FSH ELISA Kit

Catalog No. ABIN649062

Product Details FSH ELISA Kit

Target: FSH (Follicle-Stimulating Hormone (FSH))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: Immunoenzymometric assay (TYPE 3): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-FSH antibody. Upon mixing monoclonal biotinylated antibody, the enzyme-labeled antibody and a serum containing the native antigen, reaction results between the native antigen and the antibodies without competition or steric hindrance to form a soluble sandwich complex. After equilibrium is attained, the antibodybound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibodybound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen values, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytical Method: Quantitative

Components: A. FSH Calibrators (1 ml/vial). Six vials of references for FSH Antigen at levels of 0 (A), 5 (B), 10 (C), 25 (D), 50E) and 100 (F) mIU/ml. Store at 2-8°C. A preservative has been added. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the WHO 2nd IRP (78/549). B. FSH Enzyme Reagent (13 ml/vial). One vial-containing enzyme labeled antibody, biotinylated monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. C. Streptavidin Coated Plate (96 wells). One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. D. Wash Solution Concentrate (20 ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. E. Substrate A (7. 0ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. F. Substrate B (7. 0ml/vial). One bottle containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. G. Stop Solution (8ml/vial). One bottle containing a strong acid (1N HCl). Store at 2-30°C. H. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: Above reagents are for a single 96-well microplate.

Material not included: 1. Pipette capable of delivering 50µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Microplate washers or a squeeze bottle (optional). 4. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 5. Absorbent Paper for blotting the microplate wells. 6. Plastic wrap or microplate cover for incubation steps. 7. Vacuum aspirator (optional) for wash steps. 8. Timer. 9. Quality control materials.

Application Details

Application Notes: Precautions: All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA licensed reagents. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Sample Volume: 50 μL

Plate: Pre-coated

Reagent Preparation:

1. Wash Buffer: Dilute contents of wash concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature 20-27 °C for up to 60 days. 2. Working Substrate Solution: Pour the contents of the amber vial labeled Solution A into the clear vial labeled Solution B. Place the yellow cap on the clear vial for easy identification. Mix and label accordingly. Store at 2 - 8 °C. Note: Do not use the working substrate if it looks blue.

Sample Collection: The specimens shall be blood, serum in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain redtop venipuncture tube without additives or anti-coagulants. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 100 ml of the specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of follicle stimulating hormone in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding FSH concentration in mIU/ml on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Draw the best-fit curve through the plotted points. 4. To determine the concentration of FSH for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in mIU/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). Note: Computer data reduction software designed for ELISA assays may also be used for the data reduction.

Restrictions: For Research Use only

Handling

Handling Advice: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplate wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Pipette 0. 050 ml (50µl) of the appropriate serum reference, control or specimen into the assigned well. Add 0. 100 ml (100µl) of FSH-Enzyme Reagent solution to all wells. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 60 minutes at room temperature. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 7. Add 350µl of wash buffer (see Reagent Preparation Section) decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 8. Add 0. 100 ml (100µl) of working substrate solution to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 9. Incubate at room temperature for 15 minutes. 10. Add 0. 050ml (50µl) of stop solution to each well and gently mix for 15-20 seconds). Always add reagents in the same order to minimize reaction time differences between wells. 11. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 30 minutes of adding the stop solution.

Storage: 4 °C/-20 °C

Target Details for FSH

Target: FSH (Follicle-Stimulating Hormone (FSH))

Alternative Name: Follicle Stimulating hormone (FSH) (FSH Products)

Synonyms: FSH ELISA Kit, Fshbeta ELISA Kit, follicle stimulating hormone beta ELISA Kit, Plasma FSH concentration ELISA Kit, Fshb ELISA Kit, FSH ELISA Kit

Background: Summary and Explanation of the test: Follicle Stimulating hormone (FSH) is a glycoprotein consisting of two subunits with an approximate molecular mass of 35,500 daltons. The alpha subunit is similar to other pituitary hormones (luteinizing stimulating hormone (LH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG)( while the alpha subunit is unique. The alpha subunit confers the biological activity to the molecule. Stimulation by gonadotropin-releasing hormone (GnRH) causes release of FSH, as well as LH, from the pituitary and is transported by the blood to their sites of action, the testes or ovary. In men, FSH acts on the Sertoli cells of the testis, stimulating the synthesis of inhibin, which appears to specifically inhibit further FSH secretion, and androgen-binding protein. Thus, it indirectly supports spermatogenesis. In women, FSH acts on the granulosa cells of the ovary, stimulating steroidogensis. All ovulatory menstrual cycles have a characteristic pattern of FSH, as well as LH, secretion. The menstrual cycle is divided into a follicular phase and a luteal phase by the midcycle surge of the gonadotropins (LH and FSH). As the follicular phase progresses, FSH concentration decreases. Near the time ovulation occur, about midcycle, FSH peaks (lesser in magnitude than LH) to its highest level. The clinical usefulness of the measurement of Follicle Stimulating hormone (FSH) in ascertaining the homeostasis of fertility regulation via the hypothalamic - pituitary - gonadal axis has been well established (1,2). In this method, FSH calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal and enzyme labeled antibodies (directed against distinct and different epitopes of FSH) are added and the reactants mixed. Reaction between the various FSH antibodies and native FSH forms a sandwich complex that binds with the streptavidin coated to the well. After the completion of the required incubation period, the enzyme-Follicle Stimulating Hormone antibody bound conjugate is separated from the unbound enzyme-follicle stimulating hormone conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color. The employment of several serum references of known Follicle Stimulating Hormone levels permits construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with Follicle Stimulating Hormone concentration. Intended Use: The Quantitative Determination of Follicle Stimulating Hormone Concentration in Human Serum by a Microplate Immunoenzymometric assay (IEMA/ELISA ). Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met: 1. The absorbance (OD) of calibrator F should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.

Pathways: Peptide Hormone Metabolism, Chromatin Binding