CMO-HRP with 17-α-OH-P-3-O-CMO-HRP as the label showed significant displacement and led to the development of a sensitive and specific assay. The recovery of the exogenously spiked progesterone from milk pools was in the range of 94.3–97.88% for toned milk and 97.6–101% for full-cream milk. The intra-assay and interassay coefficients of variation (CVs) ranged from 4.1–7.8% and 4.4–7.0%, respectively. A high ionic strength buffer was used to obtain released progesterone from binding protein/fat. The progesterone values measured in toned and full-cream milk ranged from 1.198–9.745 ng/mL and 6.949–14.923 ng/mL, respectively. The milk progesterone values obtained by this method correlated well with those obtained by radioimmunoassay; r = 0.95 (n = 65).

DEVELOPMENT OF AN ANTIGEN HETEROLOGOUS ENZYME LINKED IMMUNOSORBENT ASSAY FOR MEASUREMENT OF CORTICOSTERONE IN RAT SERUM

Homologous and heterologous combinations of enzyme conjugate and antibody in steroid enzyme immunoassay (EIA) influences unlabeled steroid recognition by antibody that affects sensitivity of the assay. To develop corticosterone enzyme linked immunosorbent assay (ELISA), antibodies were generated against corticosterone-3-carboxymethyloxime-bovine serum albumin (corticosterone-3-CMO-BSA) and corticosterone-21-hemisuccinate-bovine serum albumin (corticosterone-21-HS-BSA). Four horseradish peroxidase (HRP) enzyme conjugates were prepared using two corticosterone derivatives (corticosterone-3-CMO and corticosterone-21-HS) and two cortisol derivatives (Cortisol-3-CMO and Cortisol-21-HS). Eight combinations of homologous and heterologous assays were evaluated. The use of antigen heterologous combination resulted in development of assay. The developed assay is simple, direct, and convenient to use, as it permits the direct addition of the serum sample in to the assay, and it requires only 1.5 hours to complete.

Kinetics Study of Thermal Degradation of Resin Derived from Salicylaldehyde, Ethylenediamine and Formaldehyde

The present paper reports the synthesis and kinetics of thermal degradation studies of resin salicylicldehyde -ethylenediamine -formaldehyde (SdEDF) derived by the condensation of salicylicldehyde and ethylenediamine with formaldehyde in the presence of catalyst hydrochloric acid in 1:1:2 molar proportions of reactants. Detailed thermal degradation studies of the SdEDF resin has been carried out to ascertain its thermal stability. Thermal degradation curve has been discussed in order to determine their mode of decomposition, order of reaction, apparent activation energy, frequency factor, free energy change, entropy change, and apparent energy change. Freeman - Carroll and Sharp- Wentworth methods have been applied for the calculation of kinetic parameters while the data from the Freeman - Carroll methods have been used to determine various thermodynamic parameters.

Synthesis, Characterization and Thermal Degradation Study of Terpolymer-derived from p-Hydroxybenzoic Acid, Diaminonaphthelein with Formaldehyde

Resin pHDNF has been synthesized by the condensation of p-hydroxybenzoic acid and diaminonaphthelein with formaldehyde in the presence of catalyst hydrochloric acid in 1 : 1 : 2 molar proportions of reactants. The purity of newly synthesized terpolymer has been tested and confirmed by the thin layer chromatography (TLC) technique. Detailed thermal degradation studies of the pHDNF resin have been carried out to ascertain its thermal stability. The thermal degradation curve was examined in order to determine their mode of decomposition, order of reaction, apparent activation energy, frequency factor, free energy change, entropy change and apparent energy change. The Freeman—Carroll and Sharp—Wentworth methods were applied for the calculation of kinetic parameters and the data from the Freeman—Carroll method was been used to determine various thermodynamic parameters. The terpolymer resin was characterized by elemental analysis, infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and UV-visible spectral studies. The number-average molecular weight of the resin was determined by non-aqueous conductometric titration.