Common problems and solutions in ELISA kit experiments - Database & Sql Blog Articles

The elisa kit experiment has been widely used in the characteristics of high sensitivity and good specificity. However, all the links in the operation have a great influence on the detection effect of the test. If not pay attention, it may lead to incomplete coloration, flower plates, etc. result. Our company summarizes the causes and solutions of the problems that often occur in each part of the operation, in order to bring some inspiration to the peers and improve the quality of the experiment. The following is an analysis of the reasons that may affect the results of the ELISA kit experimental operation, and the corresponding solutions are given.

First, select the reagent

Select a good quality test reagent, strictly follow the reagent instructions, and equilibrate the reagent at room temperature for 30-60 minutes before the operation.

Second, the sample loading

Possible Causes:

1) If the serum or plasma specimens are not well separated, the sample is loaded; 2) In the manual operation, the excessive application of the sample plate causes the waiting time to be too long before the injection into the incubator (especially when the indoor temperature is high); When the specimen is added and the enzyme reagent is added, the enzyme spills out of the pore.

Solution:

1) The specimen is serum: it is best to store the blood naturally for 1-2 hours, then centrifuge at 3000rmp for 15 minutes; the specimen is plasma: blood specimen collection tubes containing anticoagulant must be used, and the blood collection must be reversed immediately after blood collection. 5-10 times, after a period of time, centrifuge at 3000 rpm for 15 minutes; if it is tested within a few days, it can be placed in a refrigerator at 2-8 °C. If it is to be stored, it should be placed in a low temperature refrigerator at -20 °C. 2) Put it into the incubator in time after loading. 3) After adding the enzyme reagent, use a blotting paper to gently blot and dry on the surface of the microplate. 4) If using AT or other fully automatic loading, it is best to choose FAME or other post-processing instruments plus enzyme reagents. 5) When there are many specimens, please operate in batches.

Third, incubate

Possible Causes:

1) When the incubation is not attached or capped, the specimen or diluent is evaporated and adsorbed on the pore wall, which is difficult to clean thoroughly; 2) The incubation time is artificially extended, resulting in non-specific binding surrounding the reaction well, which is difficult to clean thoroughly.

Solution:

1) Attach the cover or cover; 2) Strictly control the operation time according to the instructions.

Fourth, wash the board

Possible Causes:

1) Wash the plate by hand and cross the liquid between the hole and the hole. 2) When washing the plate with a semi-automatic washer, the amount of washing liquid is insufficient, resulting in incomplete washing; the washing plate is clogged, the suction is not complete; the washing plate is not smooth, resulting in poor washing effect. 3) Excessive reaction plates cause long waiting time for washing.

Solution:

1) Ensure that the washing liquid fills the holes, and the washing plate needle is unblocked. After washing the plate, it is best to pat dry on the absorbent paper (choose a clean, no or little dust-absorbing material); 2) arrange it reasonably, or use a few more Washing machine.

Five, color development

Possible Causes:

1) The developer is left for a long time after preparation or the expired color developer is used; 2) When the developer is added, the liquid is recirculated outside the hole.

Solution: 1) The coloring agent should be prepared as far as possible before use. It should not be used without expiring the color developer. The light blue TMB developer is not visible to the naked eye; 2) Keep the color developer out of the flow when loading; 3) A. Liquid B should avoid contact with metal instruments.

Sixth, termination

Possible causes are that more bubbles are generated when the stop solution is added, resulting in an increase in false positives. Therefore, bubbles should be avoided when adding stop solution.

Seven, read the board

If the board is not clean when reading the board, etc. The microplate should be cleaned. Therefore, during the whole operation process, the enzyme labeling plate is not contacted with hypochlorous acid; the ELISA detection standard is automated as much as possible, and the detection quality is effectively improved.

In the actual operation, in addition to selecting excellent reagents, it is necessary to strictly follow the operation steps, and at the same time, make indoor quality control and inter-room quality assessment, and test each specimen with strict working style to ensure the quality of inspection. At present, a considerable number of units in China have automatic microplate readers, which plays an important role in achieving standardized detection of ELISA and improving the quality of testing.

I hope that these can help you. In the ELISA kit experiment, you must pay attention to the details and be foolproof.

It is derived from the advanced enzyme-linked immunosorbent technology Shanghai Jinma Experimental Equipment Co., Ltd., covering the experience and wisdom of domestic and foreign experts. The use of advanced technology and equipment to ensure quality while reducing costs, for more researchers in need, save experimental funds, ensure the quality of experiments, and contribute to the country's scientific and technological development. A number of bio-research base partners, the strength team is dedicated to creating a focus on

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More developments in science and technology. A number of bio-research base partners, the strength team is dedicated to creating a focus on

Elisa

Production, research and development of kits to help your research

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