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Home > Company News

ELISA experiment steps and precautions

Views : 1718
Update time : 2023-03-01 15:59:54
Experimental steps

一、 Encapsulated antigen

1. Dissolve the antigen with 50 mM carbonate coating buffer (pH 9.6) to a concentration of 10-20 μg/ml, add 100 μl/well to a 96-well enzyme plate, and leave it overnight at 4℃.
2. After discarding the coating solution the next day, wash the plate 3 times with PBST and add 150 μl of 1% BSA to each well for 1 hour at 37℃.
3. After 3 washes with PBST, add 100 μl of serum at different dilutions to each well and add control samples and incubate at 37°C for 2 hours.
4. After 5 washes of PBST, add 100 μl of diluted HRP-labeled secondary antibody and incubate at 37°C for 1 hour.
5. After 5 washes of PBST, the color developer was used for 20 min and the absorption value of A405 was read on the enzyme marker.


二、Coat the cells 

1. Inoculate the 96-well culture plate with 1 x 104 cells/well and incubate overnight at 37°C. 2.
2. Wash the plate with PBS 2-3 times on the next day. 3.
3. Add 125 μl/well of 10% Forma lin (1:10 dilution) and fix for 15 min at room temperature.
4. Wash the plate 3 times with ddH2O, dry and store at 2-8°C.
5. Wash 3 times with PBST and add 150 μl of 1% BSA per well for 1 hr at 37°C.
6. After 3 washes with PBST, add 100 μl of serum at different dilutions to each well and add control samples and incubate at 37℃ for 2 hours.
7. After 5 washes of PBST, add 100 μl of diluted HRP-labeled secondary antibody and incubate at 37°C for 1 hour.
8. After 5 washes of PBST, the color developer was developed for 20 min and the absorption value of A405 was read on the enzyme marker. 50 mM of carbonate coating buffer: 0.05 mol/L pH 9.6 carbonate buffer, 4°C, stored, Na2CO3 0.15 g, NaHCO3 0.293 g, diluted to 100 ml in distilled water. 
ABTS as substrate for color development reaction (10 ml).
 0.2M Na2HPO4 2.4ml
 0.1M Citric acid 2.6ml
ddH2O 5ml
ABTS 5mg
H2O2 (30%) 4 ul (added before use)



Precautions

Serum specimens should be tested when they are fresh. If there is bacterial contamination, the bacterium may contain endogenous HRP, which may also produce false positive reaction. If stored too long in the refrigerator, the polymerization of which can occur, can deepen the background in the indirect method ELISA. Generally speaking, the serum specimens measured within 5 days can be placed at 4℃, and those measured over a week need to be stored on ice at low temperature. After thawing the frozen serum, the protein is locally concentrated and unevenly distributed, so it should be mixed gently, avoid bubbles, mix upside down, and do not shake strongly on the mixer. Cloudy or precipitated serum specimens should be centrifuged or filtered first and then clarified before testing. Repeated freezing and thawing can cause antibody potency to fall, so if a serum specimen is to be stored for multiple testing, it should be stored on ice in small amounts. Preservation of sera should be done aseptically from the time of collection, and appropriate preservatives can be added.
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