山羊促卵泡激素（FSH）ELISA试剂盒操作方法

Goat Follicle-stimulating hormone (FSH) ELISA Kit              

• For research use only.
• For the quantitative in vitro determination of FSH concentrations in Goat serum, plasma, culture media or any biological fluid.
• Expiration date：six months .

l Storage： 2-8℃.

Principle

This ELISA kit uses Sandwich-ELISA as the method. The Microelisa stripplate provided in this kit has been pre-coated with an antibody specific to FSH. Standards or samples are added to the appropriate Microelisa stripplate wells and combined to the specific antibody. Then a Horseradish Peroxidase (HRP)- conjugated antibody specific for FSH  is added to each Microelisa stripplate well and incubated. Free components are washed away. The TMB substrate solution is added to each well. Only those wells that contain FSH and HRP conjugated FSH antibody will appear blue in color and then turn yellow after the addition of the stop solution. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm. The OD value is proportional to the concentration of FSH. You can calculate the concentration of FSH in the samples by comparing the OD of the samples to the standard curve.

Materials provided with the kit

Sample preparation

1. Serum preparation

After collection of the whole blood, allow the blood to clot by leaving it undisturbed at room temperature. This usually takes 10-20 minutes. Remove the clot by centrifuging at 2,000-3,000 rpm for 20 minutes. If precipitates appear during reservation, the sample should be centrifugated again.

2. Plasma preparation

Collect the whole blood into tubes with anticoagulant (EDTA or citrate). After incubated at room temperature for 10-20 minutes, tubes are centrifugated for 20 min at 2,000-3,000 rpm. Collect the supernatant carefully as plasma samples. If precipitates appear during reservation, the sample should be centrifugated again.

3. Urine samples

Collect urine into aseptic tubes. Collect the supernatant carefully after centrifuging for 20 min at 2,000-3,000 rpm. If precipitates appear during reservation, the sample should be centrifugated again. The preparation procedure of cerebrospinal fluid and pleuroperitoneal fluid is the same as that of urine sample.

4. Cell samples

If you want to detect the secretions of cells, collect culture supernatant into aseptic tubes. Collect the supernatant carefully after centrifuging for 20 min at 2,000-3,000 rpm. If you want to detect intracellular components, dilute the cells to 1X100/ml with PBS (pH 7.2-7.4). The cells were destroyed to release intracellular components by repeated freezing and thawing. Collect the supernatant carefully after centrifuging for 20 min at 2,000-3,000 rpm. If precipitates appear during reservation, the sample should be centrifugated again.

5. Tissue samples

Tissue samples are cut, weighed, frozen in liquid nitrogen and stored at -80℃ for future use. The tissue samples were homogenized after adding PBS (pH 7.4). Samples should be operated at 4℃. Collect the supernatant carefully after centrifuging for 20 min at 2,000-3,000 rpm. Aliquot the supernatant for ELISA assay and future use.

Notes:

• Sample extraction and ELISA assay should be performed as soon as possible after sample collection. The samples should be extracted according to the relevant literature. If ELISA assay can not be performed immediay, samples can be stored at -20℃.Repeated freeze-thaw cycles should be avoided.
• Our kits can not be used for samples with NaN3 which can inhibit the activity of HRP.

Procedure

• Dilution of Standards

Ten wells are set for standards in a Microelisa stripplate. In Well 1 and Well 2, 100μl Standard solution and 50μl Standard Dilution buffer are added and mixed well. In Well 3 and Well 4, 100μl solution from Well 1 and Well 2 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. 50μl solution is discarded from Well 3 and Well 4. In Well 5 and Well 6, 50μl solution from Well 3 and Well 4 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. In Well 7 and Well 8, 50μl solution from Well 5 and Well 6 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. In Well 9 and Well 10, 50μl solution from Well 7 and Well 8 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. 50μl solution is discarded from Well 9 and Well 10. After dilution, the total volume in all the wells are 50μl and the concentrations are36ng/ml ,24ng/ml,12ng/ml,6 ng/ml and 3ng/ml, respectively.

 54ng/ml       36ng/m     24ng/m    12ng/m   6ng/m     3ng/m

2. In the Microelisa stripplate, leave a well empty as blank control. In sample wells, 40μl Sample dilution buffer and 10μl sample are added (dilution factor is 5). Samples should be loaded onto the bottom without touching the well wall. Mix well with gentle shaking.

3. Incubation: incubate 30 min at 37℃ after sealed with Closure plate membrane.

4. Dilution: dilute the concentrated washing buffer with distilled water (30 times for 96T and 20 times for 48T).

5. Washing: carefully peel off Closure plate membrane, aspirate and refill with the wash  solution. Discard the wash  solution after resting for 30 seconds. Repeat the washing procedure for 5 times.  

6. Add 50 μl HRP-Conjugate reagent to each well except the blank control well.

7. Incubation as described in Step 3.

8. Washing as described in Step 5.

9. Coloring: Add 50 μl Chromogen Solution A and 50 μl Chromogen Solution  B to each well, mix with gently shaking and incubate at 37℃ for 15 minutes. Please avoid light during coloring.

10. Termination: add 50 μl stop solution to each well to terminate the reaction. The color in the well should change from blue to yellow.

11. Read absorbance O.D. at 450nm using a Microtiter Plate Reader. The OD value of the blank control well is set as zero. Assay should be carried out within 15 minutes after adding stop solution.

Notes:

• Store the kit at 4°C upon receipt.The kit should be equilibrat4ed to room temperature before the assay. Remove any unneeded strips from Goat FSH Antibody-Coated plate, reseal them in zip-lock foil and keep at 4°C.
• Precipitates may appear in concentrated washing buffer. Please heat the buffer to dissolve all the precipitates, which will not affect the results.
• Accurate pipette should be used to avoid experimental error. Samples should be added to the Microplate in less than 5 minutes. If a large number of samples are included, multiple channel pipette is recommended.
• Standard curve should be included in every assay. Replicate wells are recommended. If the OD value of the sample is greater than the first well of standards, please dilute the sample (n times) before test. When calculating the original FSHa concentration, please multiply the total dilution factor (XnX5).
• In order to avoid cross-contamination, Microplate sealers are for one-time use only.
• Please keep Substrate away from light.
• All the operation should be accordance with the manufacturer's instructions strictly. The results determined by the Microtiter Plate Reader.
• All the samples, washing buffer and wastes should be treated as infectious agents.
• Reagents from different lots should not be mixed.

Calculation of Results

Known concentrations of Goat FSH Standard and its corresponding reading OD is plotted on the log scale (x-axis) and the log scale (y-axis) respectively. The concentration of Goat FSH in sample is determined by plotting the sample’s O.D. on the Y-axis. The original concentration is calculated by multiplying the dilution factor. 

Kit performance

1.  Correlation coefficient (R) of linear regression of the samples is more than 0.92

2.  The difference in intra-assay and inter-assay is less than 9% and 15% respectively.

Assay range

0.6ng/ml -45ng/ml