Method for identifying a suitable type of ELISA kit for testing samples
2022-09-28 04:05:05
In addition to the cell supernatant, a large portion of the samples tested by the ELISA kit are serum and plasma samples.
For ELISA customers, different samples may be used in the experiment. How can I tell if the purchased kit can measure the sample in the hand?
For the most common cell supernatants, we all know that in the process of culturing cells, 10% of bovine serum is usually added to the medium. After culture, the cells are absorbed and consumed, and finally the protein content in the supernatant is very high. It is rare, and for the manufacturers of general bovine serum production, most of the substances that interfere with the detection have been removed during the production process, so the general ELISA does not affect the binding of the antibody antigen.
So what are the more common serum plasma samples?
Serum is one of the most commonly used ELISA specimens. Plasma in ELISA can be regarded as the same specimen as serum. Interfering substances in specimens are the main cause of high or low results after detection. Interfering substances are classified as endogenous. Sexual substances and exogenous substances:
1. Endogenous substances Common interfering substances are: rheumatoid factor, complement, heterophilic antibody, target antigen autoantibodies, iatrogenic induced anti-mouse Ig (s) antibodies, cross-reactive substances and other substances.
(1) Rheumatoid factor: IgM and IgG-type rheumatoid factor (RF) in serum can be directly combined with the capture antibody and the FC segment of the enzyme-labeled secondary antibody in the ELISA system, resulting in high detection OD value. .
(1) Rheumatoid factor: IgM and IgG-type rheumatoid factor (RF) in serum can be directly combined with the capture antibody and the FC segment of the enzyme-labeled secondary antibody in the ELISA system, resulting in high detection OD value. .
(2) Complement: In the process of solid phase primary antibody and labeled secondary antibody in ELISA system, the antibody molecule is allosteric, and the complement C1q molecular binding site of the FC segment is exposed, so that C1q can connect the two, thereby causing The detected OD value is too high.
(3) Heterophilic antibodies
Human serum contains natural heterophilic antibodies that bind to rodent (such as mice) Ig (s), which can link the primary and secondary antibodies in the ELISA system, and can also cause high OD values.
(3) Heterophilic antibodies
Human serum contains natural heterophilic antibodies that bind to rodent (such as mice) Ig (s), which can link the primary and secondary antibodies in the ELISA system, and can also cause high OD values.
In addition, serum anti-target antigen autoantibodies, iatrogenic-induced anti-mouse Ig (s) antibodies, cross-reactive substances and serum lipids, bilirubin, hemoglobin and blood viscosity are too large, etc. There is interference.
2. Exogenous substance
Exogenous substances are often caused by improper collection, storage, etc. of blood samples for ELISA assays. Such as specimen hemolysis, specimen contamination by bacteria, specimen storage for too long, specimen agglutination and additives in blood collection tubes.
(1) specimen hemolysis
Due to various human causes of hemolysis of the specimen, a large amount of hemoglobin with peroxidase activity can be released due to red blood cell destruction, which leads to non-specific color development in an ELISA assay labeled with horseradish peroxidase. , interfere with the measurement results.
Exogenous substances are often caused by improper collection, storage, etc. of blood samples for ELISA assays. Such as specimen hemolysis, specimen contamination by bacteria, specimen storage for too long, specimen agglutination and additives in blood collection tubes.
(1) specimen hemolysis
Due to various human causes of hemolysis of the specimen, a large amount of hemoglobin with peroxidase activity can be released due to red blood cell destruction, which leads to non-specific color development in an ELISA assay labeled with horseradish peroxidase. , interfere with the measurement results.
(2) The specimen is contaminated by bacteria: Since the bacteria may contain endogenous horseradish peroxidase, the specimen contaminated by bacteria may produce non-specific coloration and interfere with the measurement result, just like the hemolyzed specimen.
(3) Improper preservation of specimens: Specimens stored in the refrigerator for too long, IgG in serum can be polymerized into multimers, and AFP can form dimers, which can lead to excessive background and even detection of OD in indirect ELISA assays. High value;
(3) Improper preservation of specimens: Specimens stored in the refrigerator for too long, IgG in serum can be polymerized into multimers, and AFP can form dimers, which can lead to excessive background and even detection of OD in indirect ELISA assays. High value;
(4) Specimen agglutination
In summary, there are many factors influencing serum plasma samples in ELISA assays. In addition to considering reagent factors and operational factors, it should also be analyzed from the perspective of specimen factors.
Serum and plasma samples
At present, the research ELISA kit manufacturers claim that all kinds of specimens can be tested, but after use, we found that some kits have poor detection of serum and plasma samples, and the light value is low. If the RF factor exists, the light value High. The main reason for the above phenomenon is that there are many heteroproteins and complex components. Therefore, special reagents are needed to eliminate the effects.
How to tell if the purchased kit can measure serum plasma samples?
This is divided into several situations:
1. If the manufacturer does not provide a buffer specifically for serum plasma samples, then as long as a known concentration of the test protein is added to the serum of the test species as a sample and a known concentration of the test protein is added to the PBS. Simultaneous detection and comparison in the buffer will tell if the kit can be used directly to measure serum plasma samples.
2. If the manufacturer does not provide a buffer for serum plasma samples, but there is a general or uniform dilution, then just add the sample with the known concentration of the test protein to the serum of the tested species. The known concentration of the indicator protein to be tested is added to the uniform buffer provided by the manufacturer for simultaneous detection, and it can also be found whether the kit can be directly used for detecting serum plasma samples.
3. If the manufacturer provides a buffer specifically for serum plasma samples, the serum can be directly added by adding the known concentration of the test target protein to the serum of the tested species, and the results can be obtained by using the buffer separately according to the instructions.
If the total sample size of this type of kit is 100 μl, in general, it will be 50 μl of sample loading and 50 μl of specific buffer.
In the process of using the kit, the known concentration of protein can be adjusted to the highest value of the detection limit of the kit calibration with serum or plasma, 50 μl is added for two wells, and one well is filled with the conventional 50 μl standard dilution. A hole can be added to 50 μl of sample analysis buffer to distinguish the effect. Sometimes, some manufacturers add the target protein to be sampled in the sample analysis buffer in order to achieve the "elimination" effect. In order to identify this situation, It is also possible to add a sample analysis buffer directly to the well and make a comparison, so that the buffer for the serum plasma sample in the kit can be tested to be really effective.
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