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Common Problems and Solutions in ELISA

Common Problems and Solutions in ELISA

The test of the ELISA workstation has been widely used clinically because of its high sensitivity and good specificity. However, each link in the operation has a greater impact on the detection effect of the test. If you do not pay attention, it may lead to incomplete color rendering, patterning and other results. We summarize the causes and solutions of the problems that often occur in each link of the operation as follows, hoping to give you some inspiration and improve the quality of the experiment.


Ⅰ. Selection of reagents for fully automated ELISA


Select high-quality detection reagents for the fully automated ELISA and operate in strict accordance with the instructions. Before the operation, equilibrate the reagents at room temperature for 30-60 minutes.


Ⅱ. The sample loading of fully automated ELISA


1. Possible reasons:


(1) If the separation of serum or plasma samples is not good, add samples;


(2) In manual operation, too many sample plates will cause too long waiting time before putting the sample into the incubator (especially when the indoor temperature is high);


(3) After adding the sample and adding the enzyme reagent, the enzyme splashed out of the hole.


2. Solution:


(1) The specimen is serum: it is best to store the blood naturally for 1-2 hours, and then centrifuge at 3000 rpm for 15 minutes;

The specimen is plasma: a blood specimen collection tube containing anticoagulant must be used, and the blood collection tube must be inverted and mixed 5-10 times immediately after blood collection. 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, and if it is to be stored, it can be placed in a low-temperature refrigerator at -20°C.


(2) Put the sample into the incubator in time after adding the sample.

(3) After adding the enzyme reagent, wipe the surface of the microtiter plate with absorbent paper to dry.

(4) If AT or other automatic sample addition is used, it is best to choose FAME or other post-processing instruments to add enzyme reagents.

(5) When there are many specimens, please operate in batches.


Ⅲ. Incubation of fully automated ELISA


1. Possible reasons:


(1) There is no cover or cover during incubation, so that the specimen or diluent evaporates and adsorbs to the wall of the hole, making it difficult to clean thoroughly;


(2) The incubation time is artificially prolonged, resulting in non-specific binding tightly around the reaction well, making it difficult to clean thoroughly.


2. Solution:


(1) Attach a cover sheet or cover;


(2) Strictly control the operating time according to the instructions.


Ⅳ. The washing plate of fully automated ELISA


1. Possible reasons:


(1) Wash the plate manually, and the liquid crosses between the holes;


(2) When the semi-automatic plate washer is used to wash the plates, the amount of washing liquid is insufficient, which leads to incomplete plate washing; the clogging of the plate washing needle causes incomplete suction; the poor plate washing results in poor plate washing effect;


(3) Too many reaction plates cause long waiting time for washing plates.


2. Solution:


(1) Ensure that the lotion fills the holes and the washing needle is unblocked. After washing the plate, it is best to gently pat dry on absorbent paper (choose clean, no or less dusty absorbent material);


(2) Reasonably arrange, or use more plate washer.


Ⅴ. The color development of fully automated ELISA


1. Possible reasons:


(1) The developer has been left for too long after preparation or used an expired developer;


(2) When the developer is added, it splashes out of the hole and causes the liquid to flow back.


2. Solution:


(1) The color developer should be prepared before use as much as possible, and the expired color developer should not be used. The light blue TMB color developer is not used;


(2) Keep the developer from flowing out when adding samples;


(3) A and B liquid should avoid contact with metal equipment.


Ⅵ. Termination of fully automated ELISA


Possible cause: For example, more bubbles are generated when the stop solution is added, resulting in an increase in false positives. Therefore, avoid air bubbles when adding stop solution.


Ⅶ. The plate reading of fully automated ELISA


If the bottom of the plate is not clean when reading the plate, ensure that the microtiter plate is clean. Therefore, in the entire operation process, ensure that the ELISA plate does not contact hypochlorous acid, and realize the automation of fully automatic ELISA testing standards as much as possible, which effectively improves the quality of testing. Besides, it is important to choose a qualified ELISA workstation supplier.


In actual operation, in addition to selecting good reagents, the operation steps must be strictly followed. At the same time, we should do well in indoor quality control and external quality evaluation, and inspect each specimen with a rigorous work style to ensure the quality of the inspection. Nowadays, quite a number of units in China have fully automated microplate readers, which have played an important role in realizing fully automated ELISA standardized testing and improving the quality of testing.

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