Human mitochondrial inner membrane protein (IMMT) ELISA test kit instruction manual

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

Test principle

The IMMT kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards of known IMMT concentrations and samples of unknown concentration are added to the microplates for detection. IMMT 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 IMMT in the sample.

Kit content and its preparation
Kit components (2-8 ° C preservation) 96-well configuration 48-well configuration
96/48 person plate: one plate (96T) half plate (48T) ready-to-use plastic film cover 1 half piece ready-to-use standard: 400nmol/L 1 bottle (0.6ml) 1 bottle (0.3 Ml) According to the instructions, 1 bottle (1.0ml), 1 bottle (0.5ml), standard standard dilution buffer, 1 bottle (5ml), 1 bottle (2.5ml), ready-to-use biotin-labeled anti-IMMT antibody 1 bottle (6ml) 1 bottle (3.0ml) ready-to-use streptavidin-HRP 1 bottle (10ml) 1 bottle (5.0ml) ready-to-use washing buffer 1 bottle (20ml) 1 bottle (10ml) according to the instructions Dilute 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 stop solution 1 bottle (6.0ml) 1 bottle (3.0 Ml) Ready-to-use specimen dilution 1 bottle (12ml) 1 bottle (6.0ml) Ready-to-use

Self-contained materials
Distilled water.
Sampler: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul.
Oscillators and magnetic stirrers, etc.

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 during the experiment.
3. Do not use the mouth to absorb any ingredients in 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 packaging bag and sealed to prevent deterioration.
3. Other reagents that are not used should be packaged or covered. Do not mix reagents of different batches. Use before warranty.
4. Use disposable tips 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 thoroughly and pat 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 a long 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. Carry out the incubation operation according to the time indicated in the instruction manual, the amount of liquid addition and the order.

Sample collection, processing and storage methods
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.
Plasma-----EDTA, citrate, heparin plasma can be used for detection. The pellet was removed by centrifugation at 1000 x g for 30 minutes.
The cell supernatant - 1000 x g was centrifuged for 10 minutes to remove particles and polymer.
Tissue homogenization ----- tissue is added to the appropriate amount of physiological saline. Centrifuge at 1000 × g for 10 minutes, and take the supernatant for storage. ------ If the sample is not used immediately, it should be divided into small portions at -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
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:
400 nmol/L (Standard No. 6) The original concentration was directly added to 50 ul without dilution.
200 nmol/L (No. 5 standard) 100 ul of the original standard is added to 100 ul of standard dilution
100 nmol/L (Standard No. 4) 100 ul of Standard 5 is added to 100 ul of standard dilution
50 nmol/L (Standard No. 3) 100 ul of Standard 4 is added to 100 ul of standard dilution
25 nmol/L (Standard No. 2) 100 ul of Standard No. 3 plus 100 ul of standard dilution
12.5 nmol/L (Standard No. 1) 100 ul of Standard 2 is added to 100 ul of standard dilution
0 nmol/L (empty transfer chemotactic growth factor β1 control) The original concentration was directly added to 50 ul without dilution.

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. Determine the number of slats required based on the number of samples to be tested plus the number of standards. Multiple wells are recommended for each standard and empty metastatic growth factor β1 well. 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. Add 50 ul of the diluted standard to the reaction well and add 50 ul of the sample to be tested 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. Add 80 ul of affinity streptavidin-HRP to each well, mix gently by shaking, and incubate 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 ul of substrate 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. Determine the OD value of each well at a wavelength of 450 nm.

Recommended experimental protocol
Standard concentration (nmol/L)
A 400 400 sample sample sample sample sample sample sample sample sample
B 200 200 sample sample sample sample sample sample sample sample sample
C 100 100 sample sample sample sample sample sample sample sample sample
D 50 50 sample sample sample sample sample sample sample sample sample
E 25 25 Sample Sample Sample Sample Sample Sample Sample Sample Sample
F 12.5 12.5 Sample Sample Sample Sample Sample Sample Sample Sample Sample
G 0 0 sample sample sample sample sample sample sample sample sample
H sample sample sample sample sample sample sample sample sample sample

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

Kit performance
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 IMMT standard concentration is the abscissa (X), and the corresponding curve is obtained. The IMMT content of the sample can be converted into the corresponding concentration according to the OD value from the standard curve.

3. Detection range: 0-400nmol/L

4. Sensitivity: 1.0 nmol/L