What is antigen testing? How is it different from other detection methods? In this paper, taking novel coronavirus as an example, we will talk about the common rapid screening methods, and talk about the relevant principles and scope of application.
When we screen, we can find the diseases or substances we want to screen. The first thing we need to figure out is "where to start" and the second is "how to detect", so that the changes in the micro-world can be reflected in our eyes and help us make judgments.
Where to start? We are dealing with a virus. According to the familiar knowledge in biology class of middle school, virus is a kind of life-like body composed of genetic material and protein shell. If you want to detect the infection of the virus, you need to start with its composition. The following content, I hope you can read it with your biological knowledge in middle school.
Take novel coronavirus as an example. It belongs to Coronavirus B of Coronaviridae and is the seventh coronavirus known to infect humans. All coronaviruses are enveloped single-stranded RNA viruses, that is, their genetic material is a single RNA chain, which can directly participate in translation as mRNA (messenger RNA) to guide the synthesis of protein.
The virus species with the number of NPRC 2020.00002 was provided by the National Pathogenic Microorganism Resource Bank (China Center for Disease Control and Prevention).
Our purpose now is to detect whether there is this virus in the specimen. No matter whether it is the virus itself or the products brought by the virus, there are two directions to start with: protein shell (envelope) and genetic material.
The most obvious way to test this logic is to check whether it can be seen, but the virus itself is very small, and the diameter of novel coronavirus is 80- 120 nm. It is unrealistic to scan every specimen with an electron microscope, and manpower, material resources and financial resources can't hold up. Then the more economical and practical method is to take some measures to make the components of the virus or some special substances that appear because of the virus accumulate to a certain order of magnitude and then glow, change color and appear macroscopic.
Then our problem becomes to choose a method that can observe macro-scale changes, which is related to the composition of the virus and some substances caused by the virus. The materials we can choose are also on the table: the genetic material of the virus, here is its RNA；; The envelope of the virus, that is, the protein shell; Moreover, if you remember some basic biological knowledge, the human immune system will produce antibodies to resist invasion after being infected with the virus, which is also a good choice.
At present, several detection methods we use are born out of these substances (and their related substances), that is, nucleic acid detection for genetic material, antigen detection for envelope, and serum antibody detection for antibody.
As the genetic material of the virus, nucleic acid detection contains genetic characteristics that can identify the virus as a specific species, so the nucleic acid is positive, indicating that the virus already exists in the body.
Our current "nucleic acid detection" is actually divided into two parts. The first part is the sampling of "poking nose" and "stabbing throat" and the subsequent qualitative analysis. After obtaining the sample, because the virus amount is too small, the sample will be amplified by a certain multiple in the laboratory, and the positive or negative will be judged according to the fluorescence reaction result.
In the second part, it is necessary to determine the virus type of the sample by gene sequencing in order to trace the source. This step no longer belongs to the scope of daily screening, but it is of great significance in epidemiological investigation. If you are interested, you can refer to Wikipedia for a brief understanding.
We usually participate in nucleic acid detection as a screening tool, which refers to the first part of qualitative collection.
After being infected with novel coronavirus, viral nucleic acid can be found in throat swab, sputum, lower respiratory secretions, blood and other samples. The positive rate of nucleic acid detection in different parts of the specimen is different, and the detection rate of each part will change with the progress of the disease.
What we are used to calling "nasal swab" and "pharyngeal swab" are actually collecting secretions and tissues from the posterior wall of pharyngeal cavity. The former collects nasopharynx and the latter collects oropharynx. Other standards, such as saliva, can also be used as test specimens, and there are essential differences in different regions.
Both nasal (pharyngeal) swab and oral (pharyngeal) swab consider the positive rate and convenience. In fact, feces and urine can also be the objects of specimen collection. Moreover, according to a study of 3 1 patient, the accuracy of anal swab is higher than that of nasopharyngeal and oropharyngeal sampling, especially in the later stage of the disease, the positive rate of nasal swab is less than 30%. But obviously, due to the limitation of operation, it cannot be used as the first choice for early screening.
The next step is to extract nucleic acid from a small amount of samples. Because the virus in the sample is too small to be analyzed, it needs to be amplified and labeled. What you need to use is what you have learned in senior high school: polymerase chain reaction (PCR)-this step seems troublesome, but because its principle and process have been well learned, you only need to add reagents and send them to the machine in actual operation. The most troublesome thing in the whole process of nucleic acid detection is to get the sample safely (laughs).
Local disease control institutions or testing centers will purchase appropriate nucleic acid extraction kits and nucleic acid detection kits. The extraction kit is responsible for extracting RNA from mixed samples (cell fragments, secretions, dust and other impurities). Common methods include magnetic bead method, centrifugal column method and release agent method, and different extraction methods may have a slight impact on the accuracy of later detection. After that, the purified RNA will be handed over to the detection kit (there are also some kits that combine the two) for subsequent processing.
The process that the test kit carries the sample into the machine is the most important reaction in this test: RT-qPCR (Real-time Quantitative Reverse Transcription Polymerase Chain Reaction).
Next, I need you to pick up your knowledge of high school biology. A general PCR reaction has the following steps:
Heating: double-stranded DNA is unscrewed and deformed into two single strands; Annealing: combining the mixed single-stranded DNA with primers designed according to the fragment to be replicated; Extension: Adjust the temperature to allow DNA polymerase to start working along the primer, copy a new strand and form a new double strand. When detecting viruses, we have to do nothing more than the above steps, but we also need two things:
Before the first reaction, the complementary strand of virus single-stranded RNA was synthesized by reverse transcriptase (RNA-dependent DNA polymerase) and combined into cDNA. In the annealing and extension stages, besides primers and needed enzymes, TaqMan probes are also needed. You can understand the TaqMan probe in this way: its main part is an oligonucleotide chain, which is designed to pair with a small number of gene fragments that need to be replicated to form a double strand; One end of it is connected with a fluorescent molecule, and the other end is connected with a switch (quenching group). When they are connected to the probe, the fluorescence will be inhibited by quenching groups and cannot be detected. During annealing, the probe will bind to the single-stranded fragment to be replicated with the primer. In the process of extension, DNA polymerase will chop up the obstacles in front, including this probe, so that quenching groups and fluorescent molecules are separated and fluorescence is displayed.
With the increase of the number of cycles, more and more DNA fragments and fluorescence were amplified. By comparing the fluorescence brightness of each cycle with the reference brightness of previous cycles, the current DNA fragment amount can be obtained, or the cycle number and fluorescence brightness can be directly used for qualitative judgment.
So which part should I copy? Since we want to detect the virus, let's choose the most representative nucleic acid fragment. In the current standard, ORF gene and N gene are commonly used detection sites.
The detection kit is responsible for putting the extracted RNA (sample) in, setting the corresponding PCR temperature and duration according to the program instructions on the kit, completing the amplification process under the control of the machine, collecting the fluorescence signal at the fixed link and recording the corresponding cycle number (Ct value). The criterion for judging negative-positive/infectivity is to see what the current cycle number is when the fluorescence signal reaches the threshold. According to the current "novel coronavirus Diagnosis and Treatment Plan (Trial Ninth Edition)", the standard for lifting isolation management is Ct value ≥ 35. Compared with the previous standard of ≥40, the discharge isolation time will be greatly shortened.
Nucleic acid detection by RT-PCR is now the gold standard for diagnosis, because from the perspective of methodology, it can reach the accuracy of 100%. However, nucleic acid detection takes a long time and requires high environment and operators. When the environmental conditions are not up to standard and the materials and instruments are not complete, a large number of nucleic acid tests will bring huge human and financial consumption.
In this epidemic, we used rapid antigen detection and antibody detection. Based on the antigen-antibody reaction, through the rapid neutralization of antigen and antibody, we can detect whether the analyte exists in the sample with less time and cost. Both belong to the category of immunochromatography.
The antigen detection that appears in the form of box and can be operated by itself is very suitable as a supplement in the case of insufficient materials and self-determination.
Antigen detection nucleic acid detection examines the genetic material of the virus, that is, the "inside" of the virus. Then (rapid) antigen detection is to check the "appearance" of the virus and directly check the complete virus particles. At present, there are three approved antigen detection kits: colloidal gold method, latex method and fluorescence immunochromatography. The internal principles of the three are consistent. However, fluorescence immunochromatography kit still needs special detector or ultraviolet flashlight, which is not suitable for home self-test; Colloidal gold method and latex rule both transform the test results into strips visible to the naked eye, and the difference lies in the different substances used for marking and coloring.
Of course, antigen detection naturally has its shortcomings, that is, its false negative rate (positive but negative) is high, which may lead to missed detection and false detection. However, in the face of the time advantage that a cup of tea can produce results, the gap in accuracy can be temporarily compromised in some cases.
Source: Working principle of EUA antigen test in novel coronavirus, ASM.org.
Compared with nucleic acid detection, antigen detection increases the sampling route of "nose swab" and reduces the difficulty of personal self-test. The sample on the cotton swab is eluted in the buffer solution, and the liquid drops into the sample adding hole, and the liquid will pass through the antibody area (binding pad) preloaded with potential antigen due to capillary action.
The antibody in this region is a monoclonal antibody against the target antigen (novel coronavirus). Each antibody molecule binds to a special label, and they react with the antigen in the sample to form an antigen-antibody complex, which flows to the next test strip through capillary action.
Then there is the test line (T line), and the monoclonal antibody against the target antigen is also attached to the test line. You can understand that the things here are the same as those on the pad, but they are not marked. At this time, if the subject has been infected with novel coronavirus, the antigen-antibody complex formed by the antigen he left in the sample will be combined with the antibody fixed on the line again here. Here, these labeled compounds are constantly deposited, and eventually a deep or shallow band will be displayed. The color source of the test paper is the label attached by antibody molecules on the previous bonding pad. Colloidal gold method is colloidal gold particles, latex method is colored latex drops, and fluorescence method is fluorescent molecules. So when you use this test paper, you will find that when the sample is just added and the liquid just begins to diffuse, the front end of the diffusion will have a little bright color, which is the color of the mark that has not been fixed and precipitated.
Next, the liquid continues to diffuse, and another antibody, the monoclonal antibody against the target antigen, is attached to the control line (line C), which is called "secondary antibody" for short. This new antibody is obtained by reacting with the last antibody in the immune system of another animal. For example, the antibody binding to pad comes from rabbits, and the antibody here comes from sheep, which is a monoclonal antibody against rabbits. In other words, the antigen of the second antibody is the antibody on the previous binding pad. This line exists to detect whether the liquid diffuses normally, whether the antibody on the binding pad fails, and so on. At this time, a large number of antibodies remaining in the binding pad liquid will react with the second antibody on the quality control line as antigens to form a complex, showing obvious bands.
Because the antibody on the binding pad is rich, this quality control line will appear and develop color soon. However, because the number of antigens (viruses) is not certain, the color development speed of the detection line will be different, but generally it is enough to judge the result within 15 minutes. So don't look at the x-rays. Obviously, the T-ray vaguely feels "nothing". No matter how deep the T-ray is, as long as it exists, it is positive.
For specific operation, please refer to the teaching video released by the Medical Administration and Hospital Authority. At present, the country is gradually promoting the antigen self-test kit, which can alleviate the pressure of future medical care and streets to some extent.
In addition to the first two detection methods, there is also an equally important but seldom used detection method, that is, "serum antibody detection", which belongs to the same immunoassay method.
The kit used for antibody detection is very close to antigen detection, but the sample is more limited-because the object of detection has become an antibody, the sample must be blood (or plasma, serum) with definite antibodies. Moreover, the human body will not produce antibodies at the first time after being infected with the virus for the first time. Antibodies can definitely reach the order of magnitude of detection, usually one to two weeks after the first infection (or vaccination). These conditions limit that antibody detection cannot be used as a diagnostic test. At present, serum antibody testing is only used as a method to check whether the vaccine is effective in a specific situation or whether the subject has recently been infected with COVID-19.
There are five kinds of antibodies in human body, namely IgA, IgD, IgE, IgG and IgM. IgA is mainly responsible for mucosal immunity. IgD is related to the activation of immune response. IgE protects against parasites and is also involved in allergic reactions. The remaining IgG and IgM are the main forces sent by the immune system in the process of fighting pathogens.
Novel coronavirus, as a pathogen, is mainly secreted by human body after being stimulated. The existing antibody detection kits are mainly aimed at IgG and IgM produced by human body for novel coronavirus N protein (nucleocapsid protein) or S protein (spike protein).
The appearance of the detection device of antibody kit is the same as that of antigen detection. The difference between them lies in the substances attached to the pad, detection line and quality control line mentioned above.
This time, the sample dripped into the sampling hole may contain novel coronavirus antibody. So the binding pad should be labeled antigen-of course, it is impossible to release the virus. Generally, the antigen proteins used here are all designed and tested, such as N protein or S protein mentioned above, or recombinant virus. Whatever it is, it must contain the receptor binding domain (RBD) as the target of antibody binding. On the detection line, anti-IgM or anti-IgG antibodies are attached to capture antibody proteins that bind to antigens. Finally, the specific antibody of antigen is connected to the quality control line to capture the remaining free antigen.
Summary Generally speaking, the three detection methods have their own advantages and complement each other for different needs. As the gold standard, nucleic acid detection directly checks the RNA of the virus and is responsible for seeing whether the patient has the virus; As a rapid detection method, antigen detection is to check the protein of virus, but its accuracy is not as good as that of nucleic acid detection, and it is more effective for highly infectious people. Antibody testing checks whether the vaccine has taken effect and whether anyone has been infected with the virus recently.
Recently, the COVID-19 epidemic has made a comeback in various places. Compared with the previously popular Delta variant, the Omicron variant has shorter incubation period, faster virus replication and significantly enhanced infectivity, although the mortality and severity rate are significantly reduced. I hope everyone will stay healthy in such an environment.