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-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).

Quantitative LC–MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum

“…Immunoassays are sensitive techniques and have been used often for steroid determination [15,16]. Nevertheless, it is known that commercially available antibodies present a lack of specificity, owing to cross reactivity.…”

Magnetite Nanoparticle-Linked Immunosorbent Assay

“…Enzyme-linked immunosorbent assay (ELISA) is a versatile biochemical technique commonly used as a diagnostic tool and/or a quality control check for detection of antigens in medicine, plant pathology, and various industries. − A typical procedure of ELISA involves three steps: (i) immobilizing the antigen on a solid surface (such as a microtiter plate) either specifically or nonspecifically, (ii) binding the antigen with an antibody that is linked with an enzyme either by itself or via a secondary antibody, and (iii) adding a substrate of the enzyme and color agents that are able to produce a detectable chromogenic or fluorogenic signal via an enzymatic reaction indicating the quantity of the antigen in the sample. Most commonly used enzymes in ELISA include horseradish peroxidase (HRP) and alkaline phosphatase (AP). − Although these enzymes are very effective in converting the substrate to elicit a chromogenic or fluorogenic signal, they lose their enzymatic activities gradually accompanying long-term storage, which limits the assay’s performance. In addition, a significant fraction of the cost of the ELISA technology is involved with production and purification of the enzyme-antibody conjugation.…”

17-Hydroxyprogesterone in children, adolescents and adults

“…The free steroids are extracted into a solvent that is dried and reconstituted in a buffer for IA. Radioimmunoassay, enzyme-linked immunosorbent and time-resolved fluorescence immunoassays are in general use, [61][62][63] though there is an expected move away from this technology.…”