Human α1 microglobulin (α1-MG) ELISA detection kit

Description of human α1 microglobulin (α1-MG) ELISA detection kit

Detection value range: 0-800ng / ml

Sensitivity: 1.0 ng / ml

Human α1 microglobulin (α1-MG) ELISA detection kit test principle: α1-MG kit is a solid-phase sandwich method enzyme-linked immunosorbent assay (ELISA). Standards with known α1-MG concentration, samples with unknown concentration Add to the microplate for detection. First, α1-MG and biotin-labeled antibodies were incubated at the same time. After washing, HRP labeled with avidin was added. After incubation and washing, the unbound enzyme conjugate is removed, and then substrates A and B are added to act simultaneously with the enzyme conjugate. Produce colors. The color depth is proportional to the concentration of α1-MG in the sample.

Content and preparation of human α1 microglobulin (α1-MG) ELISA test kit:

Kit components (stored at 2-8 ° C) 96-well configuration 48-well configuration

96/48 serving microplate 1 plate (96T) half plate (48T) ready-to-use

Plastic film cover 1 piece and half piece ready to use

Standard product: 800pg / ml 1 bottle (0.6ml) 1 bottle (0.3ml) Dilute according to the instructions

Blank control 1 bottle (1.0ml) 1 bottle (0.5ml) ready-to-use

Standard Dilution Buffer 1 bottle (5ml) 1 bottle (2.5ml) ready-to-use

Biotin-labeled anti-Gastrin antibody 1 bottle (6ml) 1 bottle (3.0ml) ready-to-use

Streptavidin-HRP 1 bottle (10ml) 1 bottle (5.0ml) ready-to-use

1 bottle of washing buffer (20ml) 1 bottle (10ml) according to the instructions for dilution

Substrate A 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use

Substrate B 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use

1 bottle (6.0ml) of stop solution 1 bottle (3.0ml) ready-to-use

Bring your own materials:

1. Oscillator and magnetic stirrer, etc.

2. Distilled water.

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

safety:

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

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

3. Do not use your mouth to absorb any components in the kit.

Human α1 microglobulin (α1-MG) ELISA detection kit sample collection, processing and storage methods:

1. Serum ----- Avoid any cell stimulation during the operation. Use test tubes free of pyrogens and endotoxins. After collecting blood, centrifuge at 1000 × g for 10 minutes to separate the serum and red blood cells quickly and carefully.

2. Plasma-EDTA, citrate, heparin plasma can be used for detection. Centrifuge at 1000 × g for 30 minutes to remove particles.

3. Cell supernatant-centrifuge at 1000 × g for 10 minutes to remove particles and polymer.

4. Storage-If the sample is not used immediately, it should be divided into small parts -70 ℃ to avoid repeated freezing. If possible, do not use hemolysis or hyperlipidemia. If there are a large number of particles in the serum, centrifuge or filter before testing. Do not thaw at 37 ° C or higher. Thaw at room temperature and ensure that the sample is thawed evenly and adequately.

Preparation of reagents:

1. Standards: Serial dilutions of standards should be prepared during the experiment and cannot be stored. Before dilution, the standard was mixed by shaking. The dilution ratio is as follows:

800 pg / ml (No. 6 standard) The original concentration is added directly to 50ul without dilution.

400 pg / ml (Standard 5) 100ul of the original standard plus 100ul of standard dilution

200 pg / ml (Standard No. 4) 100ul No. 5 Standard plus 100ul Standard Diluent

100 pg / ml (Standard 3) 100ul of Standard 4 plus 100ul of Standard Diluent

50 pg / ml (No. 2 standard) 100ul of No. 3 standard plus 100ul of standard dilution

25 pg / ml (No. 1 standard) 100ul of No. 2 standard plus 100ul of standard diluent

0 pg / ml (blank control) The original concentration is directly added to 50ul without dilution.

2. Dilution of washing buffer (50 ×): 50-fold dilution with distilled water.

Steps:

1. Before use, mix all reagents thoroughly. Don't make the liquid generate a lot of foam, so as to avoid adding a large number of bubbles during sample addition, which will cause errors in sample addition.

2. Decide the number of slats required based on the number of samples to be tested plus the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used for multiple holes.

3. Add 50ul of the diluted standard to the reaction well and 50ul of the sample to be tested in the reaction well. Immediately add 50ul of biotin-labeled antibody. Cover the membrane plate, gently shake and mix, and incubate at 37 ° C for 1 hour.

4. Shake off the liquid in the hole, fill the hole with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once.

5. Add 80 ul of streptavidin-HRP to each well, gently shake and mix, and incubate at 37 ° C for 30 minutes.

6. Shake off the liquid in the hole, fill each hole with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once.

7. Add 50ul of substrate A and B to each well, gently shake and mix, and incubate at 37 ° C for 10 minutes. Avoid light.

8. Remove the enzyme labeling plate and quickly add 50ul of stop solution. After adding the stop solution, the results should be measured immediately.

9. Measure the OD value of each well at 450nm wavelength.

Operation notes:

1. Reagents should be stored according to the label instructions and returned to room temperature before use. The sparse standards should be discarded and cannot be stored.

2. The slats not used in the experiment should be immediately returned to the packaging bag, sealed and stored to avoid deterioration.

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

4. Use a disposable pipette tip to avoid cross-contamination, and avoid using a sampler with a metal part when drawing the stop solution and the substrate A and B liquids.

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

6. When washing the enzyme plate, it should be fully patted dry. Do not put the absorbent paper directly into the reaction well of the enzyme plate to absorb water.

7. Substrate A should evaporate to avoid opening the lid for a long time. Substrate B is sensitive to light and avoid prolonged exposure to light. Avoid contact with hands, it is toxic. The OD value should be read immediately after the experiment is completed.

8. The order of adding reagents should be consistent to ensure that all reaction plates are incubated for the same time.

9. Carry out the incubation operation according to the time, the amount and sequence of the liquid added in the manual.

Kit performance:

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

2. Specificity: Does not react with other cytokines.

3. Repeatability: The coefficients of variation within and between plates are less than 10%.

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

5. Take the absorbance OD value as the ordinate (Y) and the corresponding α1-MG standard concentration as the abscissa (X), and make the corresponding curve. The α1-MG content of the sample can be converted from the standard curve according to its OD value The corresponding concentration.