How does ELISA not color the whiteboard? -Huaqiang Electronic Network

In the ELISA experiment, I believe that everyone will inevitably have some conditions, just last Friday, Xiaobian is posting a bar friend to post an ELISA experiment, after the coloring step, the enzyme All holes in the target are colorless. The positive control did not develop color. So today, Shanghai Jinma asks the technical staff to analyze and analyze the cause analysis: the reagent has expired, or different kit components are mixed, check the reagent composition and batch number, confirm that the reagent has not expired and all the components belong to the corresponding kit. . Countermeasure: Different kits or reagents of different batches cannot be mixed. The yellow plate phenomenon of the whole plate may be caused by the wrong addition of other reagents. For example, when the two pairs of reagents are simultaneously operated, the HbsAb plate is used for measuring HBsAg, etc.; the components and batch numbers of the reagents are checked before the experiment, and all the components are confirmed to be corresponding. Kit. Such as the specimens of the specimens often have bloody around, easy to fall off, should stay away from the microplate. Products with an elisa kit that exceeds the expiration date may produce a weak signal; the kit is not retained as required and is affected by high temperatures; the components and lot numbers of the reagents are checked prior to the experiment to confirm that the reagents have not expired. The incubation temperature should be controlled at 37-38 ° C. The incubation time should be strictly in accordance with the instructions. It is not advisable to open the door during the heat preservation period to avoid affecting the heat preservation. Flower plate, generally due to improper collection, processing and retention methods of clinical specimens, placement time (naturally placed 1~2h) and centrifugation (3000r/min), centrifugation time (15min) should attract attention. Reagents and samples should be unbalanced to room temperature before use. Reagents and samples taken from low-temperature refrigerators should be equilibrated at room temperature for about 20 minutes. Mis-add, leaking reagent substrate, developer A or B strictly follow the steps of the manual. After adding the reagent, you can observe whether the liquid level is consistent or not to reduce the leakage. After the end of the experiment, the positive positive control was normal and the quality control was normal, but the clinical specimens felt weaker in coloration. The specimens to be tested may not contain strong positive specimens, so the results may be normal. If there is any doubt, the specimens may be re-inspected. Nitrogen sodium is used as a preservative. The specimens in the enzyme-free experiment are banned from sodium azide as a preservative. Other preservatives such as proclin and thimerosal may be used. The positive control is normal, but the quality control, reference or individual weak positive specimens are not detected. . Different kits or different batches of reagents cannot be mixed. The contamination of the tip by the enzyme label and the contamination of the container containing the developer are likely to cause yellow plate phenomenon; avoid cross-contamination between the reagent components, ensure that the tip and the container are clean, and the developer is placed under illumination. For a long time, it has turned blue before the experiment. The developers A and B are kept away from light before use. The incubation temperature is too high or the incubation time is too long. The incubation temperature should be controlled at 37-38 °C. The incubation time is strictly in accordance with the instructions. Samples used within one week can be stored at 2-8 ° C, if long-term retention, should be kept below -20 ° C. Do not store the ELISA kit at room temperature for a long time and store it according to the regulations. Randomly occurring flower plates and jumping holes are not completely centrifuged, and coagulation or residual cellular components occur in the reaction wells; fully centrifuged at 3000 rpm for more than 6 minutes. The instrument settings are incorrect, the filters do not match, and the parameters of the microplate reader are reset. In particular, check whether the filters match. The sensitivity is too high, the bottom of the plate is high, and the background is terminated. The whole plate shows a uniform yellow. Or light yellow; or negative positive control, normal quality control, specimen negative specimen, OD value is too high. In particular, each hole is not filled with washing liquid during washing, which is easy to cause yellow plate phenomenon of the flower board, and the plate is washed strictly according to the instructions. During the process of washing and loading, the enzyme label loses the ability to catalyze the color development of the coloring agent by contamination inactivation. It is confirmed that the container containing the enzyme label does not contain an enzyme inhibitor such as sodium azide, and it is confirmed that the container for preparing the washing liquid has been washed. Cross-contamination during sample loading; avoid cross-contamination when adding specimens.