Human glutamate decarboxylase autoantibody (GAD-Ab25) enzyme-linked assay analysis ELISA instructions

Human glutamate decarboxylase autoantibody (GAD-Ab25) enzyme-linked assay analysis ELISA instructions

This reagent is for research use only. Specimen: serum or plasma

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purpose of usage:

This kit is used to determine the content of glutamate decarboxylase autoantibody (GAD-Ab25) in human serum, plasma and related liquid samples.

Test principle:

The GAD-Ab25 kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards with unknown concentrations of GAD-Ab25 and samples of unknown concentration are added to the microplates for detection. GAD-Ab25 and biotinylated antibodies were first incubated simultaneously. After washing, avidin-labeled HRP was added. After incubation and washing, the unbound enzyme conjugate is removed, then the substrates A, B, and the enzyme conjugate are added simultaneously. Produce color. The depth of the color is proportional to the concentration of GAD-Ab25 in the sample.

Kit content and its preparation

Self-contained materials

1. Distilled water.

2. Sampler: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul.

3. Oscillators and magnetic stirrers, etc.

safety

1. Avoid direct contact with the stop solution and substrates A, B. Once exposed to these liquids, rinse with water as soon as possible.

2. Do not eat, drink, smoke or use cosmetics during the experiment.

3. Do not use your mouth to take any ingredients from the kit.

Operational precautions

1. Reagents should be stored according to the label instructions and returned to room temperature before use. Standards after dilution should be discarded and cannot be stored.

2. The slats not used in the experiment should be immediately put back into the bag and sealed to prevent deterioration.

3. Other reagents not used should be packaged or covered. Do not mix reagents of different batches. Use before warranty.

4. Use a disposable tip to avoid cross-contamination. Avoid pipettes with metal parts when drawing stop solution and substrate A and B.

5. Use a clean plastic container to configure the wash solution. Mix all the ingredients and samples in the kit thoroughly before use.

6. Wash the enzyme plate should be fully patted dry, do not put the absorbent paper directly into the enzyme standard reaction well to absorb water.

7. Substrate A should be volatilized to avoid opening the lid for extended periods of time. Substrate B is sensitive to light and avoids prolonged exposure to light. Avoid contact with hands and be toxic. The OD value should be read immediately after the experiment is completed.

8. The order of addition of reagents should be consistent to ensure that all wells are incubated for the same time.

9. The incubation was carried out according to the time indicated in the instructions, the amount of addition and the order.

Sample collection, processing and storage methods

1. Serum ---- Avoid any cell irritation during the procedure. Use tubes without pyrogens and endotoxins. After collecting the blood, the serum and red blood cells were quickly and carefully separated by centrifugation at 1000 x g for 10 minutes.

2, plasma ----- EDTA, citrate, heparin plasma can be used for testing. The pellet was removed by centrifugation at 1000 x g for 30 minutes.

3. Cell supernatant - 1000 x g was centrifuged for 10 minutes to remove particles and polymer.

4, tissue homogenate ----- the tissue is added to the appropriate amount of physiological saline mash. Centrifuge at 1000 × g for 10 minutes, take the supernatant

5. Storage ------If the sample is not used immediately, it should be divided into small parts - 70 °C to avoid repeated freezing. Do not use hemolysis or hyperlipemia as much as possible. If there are large amounts of particles in the serum, centrifuge or filter before testing. Do not heat thaw at 37 ° C or higher. It should be thawed at room temperature and ensure that the sample is fully thawed evenly.

Reagent preparation

1. Standard: The serial dilution of the standard should be prepared during the experiment and cannot be stored. Mix the standard shakes before dilution. The dilution ratio is as follows:

2. Dilution of wash buffer (50 x): 50-fold dilution of distilled water.

Steps

1. Mix all reagents thoroughly before use. Do not allow the liquid to generate a large amount of foam, so as to avoid adding a large amount of air bubbles during the loading, resulting in errors in the loading.

2. The number of slats required is determined by the number of samples to be tested plus the number of standards. Multiple holes are recommended for each standard and blank hole. Each sample is determined according to its own quantity, and it is possible to use the double hole as much as possible to make a double hole. The specimen was diluted 1:1 with the specimen dilution and 50 ul was added to the reaction well.

3. 50 ul of the diluted standard was added to the reaction well, and 50 ul of the sample to be tested was added to the reaction well. Immediately add 50 ul of biotinylated antibody. Cover the membrane, mix gently by shaking, and incubate for 1 hour at 37 °C.

4. Remove the liquid from the well, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once.

5. 80 ul of affinity streptavidin-HRP was added to each well, and the mixture was gently shaken and incubated at 37 ° C for 30 minutes.

6. Remove the liquid from the well, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once.

7. Add 50 μl of each of the substrates A and B to each well, mix gently by shaking, and incubate at 37 ° C for 10 minutes. Avoid lighting.

8. Remove the microplate and quickly add 50 ul of stop solution. Immediately after adding the stop solution, the results should be determined.

9. The OD value of each well was measured at a wavelength of 450 nm.

Recommended experimental protocol

Limit

The results above the No. 6 standard are non-linear and no accurate results can be obtained from this standard curve.

Performance of glutamate decarboxylase autoantibody (GAD-Ab25) ELISA kit

1. Sensitivity: The minimum detection concentration is less than the No. 1 standard. The linearity of the dilution. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value was 0.990.

2. Specificity: Does not react with other cytokines.

3. Repeatability: The coefficient of variation between the plate and the plate is less than 10%.

Result judgment and analysis

1. Instrument value: read the OD value of each hole on a microplate reader with a wavelength of 450 nm.

2. The absorbance OD value is the ordinate (Y), and the corresponding GAD-Ab25 standard concentration is the abscissa (X), and the corresponding curve is obtained. The GAD-Ab25 content of the sample can be converted from the standard curve according to the OD value. The corresponding concentration.

3. Detection range: 0-80ng/ml

4. Sensitivity: 0.1 ng/ml

Shanghai Qiao Yu specializes in the production and supply of kit products: double-anti-sand sandwich ELISA test, ELISA kit, human ELISA kit, enzyme-linked detection kit, domestic imported reagent, sub-test elisa kit, ELISA test kit, large Laboratory products such as mouse ELISA kit, mouse ELISA kit, apoptosis related factor kit, interleukin ELISA kit, VIP ELISA kit, product quality, quality assurance.

The performance of kit:

1 sensitivity: minimum detection concentration is less than 1 standard. Linearity of dilution. Sample linear regression and the expected concentration correlation coefficient R value is 0.990.

2: no specific reaction with other cytokines.

3 repeatability: plate, plate between the coefficients of variation were less than 10%.

Human type III procollagen amino ring peptide ELISA kit steps:

1 before use, all reagents and mixing. Do not allow liquid to produce a large number of bubbles, so as to avoid adding a large number of bubbles, resulting in the addition of the error.

Each sample can be made according to its own quantity, and can be used as a hole in the hole.

3 diluted after standard 50ul in reaction hole, added to the sample 50 UL in reaction hole to be measured. Immediately joined the 50 UL antibody biotin. Cover the membrane plate, gently oscillating mixing, 37 degrees Celsius for 45 minutes.

4 left hole liquid, each hole filled with washing liquid, oscillating 30 seconds off the washing liquid, pat dry with absorbent paper. Repeat this operation 4 times. If the washing machine with washing, washing times increased once.

5 per hole adding chain affinity enzyme -HRP 100ul, gently oscillating mixing, 37 degrees 30 minutes incubation.

6 left hole liquid, each hole filled with washing liquid, oscillating 30 seconds off the washing liquid, pat dry with absorbent paper. Repeat this operation 4 times. If the washing machine with washing, washing times increased once.

7 per hole adding substrate A, B 50ul, gently oscillating mixing, 37 degrees 5 minutes incubation. Avoid light.

8 remove ELISA plate, quickly add 50ul terminated liquid, adding the stop solution immediately after the determination results.

9 od determination of each hole at the wavelength of 450nm.

The result of judgment and analysis:

1, instrument value: Yu Bo 450nm ELISA od read the hole on the instrument

2, to the OD value as a vertical coordinate (y), corresponding ot standard concentrations as a horizontal coordinate (x), do have corresponding curve, sample ot content can be according to the OD value by standard curve conversion out corresponding concentration, multiplied By the dilution multiple; or with the standard concentration and the OD value calculated the regression equation of the standard curve, the sample OD value in the equation to calculate the sample concentration, multiplied by the dilution factor is the actual concentration of the sample.

3, detection range: 0-100ng/ml

4, sensitivity: 0.39ng/ml

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