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Ox-gall acid (Cholic acid) enzyme-linked immunoassay (ELISA)

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Ox-gall acid (Cholic acid)Enzyme-linked immunoassay (ELISA)

Kit instructions

This reagent for research use only

Purpose: this kit is used to measure human serum, plasma and bile acid in the liquid sample (Cholic acid) content.

The experimental principle:

This kit using double antibody method to determine the specimens ox-gall acid (Cholic acid) level.With purified ox-gall acid (Cholic acid) antibody package is microporous plate, made from solid phase antibody, the package in the sheet resistance of the microporous join Cholic acid in turn (Cholic acid), again with the HRP labeled Cholic acid (Cholic acid) antibodies, antibody antigen - enzyme mark antibody complex formation, thoroughly with the substrate TMB color after washing.TMB under the catalysis of HRP enzyme into blue, and under the action of acid into the yellow.Color shades and bile acid in the samples (Cholic acid) were positively correlated.Enzyme standard instrument under 450 nm wavelength determination of absorbance (OD value), via a standard curve to calculate samples ox-gall acid (Cholic acid) concentration.

Kit:


Kit of

48 hole configuration

96 hole configuration

save

The instructions

1

1

Sealing plate membrane

2 slices (48)

2 (96).

bags

1

1

Enzyme standard package is plate

1 x 48

1 x 96

2- 8 save

Standard: 2700 ng/L

0.5 ml x 1 bottle

0.5 ml x 1 bottle

2- 8 save

Standard diluent

1.5 ml x 1 bottle

1.5 ml x 1 bottle

2- 8 save

Enzyme standard reagent

3 ml x 1 bottle

6 ml x 1 bottle

2- 8 save

The sample diluent

3 ml x 1 bottle

6 ml x 1 bottle

2- 8 save

A liquid agent

3 ml x 1 bottle

6 ml x 1 bottle

2- 8 save

Chromogenic agent B liquid

3 ml x 1 bottle

6 ml x 1 bottle

2- 8 save

Termination of the liquid

3 ml x 1 bottle

6 ml x 1 bottle

2- 8 save

Concentrated liquid detergent

20 times (20 ml x) x 1 bottle

(20 ml x 30 times) x 1 bottle

2- 8 save

Sample processing and requirements:

1. Serum: natural blood coagulation 10-20 minutes at room temperature, centrifugal 20 minutes (2000-3000 revolutions per minute).Carefully collected supernatant, save the process such as precipitation, centrifugal again.

2. Plasma: should choose according to the requirement of the specimen of EDTA and sodium citrate as anticoagulant, mix 10-20 minutes, centrifugal 20 minutes (2000-3000 revolutions per minute).Carefully collected supernatant, save in the process of precipitation formation, if any, should be centrifugal again.

3. The urine: use a sterile tube collection, centrifugal 20 minutes (2000-3000 revolutions per minute).Carefully collected supernatant, save in the process of precipitation formation, if any, should be centrifugal again.Chest water, cerebrospinal fluid reference.

4. Cell culture supernatant: to test the composition of the secretory, gathered in sterile tube.Centrifugal 20 minutes (2000-3000 revolutions per minute).Carefully collected supernatant.When testing the components inside the cell, with PBS (PH7.2 7.4) diluted cell suspension, cell concentration reached 1 million/ml.By repeated freezing and thawing, in order to make cell destruction and release components in the cell.Centrifugal 20 minutes (2000-3000 revolutions per minute).Carefully collected supernatant.Preservation, such as sediment is in the process of centrifugal again.

5. Tissue samples: after cutting specimens, weigh and weight.Add a certain amount of PBS, PH7.4 slightly.Using liquid nitrogen cryopreservation standby quickly.Specimen melts 2 remain the same- 8 The temperature.Add a certain amount of PBS (PH7.4 slightly), by hand or homogenate specimen homogenate.Centrifugal 20 minutes (2000-3000 revolutions per minute).Carefully collected supernatant.A detection after repackaging, frozen for later use.

6. As soon as possible after specimen collection for extraction, extraction, according to the literature should be as soon as possible after extraction experiments.If not immediately, can be put in the specimen- 20 Save, but should avoid repeated freezing and thawing.

7. Can't detect samples containing NaN3, by NaN3 inhibition of horseradish peroxidase (HRP) activity.

steps

1. The standard sample dilution and add: standard is set on the enzyme standard package is plate hole 10 hole, in the first and second hole with standard 100 mu l respectively, and then add standard diluent to the first and second hole 50 mu, l blending;Bore and then from the first, second, take 100 mu in l bore to the third and fourth respectively, and then in the third and fourth hole with standard diluent 50 mu l respectively, blending;Bore and then in the third and fourth in the first 50 mu l from each rejected, then each 50 mu l respectively to the fifth, sixth hole, and then in the fifth and sixth holes respectively add 50 ul standard diluent, blending;After blending in the fifth and sixth holes took 50 mu l respectively to the seventh and eighth in the hole, and then in the seventh and eighth hole with standard diluent 50 mu l respectively, after blending, respectively from the seventh and eighth hole 50 mu l added to the ninth and tenth hole, and then in the first 9 hole 10 plus standard diluent 50 mu l respectively, from the ninth in the tenth hole after blending 50 mu l rejected.(diluted every hole and sample amount to 50 mu l, the concentration of 1800 ng/l respectively, 1200 ng/l, 600 ng/l, 300 ng/l, 150 ng/l).

2. Add the sample: a blank hole (ck hole without samples and enzyme reagent, the rest of the same step operation), sample under test.On the enzyme standard package is board under test sample in the hole and 40 mu l sample diluent, and then add 10 mu l sample under test (final sample dilution degree for 5 times).Plus sample will be the sample and the enzyme label plate at the bottom of the hole, try not to touch the wall of hole, gently shake the blender.

3. Incubate: rear sealing plate seal plate with film37 Incubate for 30 minutes.

4. Liquor: 30 (48 t 20 times) times concentrated washing liquid with distilled water (20 times of 48 t) 30 times dilution backup.

5. Washing: carefully remove the seal plate membrane, abandon to liquid, dry, every hole filled with liquid detergent. Let stand for 30 seconds to refuse to repeat 5 times, pat dry.

6. Add enzyme: 50 mu, l per hole plus enzyme standard reagent blank except for hole.

7. Incubate: operation with 3.

With 5:8. Washing operation.

9. Color: each Kong Xian adding chromogenic agent A50 mu l, add the chromogenic agent B50 mu l, gently shake blending,37 Avoid light color for 15 minutes.

10. Termination: 50 mu, l per hole and end termination reaction (the blue turn yellow).

11. Determination: the zero blank air conditioning, 450 nm wavelength in order to measure the absorbance (OD value) of each hole.15 minutes after the determination shall be terminated with liquid.

Note:

1. The kit should be at room temperature out of the cold storage environment balance can be used only for 15 to 30 minutes, and after opening, such as enzyme standard package is board, not use up slats should be kept in sealed bag.

2. There may be crystallization precipitation concentrated washing liquid, when diluted in water bath heating to dissolve and washing does not affect the results.

Step 3. Each plus sample shall be used and pipette, and often check its accuracy, to avoid the test errors.A best sample time control within 5 minutes, such as specimen number, it is recommended to use volley and sample.

4. Please do standard curve of each measurement at the same time, it's best to do multiple holes.Such as excessive amounts of material under test in the specimen (the sample OD value is greater than standard hole first hole OD value), please use the sample diluent dilution multiples (n times) must be measured again after, when calculating the last times the total diluted multiples (* n * 5).

5. Sealing plate membrane only for one-time use, in order to avoid cross contamination.

6. The substrate please avoid light preservation.

7. In strict accordance with the manual operation, the test result determination must take enzyme mark meter readings shall prevail.

8. All samples, washing liquid and all kinds of waste should be handled according to the infection.

9. The reagent for different batch number components shall not be mixed.

10. If there are different with English instruction, the English instruction manual shall prevail.

Calculation:

In the concentration of the standard for horizontal, OD value of the vertical, draw the standard curve on graph paper, according to the sample OD value detected by the standard curve of the corresponding concentration;Times diluted multiples;Or with the concentration of the standard content of standard curve and the OD value calculated linear regression equation, the sample OD value into the equation, to calculate the concentration of sample, then multiplied by the dilution ratio, which is the actual concentration of the sample.

Kit performance:

1. The sample linear regression and correlation coefficient R expected concentration value is more than 0.95.

2. Batch to batch should be less than 9% and 9% respectively

Detection range:

100ng/L -2000ng/L

Preservation conditions and the period of validity:

1. Kit to save:;2- 8

2. The period of validity: 6 months


Delivery time:Source: China encyclopedia net author:
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