1. How to take and store the samples used for ELISA test?
Explanation: Most ELISA tests take serum or plasma as samples. Serum samples can be collected by conventional methods. Attention should be paid to avoid hemolysis. When red blood cells are dissolved, substances with peroxidase activity will be released. In the HRP-labeled ELISA assay, hemolytic samples may increase non-specific color. Serum samples should be tested when fresh. If there is bacterial contamination, the bacterial body may contain endogenous HRP, and may also produce false positive reaction. Generally speaking, the serum samples measured within 5 days can be placed at 2~8 ℃. The samples stored in the refrigerator for a long time lead to the aggregation of serum IgG, especially the deepening of the background of indirect method reagents; The samples measured for more than one week should be stored at - 20 ℃ or below. After the frozen serum is dissolved, the protein is partially concentrated and unevenly distributed, and should be fully mixed to avoid bubbles. The turbid or precipitated serum samples should be centrifuged or filtered first, and then tested after clarification. Repeated freezing and thawing will cause the antibody titer to drop, so if the serum samples for antibody testing need to be stored for multiple tests, a small number of sub-packs should be stored in ice. When storing serum, we should pay attention to aseptic operation, or add appropriate preservatives. The samples with incomplete anticoagulation are false-positive due to the interference of fibrinogen. It is recommended that anticoagulants, especially heparin anticoagulants, should not be used as much as possible.
2. How to ensure effective washing during ELISA testing?
Explanation: Washing is to separate the antigen or antibody specifically bound to the solid phase from the non-specific components adsorbed in the reaction incubation process to ensure the specificity of ELISA determination. Therefore, plate washing is a crucial step for ELISA determination.
1) It is strongly recommended to use automatic plate washing machine for washing. Manual washing is easy to cause cross contamination;
2) The washing machine shall be regularly maintained. Before use, check whether there is hole plugging, and whether the operation of suction, injection, and walking are normal. Generally, the residual amount of each hole after drying shall not exceed 2ul, and the injection amount of each hole shall be greater than 250ul, but it shall not overflow. At the same time, set a certain soaking time, 60 seconds/time, and wash at least 5 times.
3. What is the difference between single wavelength and dual wavelength in ELISA colorimetry?
Explanation:
1) The enzyme marker above the middle level basically has both single-wavelength and dual-wavelength colorimetric functions. The so-called single-wavelength measurement is to select the wavelength 450nm that has the maximum absorption for the liquid with yellow color after termination for colorimetric determination, and obtain the A value of each hole, wherein the A value includes the absorption value (W1) and non-specific absorption value (W2) of its own liquid color, and the general value of non-specific absorption value is the fingerprint, scratch If the absorption value of dirt such as dust is W1=A-W2, we set the blank zero adjustment during color comparison, which is to subtract it as the non-specific absorption value. In fact, the non-specific absorption value of each hole is not the same, so the single-wavelength measurement has a certain degree of uncertainty, that is to say, different blank hole positions may lead to different absorbance values, which is only within the acceptable range.
2) The wavelength dual-color is determined by the enzyme marker at the sensitive wavelength of 450 nm and the non-sensitive wavelength of 630 nm respectively. The absorbance of the sensitive wavelength is the sum of the absorbance of the specific color and the absorbance caused by fingerprints, scratches, dust and other dirt on the plate hole (M1); When measured at the non-sensitive wavelength, the absorbance value of the specific color is zero, and the measured absorbance is the absorbance value of the dirt (M2). Finally, the value given by the microplate is the difference between the absorbance value at the sensitive wavelength and the absorbance value at the non-sensitive wavelength (A=M1-M2). Therefore, it is better to use dual-wavelength colorimetry when determining the color.
3) When selecting dual-wavelength colorimetry, it is not necessary to set the blank hole for zeroing. If the blank hole is still set for zeroing colorimetry, the absorbance may be negative.
4. How to deal with the low sensitivity of ELISA reagent?
Explanation:
1) Re-experiment, and pay attention to the control of temperature balance, incubation time, color development time and other influencing factors of the kit in the experiment;
2) Check whether the quality control products have repeated freezing and thawing, whether the quality control products have intra-batch differences, and whether the quality control products are stored at 2~8 ℃ for a long time;
4) Re-evaluate the reliability of quality control products;
5) The sample itself is weakly positive due to various factors, but is actually negative;
6) Due to the low content of the sample itself, it is easy to fluctuate with the experimental level. This part of the sample should be handled with great care, and qualified users should conduct confirmatory experiments;
7) Pay attention to check whether the instruments and equipment used in the experiment are calibrated regularly;
8) Pay attention to check whether the batch numbers of plate and enzyme label reagents used in the experiment are the same. Reagents with different batch numbers shall not be mixed.
5. What are the main reasons for the high background or "pattern" phenomenon in ELISA product testing?
Explanation:
Washing reason:
1) The washing solution formula used by various manufacturers is prepared according to the conditions of their respective reagents. The use of mismatched washing solution often fails to achieve the normal washing results, which will mainly lead to high background and even the phenomenon of flower board. The washing system of our company is not recommended to be mixed with reagent kits of other companies.
2) Fresh distilled water or deionized water should be used to prepare the washing solution. If tap water is used, the water contains too much Ca2+and Mg2+, which will occupy the surfactant and affect the washing effect. At the same time, the endogenous substances released by some microorganisms in the water will cause interference and high non-specific reaction.
3) When washing the plate, ensure that the liquid injection volume and liquid absorption residue of each hole of the plate washer are normal before use, otherwise the washing effect will be affected. Generally, the liquid injection volume is required to be 300~350 μ L/hole, but it should not overflow to avoid cross contamination, and the residual amount of liquid absorption ≤ 2 μ l. Wash at least 5 times.
Color rendering system reason:
1) The substrate liquid B has slightly changed color, resulting in a high background.
2) Improper cleaning of the repeatedly used gun head and sampling tank will also interfere and cause non-specific reaction of substrate A and B solution.
Reasons for the sample itself:
1) When hemolytic samples are used, red blood cells will release substances with peroxidase activity when dissolved, which will increase non-specific color rendering; The serum sample is relatively old. If there is bacterial contamination, the bacterial body may contain endogenous HRP, which will also increase non-specific color rendering.
2) The samples with incomplete anticoagulation are used, and the non-specific color development is caused by the interference of fibrinogen. It is recommended to avoid anticoagulation as much as possible, especially heparin anticoagulation samples.