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Human Inhibin A (INH-A) ELISA Kit
Catalog No. CSB-E08237h
(96T)
This immunoassay kit allows for the in vitro quantitative determination of human INH-A concentrations in serum, plasma a-n-d other biological fluids.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with goat-anti-rabbit antibody. Sta-n-dards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated INH-A a-n-d antibody preparation specific for INH-A, a-n-d incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution a-n-d the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of INH-A in the samples is then determined by comparing the O.D. of the samples to the sta-n-dard curve.
DETECTION RANGE
0.32 pg/ml-80 pg/ml. The sta-n-dard curve concentrations used for the ELISA’s were 80 pg/ml, 20 pg/ml, 5 pg/ml, 1.24 pg/ml, 0.32 pg/ml.
SPECIFICITY
This assay recognizes human INH-A. No significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human INH-A is typically less than 0.2 pg/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1 Sta-n-dard 5 x 0.5 ml HRP-Conjugate 1 x 6ml Antibody 1 x 6 ml
1 x15 ml
Wash Buffer
(20×concentrate) Substrate A 1 x 7 ml Substrate B 1 x 7 ml Stop Solution 1 x 7 ml
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STORAGE
1. Unopened test kits should be stored at 2-8°C upon receipt a-n-d the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date.
2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
3. A microtiter plate reader with a ba-n-dwidth of 10 nm or less a-n-d an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.
REAGENT PREPARATION
1 Bring all reagents to room temperature before use.
2 Wash Buffer If crystals have formed in the concentrate, warm up to room temperature a-n-d mix gently until the crystals have completely dissolved. Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, ha-n-d, face, a-n-d clothing protection when using this material.
OTHER SUPPLIES REQUIRED
1 Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
2 Pipettes a-n-d pipette tips.
3 Deionized or distilled water.
4 Squirt bottle, manifold dispenser, or automated microplate washer.
SAMPLE COLLECTION A-N-D STORAGE
? Serum Use a serum separator tube (SST) a-n-d allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum a-n-d assay immediately or aliquot a-n-d store samples at -20° C. Avoid repea ted freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents a-n-d samples to room temperature before use. It is recommended that all samples, sta-n-dards, a-n-d controls be assayed in duplicate.
1 Set a Blank without any solution. Add 50µl of Sta-n-dard or Sample per well.
2 Add 50µl of HRP-Conjugate a-n-d 50µl of Antibody to each well. Not to Blank well!
3 Cover with the adhesive strip. Incubate for 1 hour at 37°C.
4 Aspirate each well a-n-d wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (200µl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate a-n-d blot it against clean paper towels.
5 Add 50µl of Substrate A a-n-d 50µl of Substrate B to each well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts a-n-d other temperature fluctuations in the dark.
6 Add 50µl of Stop Solution to each well when the first four wells containing the highest concentration of sta-n-dards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
7 Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Average the duplicate readings for each sta-n-dard, control, a-n-d sample a-n-d subtract the average zero sta-n-dard optical density. Create a sta-n-dard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a sta-n-dard curve by plotting the mean absorbance for each sta-n-dard on the y-axis against the concentration on the x-axis a-n-d draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the INH-A concentrations versus the log of the O.D. a-n-d the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the sta-n-dard curve must be multiplied by the dilution factor.
LIMITATIONS OF THE PROCEDURE
1 The kit should not be used beyond the expiration date on the kit label.
2 Do not mix or substitute reagents with those from other lots or sources.
3 It is important that the Calibrator Diluent selected for the sta-n-dard curve be consistent with the samples being assayed.
4 If samples generate values higher than the highest sta-n-dard, dilute the samples with the appropriate Calibrator Diluent a-n-d repeat the assay.
5 Any variation in operator, pipetting technique, washing technique, incubation time or temperature, a-n-d kit age can cause variation in binding.
6 This assay is designed to eliminate interference by soluble receptors, binding proteins, a-n-d other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
TECHNICAL HINTS
1 Centrifuge vials before opening to collect contents.
2 When mixing or reconstituting protein solutions, always avoid foaming.
3 To avoid cross-contamination, change pipette tips between additions of each sta-n-dard level, between sample additions, a-n-d between reagent additions. Also, use separate reservoirs for each reagent.
4 When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, a-n-d/or rotating the plate 180 degrees between wash steps may improve assay precision.
5 To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
6 Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue.
7 Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.
