Rat gonadotropin-releasing hormone(GnRH)ELISA Kit
Catalog No. CSB-E08037r
(96T)
This immunoassay kit allows for the in vitro quantitative determination of rat GnRH concentrations in serum, plasma.
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 a goat-anti-rabbit antibody. Sta-n-dards or samples are then added to the appropriate microtiter plate wells with a HRP-conjugated GnRH a-n-d antibody preparation specific for GnRH 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 GnRH in the samples is then determined by comparing the O.D. of the samples to the sta-n-dard curve.
DETECTION RANGE
3.2 pg/ml-800 pg/ml. The sta-n-dard curve concentrations used for the ELISA’s were 800 pg/ml, 240 pg/ml, 40 pg/ml, 8 pg/ml, 3.2 pg/ml.
SPECIFICITY
This assay recognizes rat GnRH. No significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rat GnRH is typically less than
3.2 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 STORAGE
| Reagent | | | | Quantity | | Assay plate | | | | 1 | | Standards (S0-S5) | | | | 6 x 0.5 ml | | HRP-conjugate | | | | 1 x 6 ml | | Antibody | | | | 1 x 6 ml | | Wash Buffer | | | | 1 x 15 ml (20×concentrate) | | Substrate A | | | | 1 x 7 ml | | Substrate B | | | | 1 x 7 ml | | Stop Solution | | | | 1 x 7 ml | | Standard | S0 | S1 | S 2 | S3 | S4 | S5 | | Concentration (pg/ml) | 0 | 3.2 | 8 | 40 | 240 | 800 | |
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, provided it is stored as prescribed above. Refer to the package label for the expiration date.
2. Opened test plate should be stored at 2-8?C in the aluminum foil bag with desiccants to minimize exposure to damp air. The 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 a-n-d plate to room temperature for at least 30 minutes before use. Unused wells need store at 2-8℃a-n-d avoid sunlight.
2 Wash Buffer If crystals have formed in the concentrate, warm to room temperature a-n-d mix gently until the crystals have compley 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.
5 An incubator which can provide stable incubation conditions up to 37°C±0.5°C.
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 g. Remove serum a-n-d assay immediay or aliquot a-n-d store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
? Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediay or aliquot a-n-d store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated 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. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well.
1 Set a Blank well without any solution. Add 50μl of Sta-n-dard or Sample per well. Sta-n-dard need test in duplicate.
2 Add 50μl of HRP-conjugate to each well (not to Blank well), then 50μl Antibody to each well. Mix well a-n-d then incubate for 1 hour at 37°C.
3 Fill each well with Wash Buffer (about 200μl), stay for 10 seconds a-n-d Spinning. Repeat the process for a total of three washes. 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.
4 Add 50μl of Substrate A a-n-d 50μl Substrate B to each well, mix well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts a-n-d other temperature fluctuations in the dark.
5 Add 50μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
6 Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Using the professional soft "Curve Exert 1.3" to make a sta-n-dard curve is recommended, which can be downloaded from our web.
Average the duplicate readings for each sta-n-dard, Blank, a-n-d sample a-n-d subtract the optical density of Blank. 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 x-axis against the concentration on the y-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 GnRH 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 If samples generate values higher than the highest sta-n-dard, dilute the samples with the appropriate Diluent a-n-d repeat the assay.
4 Any variation in operator, pipetting technique, washing technique, incubation time or temperature, a-n-d kit age can cause variation in binding.
5 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 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.
3 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.
4 To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Sealers can not be reused.
5 Substrate Solution should remain colorless or light blue until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless or light blue to gradations of blue.
6 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.
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