ELISA reagent evaluation (evaluation) is divided into two aspects: one is the quality evaluation of the reagent itself, can only be produced and supplied after meeting certain requirements; one is the evaluation of the effect in clinical application. Taking the hepatitis ELISA diagnostic reagent as an example, first of all, it must pass the inspection of Chinese pharmaceutical and biological products to obtain a production license. In addition to packaging, labels, instructions, etc., the performance of the test must be verified item by item, such as specificity, sensitivity, precision, and linearity. Only by passing a series of reference products can the test be qualified. . The clinical quality evaluation of ELISA reagent is to use the reagent to test clinical samples to observe its practical application value. The Ministry of Clinical Examination Center carried out work on the hepatitis B ELISA diagnostic reagents in this regard, and promoted the improvement of reagent quality through quality evaluation.
1. Key points of clinical quality evaluation of diagnostic reagents
The reliability of the test reagent from the perspective of clinical application is based on its ability to distinguish between health and disease. It is still difficult to find a 100% reliable test, any test will have false positives or false negatives. Judgment of the reliability of the test is often based on its sensitivity and specificity. The sensitivity of clinical application is expressed by the percentage of patients with positive tests of disease, and the specificity is expressed by the percentage of negative tests of patients without disease.
For this evaluation, the relevant patient sera need to be collected first, and then tested with the most reliable reagent for testing the marker to determine whether it is positive or negative. This group of sera indicating whether the test substance is positive or negative constitutes a "serum panel". The relationship between the results obtained by measuring the serum of the evaluated reagent and the results indicated on the serum disk is as follows:
Serum dish results
total
It is generally considered that sensitivity or specificity> 90% is good. The coincidence rate is an index of comprehensive sensitivity and specificity.
Second, the preparation requirements of clinical assessment serum plate
1. Use human original serum;
2. The serum plate should have corresponding stability;
3. The samples in the serum dish do not contain preservatives, or only contain traces of preservatives that do not affect the test results;
4. The negative samples and positive samples contained in the serum tray account for about half each;
5. Among the positive samples, there should be a certain number of strong positive and weak positive samples;
6. There should be a certain number of samples with upper and lower threshold values ​​in the serum dish to test the sensitivity of the reagents.
7. Serum trays should contain samples of diseases related to this test and samples known to have interfering substances (RF factors) to test the specificity of the reagents.
6.3 Recommendations for clinical assessment of serum trays
Taking anti-HBc-IgM as an example, the Ministry of Clinical Examination Center collected samples of nearly 100 clinical hepatitis patients, which were repeatedly tested and screened by anti-HBc-IgM reagents of American abbott company. 70 sera were selected, of which 29 were positive and 41 were negative, forming an anti-HBc-IgM clinical assessment serum plate. Among the 70 samples, in addition to 7 quality control sera without medical history, 22 samples positive for anti-HBc-IgM included 16 cases of clinically diagnosed acute hepatitis, 5 cases of chronic active hepatitis, and 1 case of severe hepatitis; -HBc-IgM negative 40 samples, including 24 cases of clinically diagnosed chronic persistent hepatitis, acute hepatitis (both blood samples collected during the recovery period), 8 cases of chronic active hepatitis (of which 5 cases were recovered blood samples).
Therefore, this set of antiserum plate is used for clinical evaluation of commercial reagents, which can distinguish clinically patients with acute hepatitis B and chronic active phase, and has clinical diagnostic significance.
Quality Control
ELISA is an experimental diagnostic method with high sensitivity, strong specificity, and good repeatability. Due to its stable reagents, easy storage, easy operation, and objective judgment of results, it is suitable for large-scale screening tests and can be used. For the detection of a small number of specimens, it can be used for both qualitative testing and quantitative analysis. It has been widely used in the fields of microbiology, parasitology, oncology and cytokines. There are many influencing factors of ELISA. Only by strengthening quality management can the advantages of its methodology be fully utilized.
Quality control before analysis
â— The personnel training experiment is operated by humans, so the inspectors need to be trained to master the technical knowledge of the following aspects of this major:
â—Ž Basic principles of inspection items (ELISA principle);
â—Ž Clinical significance;
â—Ž Familiar with testing techniques, understand the links and difficulties that are prone to errors;
â—Ž Familiar with the performance of detection reagents (including kit composition, coated fragments and their composition);
â—Ž Familiar with the principle and performance of testing instruments; master the data processing ability and quality control knowledge.
â—Ž The testing of some special items such as anti-HIV requires special training courses organized by relevant departments.
Quality control before analysis
â—Ž Principle of ELISA: In 1971, Engvall et al. Applied immunohistochemical EIA technology to clinical testing to develop ELISA technology.
Features: Assemble immunoactive substances on the surface of solid phase to form "immunosorbent"
Enzyme-labeled immunologically active substances for signal amplification;
There is a two-step incubation reaction and a second washing process;
The sensitivity and specificity are relatively high.
Limitations: only ng levels can be detected
Poor precision (CV15-20% in batch);
Narrow linear range (one order of magnitude);
There is a "HD-HOOK" effect.
ELISA principle
â— There are three basic methods according to the detection purpose and operation steps.
â— The common method for detecting antigen (body) by the double antibody (original) sandwich method, using two monoclonal antibodies against two different determinants of the antigen as a solid phase carrier and an enzyme-labeled antibody to detect the antigen in the solution (applicable to more than two valences) Antigen, can not measure small molecule hapten).
â— Indirect method for detecting antibodies, using enzyme-labeled anti-antibodies (second antibodies) to detect antibodies that have bound to solid-phase antigens.
â— The competition method detects antigens or small molecule haptens and antibodies. Taking the test antigen as an example, the test antigen and the enzyme-labeled antigen compete with the solid phase antibody. As a result, the more the test antigen content, the enzyme that binds to the solid phase antibody The less target antigen is, the lighter the color is, and the two are negatively correlated.
The future direction of ELISA
â— Advanced preparation of polystyrene surface. The existing technologies are hydrazide (polydifluoroethylidene, PVDF); biotinylation; pre-coated polylysine, etc., all of which are patented products.
â— Changes in enzymatic signal amplification, such as enzymatic fluorescence, enzymatic chemiluminescence, etc.
ELISA clinical significance
â— The characteristics and markers of various types of hepatitis
The mode and clinical significance of two-half hepatitis B
HBsAg HBeAg HBcAb-IgG HBcAg-IgM HBeAb HBsAb & n bsp; clinical significance
---- HBV replication is active early in acute HBV infection
-Acute and chronic HBV infection with active HBV replication
--Acute and chronic HBV infection, leap in HBV replication
--Acute and chronic HBV infection, low jump in HBV replication
---HBV replication stopped or extremely low
--Calm HBV carrying status, HBsAg
---- HBV previous infection, no anti-HBs produced
--Anti-HBs appeared early, HBV replication was low
--HBV infection recovery period
--HBV infection recovery period
-HBV reinfection of different subtypes
----- HBV-DNA integration
----- + Get immunized after illness or vaccination
Kit selection
â—Ž The Ministry of Health stipulates that hepatitis B reagents, hepatitis C reagents, AIDS reagents, syphilis reagents and blood group reagents must use products that have passed the certification of the Ministry of Health Biological Products Inspection and have anti-counterfeit labels. , Simplicity, safety and economy make a comprehensive evaluation.
â—Ž Sensitivity: It has two meanings: 1) the ability of the reagent to detect the minimum amount of the tested substance; 2) the ability of the reagent to detect positives in the crowd or a large number of samples (the fewer false negatives the better), depending on The comprehensiveness of the object.
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