Problems in the operation of ELISA reagents

Problems in the operation of ELISA reagents

problem

possible reason

Prevention method

(One)
Low color rendering sensitivity

1. The kit is too long in transit and the temperature is too high

The kit is transported in an incubator, and a sufficient number of ice cubes are placed to minimize the transport time

2. Kit expired

Do not use the kit after the expiration date

3. Reagents and samples are not balanced before use

Allow reagents and samples to equilibrate at room temperature for about 30 minutes before use

4. Reagents opened too long to grow bacteria

After the reagent is turned on, please try to use it within a short time

5. The pipette has insufficient suction, the pipetting and suction discharge is too fast, too much liquid is hanging on the inner wall of the tip or the inner wall is not clean

To calibrate the pipette, the tips should be matched, and the tips should be tightly fitted each time. Pipetting should not be too fast, the discharge should be complete. The inner wall of the tip should be cleaned, and it is best to use it once.

6. (The temperature of the thermostat is less than 37 ℃ or the temperature of the water bath is less than 37 ℃)

Pay attention to the temperature of the incubator when placing it in the reaction plate, and adjust it in time. It should not be opened too much during the incubation reaction, which affects the temperature balance.

7. Insufficient reaction time

Accurate timing of fixed clock

8. The impact force is too large during washing, the washing liquid is soaked for too long, and the number of washing times increases

Reduce the washing impact, keep the washing time according to the instructions, and remember the washing times accurately

9. Insufficient amount of developer is added or the order is reversed, or added after mixing

When adding the color developing solution, the drop bottle is vertically downward, the holding force is uniform, and the dripping speed should not be too fast.

10. Insufficient reaction time of developer

Accurate timing

11. Distilled water quality problem

Determination of the influence of reagents prepared by distilled water on enzyme immunoassay

12. The sample and NaN 3 are preserved, inhibiting the enzyme reaction

The sample cannot be added with NaN 3 for corrosion protection

(two)
Poor repeatability

1. For quantitative products, without standard products and quality control products, the previous standard line is used

Each test must bring standard products and quality control products

2. The number of samples varies, and the sample addition time varies

When repeating a sample, the sample addition time should be as close as possible to the first time

3. Samples were not mixed after addition

Mix well on the mixer after loading

4. Pipette sample loading volume is inconsistent

Use the same pipette as much as possible and use the same force and suction speed when pipetting

5. The filter of the microplate reader is wrong or the input wavelength is wrong

TMB color rendering with 450 or 450 / 630nm dual wavelength

6. Poor repeatability of microplate reader

Calibration microplate reader

7. Liquid splashing in individual holes during operation

Hold steady and prevent splashing

8. Confusion of components in different batch kits

Reagents of different batches cannot be mixed

9. Bottle caps cross each other

Do not cross use between caps of different components

10. Blood donor impostor

Strictly manage blood donors

11. Inconsistent holding time

Repeat the measurement of the specimen, and the reaction conditions, personnel, etc. should be consistent with the last time as much as possible to eliminate the possibility of inconsistency caused by these factors.

12. Inconsistent washing conditions

13. Color development condition time is inconsistent

14. Operators are inconsistent

15. Incorrect washing

Fill the holes with lotion, but do not overflow. When flicking the plate, the reaction plate is facing downwards, and the contents are flicked vertically (can be flicked several times), and patted dry on a clean, no-dust or less-absorbent material. The plate washer should not have holes when washing the plate, and the washing should be sufficient

16. Inconsistent incubation conditions (once in a water bath and once in a thermostat)

The incubation of the thermostat is deeper than that of the water bath, and the OD value is higher than 0.10-0.15. It is best to use a thermostat. If there is no thermostat, the water temperature of the water bath should be controlled at 37 ℃

17. Saliva or other debris falls into the hole

Straight circle with a marker for easy analysis

18. Inadvertently add enzyme or substrate solution to the well wall, there is residue

Absorb remaining enzyme or substrate liquid on the wall with absorbent paper

19. Inadvertently adding more or less enzyme or developer B

When adding more, the color is deeper, and when adding less, it is lighter.

20. Yin and yang

Repeat the odd number of times, whichever is the even number of times

twenty one. Incomplete centrifugal processing of specimens, resulting in coagulation or interference of residual cellular components in the well of the reaction plate during detection

Serum or plasma should be fully centrifuged, 3000rpm for more than 6 minutes



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