On March 15, the National Health Commission issued the "novel coronavirus Pneumonia Program (Trial Version 9)", which mentioned that "on the basis of detecting cases by nucleic acid testing, the method of antigen detection is added". Detection is crucial for coping with the COVID-19 epidemic. At present, there are three methods to detect COVID-19 infection, including nucleic acid detection, antigen detection and antibody detection. Today, I would like to introduce the detection principle and operation method of COVID-19 pathogen and antibody.
Rapid antigen testing can be used to diagnose patients with acute infections who develop symptoms within 2 weeks. Its operation is simple, the results are fast, and the cost is low. Although the detection limit is 105-106 genome copies per milliliter, individuals with viral loads below 106 genome copies per milliliter are unlikely to transmit the virus, thus enabling rapid identification of the most likely carriers of the virus. Almost all antigen tests use the nucleocapsid protein of COVID-19 as the target, so it is less likely to be affected by the mutant strain. There are three types of detection methods: colloidal gold immunochromatography, latex method, and fluorescence immunochromatography.
Principle:Colloidal gold is formed by the polymerization of chloroauric acid (HAuCl4) into gold particles of a certain size under the action of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, etc. Under the action of static electricity, it becomes a stable colloidal state and forms a negatively charged hydrophobic colloidal solution. Colloidal gold carries a negative charge in weak alkaline environments and can form strong bonds with positively charged groups of protein molecules.
Testing process:
1. Sampling. Open the reagent kit packaging and take out the swab for sampling.
2. Dilute the sample. Put the swab into the diluent, squeeze the swab head several times, and fully dissolve the sample.
3. Testing. Take out the test card, place it flat on the table, and drop 3 drops of the sample into the sampling hole.
4. Observe. Observe the results after 15 minutes of timing.
The results show
Principle:The markers used in latex method and colloidal gold method are different. Latex method is an indirect agglutination test using latex particles as carriers. Adsorbing soluble antigens on its surface, specific antibodies can bind to it and produce agglutination reactions.
The detection process is similar to colloidal gold immunochromatography. The characteristic of this method is that it does not require the use of special instruments, can be judged with the naked eye, and is easy to operate with short detection time.
principle：The detection target is viral protein (such as nucleocapsid or spike protein), usually collected from nasopharyngeal swabs.
Testing method:A handheld fluorescence immunoassay analyzer is required. The testing personnel use a throat swab to take samples, then insert the throat swab into the extraction tube, rotate it 10 times, squeeze and extract the swab, so that the liquid stays in the tube as much as possible, and cover it with a dropper. Add 3 drops of extract to each detection card in sequence, let it stand for 15 minutes, insert the detection card into the instrument detection port, fill in the sample information or scan information, click the quick test button, and the device will automatically interpret the results. Directly detecting the unique proteins (i.e. antigens) in the virus can quickly detect whether the human body contains the virus during the acute infection period, allowing for early triage and management of suspected populations.
COVID-19 antibody detection
At present, colloidal gold immunochromatography and magnetic particle chemiluminescence are the main methods to detect antibodies to COVID-19. Antibody testing, as an important auxiliary method for the diagnosis of COVID-19, can complement and verify with nucleic acid testing to improve the accuracy of diagnosis, and can also be used to screen asymptomatic or mildly infected people, helping the country to judge and track the overall situation of the epidemic. It mainly detects whether there are antibodies that specifically bind to COVID-19 in human body, so as to prove whether human body has been infected with novel coronavirus. Antibody testing only provides indirect evidence 2 weeks after symptom onset or vaccination, and is recommended for monitoring purposes.
Colloidal gold immunochromatography
Principle:After COVID-19 infects human body, it stimulates plasma cells to produce specific antibodies. In addition to the phospholipid bilayer, COVID-19 can induce the immune response of the body and generate specific antibodies with prefabricated binding. LgG is a monomeric structure widely distributed in tissue fluid, intravascular and extracellular spaces, and is the main antibody in serum. IgM is called macroglobulin. The colloidal gold method uses colloidal gold test strips for detection, namely rapid detection test strips. There are two lines on the surface of colloidal gold test paper, one is the detection line and the other is the quality control line. If both lines show color, it represents a positive result; If only the quality control line shows color, it represents negative; All others are invalid. The test strips are arranged from left to right as sample pad, binding pad, nitrocellulose membrane, and absorbent pad. If the sample contains COVID-19 antibody lgG, it will combine with the COVID-19 antigen labeled with colloidal gold at the binding pad, and then continue to move. It will combine with the mouse anti human lgG antibody at the lgG detection line to form a sandwich complex. At this time, aggregation precipitation occurs and displays red. At the line of accusation, colloidal gold labeled mouse IgG binds to sheep anti mouse IgG antibodies, causing aggregation precipitation and displaying a red color, indicating the successful completion of the chromatography process. When the detection object is COVID-19 IgM, just replace the coating at the detection line with mouse anti human IgM antibody. When detecting lgM/lgG simultaneously, two detection lines, lgM/lgG, can be set separately.
Testing method:
1. Draw a blood sample. Fingertip blood needs to be collected.
2. Add samples. Use a straw to draw blood and drip it into the sample well of the detection card along with diluent.
3. Place horizontally for 10 minutes.
4. Observe. Check for the presence of a red strip in the observation hole of the detection card.
Magnetic particle chemiluminescence method is a highly sensitive immunoassay, and any substance with antigenicity can be determined using this method. On the basis of chemiluminescence detection, it uses magnetic nanoparticles, which gives the detection the advantages of high sensitivity and fast speed. However, the magnetic particle chemiluminescence method has poor selectivity and can react with a range of compounds rather than specific compounds. Therefore, the accuracy is not good. Moreover, the environment has a significant impact on detection, which can easily lead to errors.
Principle:The antigen protein of COVID-19 was coated on the particles, and the antigen protein combined with the IgG/IgM in the serum of patients with COVID-19, the chemically labeled human IgG/IgM secondary antibody, and the free chemical markers were removed after cleaning. The luminescent substrate generates chemiluminescence, and qualitative or quantitative detection of IgG/IgM in serum is achieved by measuring the light signal. Add reagent 0, reagent 1, and sample into the reaction tube. If the sample contains COVID-19 IgM antibody, it will form a complex with the recombinant antigen in the above reagents. At the same time, it will bind to the magnetic particles to wash off the free components. Add reagent 2 to the reaction tube, and use the alkaline phosphatase labeled antibody as the secondary antibody to bind with the IgM antibody in the sample, forming an alkaline phosphatase labeled antibody IgM antibody recombinant antigen magnetic particle complex to wash away free components. Add the substrate solution of the fully automated immunoassay system, catalyze the luminescence of the substrate solution with alkaline phosphatase, and measure the luminescence values of each sample tube. The luminous value of the sample is positively correlated with the concentration of COVID-19 IgM antibody, so as to detect COVID-19 IgM antibody in human serum.
Testing process:
1. Sample processing. Take 1ml of sample diluent, add 20ul of sample, mix well for 5 seconds, and let it stand for 15 minutes.
2. Set 2 tubes of negative and positive samples and treated samples, and add 50ul COVID-19 IgM antibody reagent 1, 75ul treated samples, 50ul COVID-19 IgM antibody reagent 0, 75ul COVID-19 IgM antibody reagent respectively. Two step method with a reaction time of 35 minutes. After secondary cleaning, add 200ul of substrate solution from the fully automated immunoassay system to each tube, perform light avoidance detection, and read the reaction for 5 seconds.
With the issuance of the Notice of the State Council on the Application Scheme of COVID-19 Antigen Testing (Trial), more and more enterprises have obtained the registration certificate of COVID-19 Antigen Testing, and their IVD market has also burst into vitality. Although the State Council pointed out that the price of testing reagents can be reduced through provincial centralized recruitment, the domestic COVID-19 antigen testing market is still booming. Due to the huge demand for antigen reagents, multiple reagent manufacturers have announced active production to ensure supply capacity. The increase in demand will also continuously promote the development of upstream enterprises in the industrial chain.
The above article is excerpted from the internet
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