This assay employs the competitive inhibition enzyme immunoassay technique. The micro-plate provided in this kit has been pre-coated with 17-Hydroxyprogesterone (17-OHP) protein. Standards or samples are then added to the appropriate micro-plate wells with a biotin-conjugated antibody specific to 17-Hydroxyprogesterone (17-OHP). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm±10nm. The concentration of 17-Hydroxyprogesterone (17-OHP) in the samples is then determined by Fine Test Ltd comparing the OD of the samples to the standard curve.
The 17-Hydroxyprogesterone Competitive ELISA quantitates 17-Hydroxyprogesterone in serum, plasma, urine, dried fecal extracts or cell culture medium.
Principle of the method
The 17-Hydroxyprogesterone Competitive ELISA research-use-only kit uses a specifically generated antibody to measure 17-Hydroxyprogesterone (17HO-P) and its metabolites in reconstituted buffer samples, tissue culture media samples, urine and fecal samples, or in extracted serum and plasma. This kit is not recommended for serum or plasma samples without extraction. A 17HO-P standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. Standards or diluted samples are pipetted into the provided plate coated with a capture antibody. A 17HO-P-peroxidase conjugate is added to the standards and samples in the wells. The binding reaction is initiated by the addition of a polyclonal antibody to 17HO-P to each well. After a 1-hour incubation the plate is washed and substrate is added. The substrate reacts with the bound 17HO-P-peroxidase conjugate. After a short incubation, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measuring at 450 nm.
17-Hydroxyprogesterone (4-pregen-17-ol-3, 20-dione, 17HO-P, OHPG) is a steroid hormone from the androgen group that is found in mammals, reptiles, birds, and other vertebrates. 17HO-P is identical across all species and so this kit should measure 17HO-P from all sources. In humans, it is primarily synthesized in the adrenal glands, the gonads, testes, and placenta.
Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.
The 17-OH-Progesterone ELISA kit is intended for quantitative determination of 17-OH progesterone in human serum and human plasma.
17-OH-progesterone (17-OHP) is a steroid hormone produced by suprarenal gland cortex and sex glands. The measurement of 17 OHP in serum can be used to monitor the activity of 21-hydroxylase of the suprarenal gland cortex. Deficiencies in 21-hydroxylase that are most commonly found in congenital adrenal hyperplasia result in excessive secretion of 17-OHP. However, deficiencies in 11-hydroxylase lead to moderately increase of the 17-OHP level. Therefore, the analysis of 17-OHP plays a significant role in the differential diagnostics of congenital hyperplasia of suprarenal gland cortex.
In addition, levels of 17-OHP are influenced by circadian rhythm and correlate with the adrenal secretion of cortisol. Maximal levels are found in samples collected in the morning.
|Kit volume, wells (including controls)||96|
|Analitical sensitivity, nmol/L||0.3|
|Range of evaluated concentrations, nmol/L||0.3-60|
|Incubations||30 min + 10 min at 37°C
30 min at 37°C + 15-30 min at RT
|Shelf life, months||18|
The principle of the 17OH Progesterone ELISA Assay Kit enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabeled antigen (present in standards, controls and patient samples) and an enzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microplate. The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the color formed is inversely proportional to the concentration of 17α-OHP in the sample. A set of standards is used to plot a standard curve from which the amount of 17α-OHP in patient samples and controls can be directly read.
SPECIMEN COLLECTION AND STORAGE
Approximately 0.1 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.
POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that may be used in the preparation of the standards and controls has been tested and found to be nonreactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCV and Human Immunodeficiency Virus (HIV) and found to be negative. No test method however, can offer complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. The reagents should be considered a potential biohazard and handled with the same precautions as applied to any blood specimen.