In the process of doing ELISA kit experiments, it is inevitable that you will encounter various problems, such as poor standard curve, kit recovery rate, false negative, false positive, poor repeatability of the experiment and so on. Now come to answer all the questions encountered by users during the experiment!
ELISA kit curve problem:
1. The OD value is abnormal
The kit is not warmed, the kit is warmed to 25 ℃
Poor temperature control, correct temperature control
Incubation time is not accurate, control incubation time
When washing, the impact force is too large, the soaking time is too long, the washing frequency is increased, washing 4-5 times, 250ul per hole, then pat dry
Direct sunlight, blowing directly against the wind, avoid the wind
The wavelength of the microplate reader is wrong, the wavelength of the microplate reader is 450nm
The antibody was diluted incorrectly, the antibody was diluted correctly
Water quality problems, use Wahaha mineral water instead
2. Whiteboard
Wrong preparation of washing solution, correct preparation of washing solution
Add less liquid (add liquid by mistake), add liquid in the right way
3. No gradient
The standard product is also contaminated.
The OD value is abnormal, the temperature control time is done once
Do not click the board artificially
4. Linearity is not good
The OD value is abnormal, the temperature control time is done once
ELISA kit recovery rate
ELISA kit 1. Blank tube positive high
The sample itself is a positive sample, change the negative sample for recovery
The blank tube was contaminated during the experiment, preventing cross-contamination during the experiment
For water quality reasons, Wahaha mineral water is used instead
2. On the high side
Inaccurate addition amount, precise addition amount
Add concentration is not suitable, add appropriate concentration
Large amount of liquid to be blown dry, accurate liquid to be blown dry
Take other phase layer liquid, and use phase to blow dry
The double solution is undiluted or the added amount is small, the correct double diluted solution
ELISA kit 3. Low
Inaccurate addition amount, precise addition amount
Add concentration is not suitable, add appropriate concentration
The amount of liquid extraction and drying is small, and the accurate liquid extraction and drying
Dilute the double solution incorrectly or add too much, correct the double solution
False negative, positive
1. Water quality reason: point complex solution verification, use Wahaha mineral water instead
2. Reasons for experiment operation
Absorb to other phases and blow dry, accurately take the liquid and blow dry
Incomplete centrifugation, extend the centrifugation time
Sample contamination, replace sample
The emulsification is not treated completely, and the emulsification phenomenon is treated in a warm bath (70 ℃)
3. Reasons for instrument and reagent
The centrifuge tube is not cleaned, the correct washing utensils
The influence of organic solvents, ELISA kit as a reagent blank to see the impact results
4. Reasons for derivatization reagent (long-term derivatization reagent deterioration), change derivatization reagent
5, related to the quality of the curve, to ensure the normality of the curve
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