Human 6-keto prostaglandin F1α (6-K-PGF1α) ELISA

Kit instruction manual

This reagent. Purpose: The kit for determination of human serum, blood plasma, and the content of the liquid sample of 6-keto prostaglandin F1 α (6-K-PGF1 α) is.

Experimental principle:

    The kit uses a double antibody sandwich method to determine the level of human 6- keto prostaglandin F1 α (6-K-PGF1 α ) in the specimen . Purified human 6-keto Prostaglandin F1 α (6-K-PGF1 α) antibody-coated microtiter plate wells the immobilized antibody, to coat wells, were added 6-keto-prostaglandin F1 α ( 6-K-PGF1 α ) , and then combined with HRP- labeled PGF1 α antibody to form an antibody - antigen - enzyme - labeled antibody complex, which was thoroughly washed and then added with substrate TMB for color development. TMB in HRP enzyme blue, yellow and eventually converted to the action of an acid. The color depth is positively correlated with the 6- keto prostaglandin F1 α (6-K-PGF1 α ) in the sample . The absorbance ( OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human 6- keto prostaglandin F1 α (6-K-PGF1 α ) in the sample was calculated from a standard curve .

Kit composition :

Sample processing and requirements :

1. Serum: coagulation at room temperature for 10-20 minutes, 20 minutes, centrifugation (2000-3000 rev / min). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.

2. Plasma: should be selected EDTA or citrate as an anticoagulant, mix 10-20 minutes, about 20 minutes centrifugation (2000-3000 rpm / min) according to the requirements of the specimen. The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.

3. Urine: a sterile collection tube, centrifuged at about 20 minutes (2000-3000 rev / min). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.

4. Cell culture supernatant: When detecting secreted components, collect them with a sterile tube. Centrifugation 20 min (2000-3000 rev / min). Collect the supernatant carefully. When measuring the components in the cells, the cell suspension was diluted with PBS ( pH 7.2-7.4 ), and the cell concentration reached about 1 million /ml . By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifugation 20 min (2000-3000 rev / min). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS , pH 7.4 . It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 °C after melting . Add a certain amount of PBS ( pH 7.4 ) and homogenize the specimen by hand or homogenizer. Centrifugation 20 min (2000-3000 rev / min). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.

6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided .

7. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase ( HRP ) activity.

Steps

1.          Dilution and loading of standard products: 10 wells of standard wells were placed on the enzyme-labeled plate , 100 μl of standard was added to the first and second wells, and standard dilutions were added to the first and second wells. 50μl , mix; then add 100μl from the first hole and the second hole to the third hole and the fourth hole respectively, and then add 50μl of the standard dilution solution in the third and fourth holes, and mix; Take 50 μl of each of the third and fourth wells, discard them, add 50 μl to each of the fifth and sixth wells, and add 50 μl of the standard dilution solution to the fifth and sixth wells . Mix well; mix well. Then, 50 μl of each of the fifth and sixth holes are respectively added to the seventh and eighth holes, and then 50 μl of the standard dilution solution is added to the seventh and eighth holes, respectively, and the seventh and eighth holes are mixed. were taken in 50μl was added to the ninth, tenth hole, and then add standard dilution 50μl ninth tenth hole, after mixing 50μl from each well was discarded from the ninth tenth. (The amount of each well after dilution is 50μl , the concentration is 1200 pg/ml , 800 pg/ml , 400 pg/ml , 200 pg/ml , 100 pg/ml ).

2.          Adding samples: Set blank holes separately (the blank control holes are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Coated ELISA plates testing sample well add Sample dilution 40 μ l, was then combined with the test sample 10 μ l (final dilution is 5-fold). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.

3.          Incubation: After sealing plate sealing film and incubated at 37 ℃ board 30 minutes.

4.          Dosing: 30 (20 times the 48T) fold concentrated fold diluted solution was washed with distilled water and 30 alternate (48T of 20 times).

5.          Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.

6.          Add enzyme: 50 μl of enzyme labeling reagent was added to each well , except for blank wells.

7.          Incubation: The operation is the same as 3 .

8.          Washing: The operation is the same as 5 .

9.          Color development: Add 50 μl of the developer to each well , then add 50 μl of the developer , gently shake and mix, and color at 37 °C for 15 minutes .

10.      Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow).

11.      Measurement: The absorbance ( OD value) of each well was measured in sequence with a blank air conditioner of zero and a wavelength of 450 nm . The measurement should be carried out within 15 minutes after the addition of the stop solution .

Precautions:

1.   The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.

2.   Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.

3.   The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.

4.   Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well ), please first dilute the sample dilution with a certain multiple ( n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple ( × n × 5 ).

5.   The sealing film is intended for single use only to avoid cross-contamination.

6.   Please keep the substrate away from light.

7.   Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading .

8.   All samples, washings and various wastes should be treated as infectious materials.

9.   The different batch components of this reagent must not be mixed.

10. If it is different from the English manual, the English manual shall prevail.

Calculation:

Take the standard density as abscissa, OD value of the ordinate, a standard curve is plotted on graph paper, the concentration of the corresponding OD values of samples isolated according to the standard curve; multiplied by the dilution factor; or a standard concentration OD values calculated from the linear regression equation of the standard curve, the sample OD value in the equation to calculate the concentration of the sample, then multiplied by the dilution factor, the actual concentration of the sample.                   

Kit performance:

1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is 0.990 or more.

2. Within and within the batch should be less than 9% and 11% respectively

examination range:    

35 pg/ml – 1600 pg/ml         

Storage conditions and expiration date:

1. The kit of preservation:; 2-8 ℃.

2 . Validity: 6 months

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