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.

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

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-alpha-hydroxy-progesterone-3-O-carboxymethyloxime (17-alpha-OH-P-3-O-CMO) with Horseradish Peroxidase (HRP) to form 17-alpha-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 100microL enzyme conjugate along with 50microL of standards in the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using tetramethyl benzidine/hydrogen peroxide (TMB/H(2)O(2)) as substrate. The enzyme substrate reaction was terminated with 100microL of 0.5 M H(2)SO(4) 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-alpha-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).

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.

A novel enzyme-linked immunosorbent assay for cortisol using a long-chain biotinylated cortisol-3-CMO derivative.

An enzyme-linked immunosorbent assay (ELISA) using streptavidin-biotin system as a bridge between antibodies bound antigen and reporter molecule (horseradish peroxidase enzyme) has been described. The cortisol antiserum was generated against cortisol-3-O-carboxylmethyl oxime-bovine serum albumin (F-3-CMO-BSA). We have prepared biotin-labelled cortisol as a primary probe and utilized streptavidin-labelled horseradish peroxidase (SA-HRP) as secondary probe to monitor the antigen-antibody interaction. To the cortisol antibody coated micro wells, 25 microL of standard or samples, along with 100 microL of biotinylated cortisol, were kept for 1 h at room temperature. Thereafter, wells were washed and 100 microL of SA-HRP was added to all wells and kept again for 20 min at room temperature. Bound enzyme activity was measured using tetramethyl benzidine/hydrogen peroxidase (TMB/H2O2) as substrate. The incorporation of streptavidin-biotin system as a bridge between antibody bound antigen and reporter molecule (horseradish peroxidase enzyme) increased sensitivity and specificity of the cortisol assay. The use of low molecular weight primary label (F-3-CMO-biotin) might have facilitated the easy and selective access of the analyte present in serum to compete with the antigen-binding pocket of antibody, thereby detecting as low as 3.42 ng/mL of analyte specifically.

Development of rapid and sensitive one-step direct enzyme linked immunosorbent assay for 17-alpha-OH-progesterone in serum.

Using a homologous combination of immunogen and enzyme conjugate, a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) was developed to measure 17-alpha-hydroxy-progesterone (17-alpha-OH-P) in human serum. The antiserum was raised against 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime bovine serum albumin (17-alpha-OH-P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-alpha-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 microL enzyme conjugate along with 50 microL 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 microL 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-alpha-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).

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay.

Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody.

The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten.

BACKGROUND: In hapten enzyme immunoassays (EIA), there is an increase or decrease of labeled hapten recognition by antibody that affects sensitivity of the assay. We incorporated a spacer between a hapten derivative and enzyme to test its influence on the sensitivity and specificity of enzyme immunoassays. METHOD: Antibodies were generated against cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) and cortisol-21-hemisuccinate-bovine serum albumin (cortisol-21-HS-BSA) as an immunogen. Four cortisol horseradish peroxidase (HRP) enzyme conjugates were prepared using 2 cortisol derivatives (cortisol-3-O-CMO and cortisol-21-HS) with and without adipic acid dihydrazide (ADH) as a spacer. Eight combinations of homologous and heterologous assays were evaluated. RESULT: The incorporation of ADH spacer in cortisol-enzyme conjugate improved the sensitivity in heterologous (bridge and site plus bridge) EIA systems. In heterologous assays (site plus bridge), the presence of spacer in enzyme conjugate reduced the cross-reactivity with cross-reacting steroids. CONCLUSION: Spacer in the enzyme conjugate for hapten ELISA can improve the sensitivity of heterologous assay of hapten-like steroids. It may also reduce the cross-reactivity for some assays.