CN111505286A - Novel coronavirus specific antibody double-antigen sandwich E L ISA detection kit and preparation method thereof - Google Patents
Novel coronavirus specific antibody double-antigen sandwich E L ISA detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, and discloses a novel coronavirus specific antibody double-antigen sandwich E L ISA detection kit and a preparation method thereof, wherein the kit comprises an enzyme-linked plate coated with NP antigen and S1 antigen of novel coronavirus SARS-CoV-2, and enzyme-labeled NP antigen and S1 antigen of novel coronavirus SARS-CoV-2. the invention adopts glycoprotein post-modified S1 protein and NP protein expressed by genetic engineering as well as establishes an E L ISA detection kit based on a double-antigen sandwich together as antigens, and because serum does not need to be diluted, the sensitivity is higher than that of indirect E L ISA, and the double-antigen sandwich does not need to use enzyme-labeled secondary antibody, the specificity of the reagent is higher than that of an E L ISA indirect method.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a novel coronavirus SARS-CoV-2 specific antibody double-antigen sandwich E L ISA detection kit and a preparation method thereof.
Background
An unknown coronavirus, belonging to the genus β and designated SARS-CoV-2, was discovered by sequencing a sample of the virus isolated from a patient with pneumonia, and the sequence alignment studies showed that the virus has 79.5% sequence similarity to SARS-CoV.
Coronavirus is a kind of enveloped RNA single-stranded virus, which is widely present in various mammals such as human, pig, cow, cat, camel, mouse, bat, etc. and various birds, and causes respiratory, liver, intestinal and nervous system diseases.Coronavirus is very common in animal bodies, but animal coronavirus infects human and rarely spreads between human and human.6 kinds of coronavirus are currently known to be pathogenic to human, of which 229E, OC43, N L63 and HKU 14 can cause common cold symptoms, 2 kinds of coronavirus can cause fatal respiratory diseases are SARS and MERS, and SARS-CoV-2 in this outbreak is known as the seventh kind.
The results of the study of the viral genome show that SARS-CoV-2 is a positive-strand RNA virus with envelope, the genome is close to 30Kb, and the encoded structural proteins, in addition to encoding polymerase, mainly comprise envelope spike protein S (spike), membrane protein M (membrane), and E (envelope), and nucleocapsid protein N (nucleocapsid).
The S protein of coronaviruses is primarily responsible for binding, membrane fusion and entry of the virus to cellular receptors. The results of the sequence alignment with the SARS-Spike protein show that the S protein of SARS-CoV-2 is also a type I transmembrane glycoprotein and consists of two domains, S1 and S2. S1 is located at the N-terminus of the entire protein and is responsible for binding of the virus to the receptor of the target cell. Angiotensin converting enzyme (ACE2) has been shown to be a functional receptor for SARS-CoV-2, in which the fragment Receptor Binding Domain (RBD) located in the middle region of S1 is responsible for binding to ACE 2. The S protein is also a main immunogen for inducing an organism to generate a neutralizing antibody, but the S protein is glycoprotein, and is modified after the sugar-free protein is expressed by a pronucleus, so that the biological activity of an expression product is lower; and the target protein is often expressed in the form of inclusion bodies, which causes difficulty in product purification.
The combination of the nucleocapsid protein NP and the packaging signal of the virus RNA leads the formation of the spiral nucleocapsid, which has important significance for the correct assembly of the virus, and the NP antigen is the main immunoreaction source of the coronavirus, induces the organism to generate strong immune response, and contains anti-N protein antibody in more than 90% of serums from SARS patients. In addition, because the NP protein does not contain glycosylation, the NP protein can be expressed in a large amount in pronuclei and can generate stronger immunogenicity, so that the NP protein is an ideal choice for researching an enzyme-linked immunosorbent assay for detecting SARS-CoV-2 infection, namely a method suitable for detecting an N protein antibody or directly detecting an antigen. In the detection of SARS, the U.S. CDC and the national center for microbiological research in Canada both have studied antibody detection reagents using recombinant N protein as an antigen.
The laboratory detection method for SARS-CoV-2 virus infection mainly includes ① virus separation culture, which needs to be carried out in the laboratory over P3 or P3, and has high technical requirements, great danger and difficult wide-range popularization, ② molecular biology detection, which mainly uses qPCR to detect SARS-CoV-2 nucleic acid, wherein the sample is generally a throat swab, has higher risk to medical personnel, more interference factors and easy occurrence of false positive/negative results, and ③ specific antibody detection, such as E L ISA.
The E L ISA system for detecting the antibody is simple and convenient to operate and rapid, and has great advantages for detecting epidemic diseases such as COVID-19, but the common indirect E L ISA method has inevitable defects in detection, namely, an enzyme label used in the ① indirect E L ISA method is a secondary anti-human IgG or IgM antibody, the sample is usually required to be diluted to avoid interference of other antibodies in a serum sample, so that the sensitivity is reduced, and the other antibodies in ② serum have non-specific adsorption, so that the secondary enzyme label antibody cannot be distinguished, so that a detection result has false positive.
Disclosure of Invention
In view of the above, the present invention aims to provide a novel coronavirus SARS-CoV-2 specific antibody double-antigen sandwich E L ISA detection kit and a preparation method thereof, so that the kit can significantly improve the detection rate and accuracy of the novel coronavirus SARS-CoV-2.
In order to achieve the above purpose, the invention provides the following technical scheme:
a new coronavirus SARS-CoV-2 specific antibody double antigen sandwich E L ISA detection kit, including the enzyme linked plate coated with new coronavirus SARS-CoV-2 NP antigen and S1 antigen, and enzyme labeled new coronavirus SARS-CoV-2 NP antigen and S1 antigen;
the NP antigen is obtained by prokaryotic expression preparation of a sequence shown in SEQ ID No.1, and the S1 antigen is obtained by eukaryotic expression preparation of a sequence shown in SEQ ID No. 2.
The invention adopts an optimized coding sequence to carry out prokaryotic expression of NP protein, eukaryotic expression of S protein and preferably selection of an S1 fragment with high antigen activity, and adopts an NP protein and S1 protein double-antigen sandwich E L ISA method to carry out serum antibody detection on a novel coronary pneumonia patient.
Preferably, the concentration ratio of the NP antigen to the S1 antigen in the enzyme linked plate is 1:1-3: 1; the concentration ratio of the NP antigen and the S1 antigen of the novel enzyme-labeled coronavirus SARS-CoV-2 is 1:1-3: 1.
Preferably, the kit of the invention further comprises one or more than two of the following components:
sample diluent, washing liquid, enzyme-labeled diluent, color developing agent, color development reaction stopping liquid, negative serum and positive serum.
In a specific embodiment of the invention, the sample diluent is calcium-coated human plasma added with sodium azide, the washing solution is PBST (PBS of 0.1% Tween-20), the enzyme-labeled diluent is Tris containing 0.9% NaCl, 5% BSA and P300, the color developers are H2O2 and TMB, and the color reaction termination solution is sulfuric acid.
Meanwhile, the invention also provides a preparation method of the kit, which comprises the following steps:
step 1, adding a screening label on a sequence shown in SEQ ID.No.1-2 and enzyme cutting sites at two ends of the sequence, respectively, then transferring the sequences to a bacterial expression plasmid and a eukaryotic expression plasmid, and expressing NP antigen and S1 antigen of novel coronavirus SARS-CoV-2 through a prokaryotic expression system and a eukaryotic expression system;
step 2, coating the NP antigen and the S1 antigen on an enzyme linked plate, and sealing by adopting a sealing solution to obtain the enzyme linked plate coated with the NP antigen and the S1 antigen of the novel coronavirus SARS-CoV-2; the NP antigen and the S1 antigen are coupled with HRP by a sodium periodate method, after the labeled antigen is dialyzed, BSA and glycerol are added after subpackaging, and the NP antigen and the S1 antigen of the novel enzyme-labeled coronavirus SARS-CoV-2 are obtained.
Wherein, the bacterial expression plasmid is pET-28a, and the eukaryotic expression plasmid is pCMV 5. In the specific embodiment of the invention, His screening label is added on the sequence shown in SEQ ID No.1, meanwhile, enzyme cutting site BamHI is added at 5 'and enzyme cutting site NotI is added at 3'; a His screening tag is added to the sequence shown in SEQ ID No.2, and a restriction site HindIII is added at the 5 'end and a restriction site XbaI is added at the 3' end.
In a specific embodiment of the invention, the blocking solution is 5% calf serum; the coating concentration of the antigen is 1ug/ml, and the concentration of the enzyme label is 1 mg/ml.
The invention simultaneously carries out 3 methodological detections on 50 serum samples of novel coronavirus pneumonia patients and serum samples of 24 healthy people, and the result shows that the detection rate of the serum antibody detected by the double-antigen sandwich method reaches 92 percent, the specificity is 100 percent, and the overall coincidence rate is 94.6 percent, which is superior to that of an E L ISA indirect method and a capture method.
According to the technical scheme, the detection kit for the E L ISA based on the double-antigen sandwich is established by adopting the S1 protein expressed by genetic engineering and modified by glycoprotein and the NP protein as antigens, the serum does not need to be diluted, the sensitivity is higher than that of an indirect E L ISA, and the double-antigen sandwich does not need an enzyme-labeled secondary antibody, so that the specificity of the reagent is higher than that of an E L ISA indirect method.
Drawings
FIG. 1 shows the SDS-PAGE detection result after the purification of SARS-CoV-2-NP recombinant protein;
FIG. 2 shows the SDS-PAGE results of the S1 recombinant protein supernatant after washing filtration;
FIG. 3 shows the SDS-PAGE results of the purification of S1 recombinant protein.
Detailed Description
The kit and the preparation method thereof are described by the preferred embodiments, and the related personnel can obviously modify or appropriately change and combine the kit and the preparation method thereof to realize and apply the technology of the invention without departing from the content, the spirit and the scope of the invention.
The detection kit for detecting the novel coronavirus specific antibody double-antigen sandwich E L ISA provided by the invention and the preparation method thereof are further described below.
Example 1: antigen preparation
1. NP antigens
The sequence in SARS-CoV-2 virus (GenBank: MN908947.3) is optimized, the reading frame of NP protein is synthesized by the sequence shown in SEQ ID No.1, the restriction site BamHI is added at 5 'and the restriction site NotI is added at 3', the reading frame is inserted into bacterial expression plasmid pET-28a by adopting the conventional molecular biology method to obtain bacterial expression plasmid pET-28 a-NP., the plasmid is transformed into E.coli B L21 DE3(novagen) conventionally, the SARS-CoV-2 NP recombinant protein is induced and expressed by the method recommended by manufacturers, and the SARS-CoV-2 NP recombinant protein is purified by His, the protein molecular weight is in accordance with the expected purity and is higher than 90% by SDS-PAGE detection (figure 1), and the conventional protein is quantified for standby.
2. S1 antigen
The reading frame of the S1 protein is synthesized by referring to the sequence in SARS-CoV-2 virus (GenBank: MN908947.3), and the reading frame is synthesized by the sequence shown in SEQ ID No.2, and the digestion site HindIII is added at the 5 'position and the digestion site XbaI is added at the 3' position respectively. The reading frame is inserted into a eukaryotic expression plasmid pCMV5 by adopting a conventional molecular biological method to obtain a bacterial expression plasmid pCMV-S1. The plasmid is transformed into HEK293 cells by a conventional method to express S1 recombinant protein, the soluble expression is analyzed by SDS-PAGE (figure 2) after the upper cleaning and filtering, the His purification is analyzed by His purification (figure 3) after the cleaning and filtering, the molecular weight detection is larger than the theory, the glycosylation modification is carried out, and the purity is more than 90 percent; and quantifying the conventional protein for later use.
Example 2: preparation of the kit
Diluting NP antigen and S1 antigen to 1ug/ml with carbonate buffer solution of pH9.6, respectively, coating 96-well enzyme-linked plate at 100 ul/well, coating overnight at 4 deg.C, sealing with 5% calf serum for 4h, washing with TBS containing 0.1% Tween-20 for three times, each time for 10min, drying at 37 deg.C for 2h, and packaging and storing at 4 deg.C; the concentrations of NP antigen and S1 antigen were adjusted to 1mg/ml, and they were coupled with HRP by the sodium periodate method, and after dialysis of labeled antigen, they were separately dispensed, and then, 2% BSA and 50% glycerol were added to the mixture, and they were stored at-20 ℃.
Example 3: sample detection
During detection, 50ul of sample diluent (calcium-enriched human plasma and sodium azide) is added into a coated reaction hole, then 50ul of serum to be detected, negative serum and positive control are added, incubation is carried out for 15min at 37 ℃, a washing solution PBST (PBS of 0.1% Tween-20) is used for washing a plate for 5 times, after drying, 1:1000 enzyme diluent (Tris + 0.9% NaCl + 5% BSA + P300) diluted SARS-CoV-2 enzyme-labeled antigen is added, incubation is carried out for 15min at 37 ℃, washing is carried out for 5 times by the washing solution PBST, drying is carried out, color developing agents A (H2O2) and B (TMB) are added, after color developing is finished for 10min at 37 ℃, 1 drop of 2M sulfuric acid is added into each hole to stop reaction, the result at the wavelength of OD450nm is read by an enzyme reader, and the critical value is 2.1 times of the negative average value as a standard (0.113).
Meanwhile, as a comparison, IgG antibodies in serum were identified using an indirect E L ISA using an enzyme-linked plate coated with NP antigen and S1 antigen, with a cut-off value of 2.1 times the negative average value as a standard (0.139), and IgM antibodies in serum were evaluated using an IgM capture method using HRP-labeled NP and S1 antigens, with a cut-off value of 2.1 times the negative average value as a standard (0.121).
In this example, 50 serum samples of patients with the novel coronavirus pneumonia and 24 serum samples of healthy persons were simultaneously tested by 3 methodologies, and the results are shown in table 1.
TABLE 1
The result shows that the detection rate of the serum antibody detected by the double-antigen sandwich method based on the kit reaches 92%, the specificity is 100%, and the overall coincidence rate is 94.6%, which is superior to that of an E L ISA indirect method and a capture method.
The foregoing is only for the purpose of understanding the method of the present invention and the core concept thereof, and it will be understood by those skilled in the art that various changes and modifications may be made without departing from the principle of the invention, and the invention also falls within the scope of the appended claims.
Sequence listing
<110> Zhengzhou Yimeino Biotechnology Ltd
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Claims (9)
1. A new coronavirus SARS-CoV-2 specific antibody double antigen sandwich E L ISA detection kit, characterized in that, it includes the enzyme linked plate coated with new coronavirus SARS-CoV-2 NP antigen and S1 antigen, and enzyme-labeled new coronavirus SARS-CoV-2 NP antigen and S1 antigen;
the NP antigen is obtained by prokaryotic expression preparation of a sequence shown in SEQ ID No.1, and the S1 antigen is obtained by eukaryotic expression preparation of a sequence shown in SEQ ID No. 2.
2. The kit of claim 1, wherein the concentration ratio of the NP antigen and the S1 antigen in the enzyme-linked plate is 1:1-3: 1.
3. The kit of claim 1, wherein the concentration ratio of the NP antigen and the S1 antigen of the enzyme-labeled novel coronavirus SARS-CoV-2 is 1:1 to 3: 1.
4. The kit of claim 1, wherein the prokaryotic expression is expressed by a B L21 DE3 prokaryotic expression system.
5. The kit of claim 1, wherein the eukaryotic expression is expression via a HEK293 eukaryotic expression system.
6. The kit according to any one of claims 1 to 5, further comprising one or more than two of the following components:
sample diluent, washing liquid, enzyme-labeled diluent, color developing agent, color development reaction stopping liquid, negative serum and positive serum.
7. The method for preparing the kit according to claim 1, comprising:
step 1, adding a screening label on a sequence shown in SEQ ID.No.1-2 and enzyme cutting sites at two ends of the sequence, respectively, then transferring the sequences to a bacterial expression plasmid and a eukaryotic expression plasmid, and expressing NP antigen and S1 antigen of novel coronavirus SARS-CoV-2 through a prokaryotic expression system and a eukaryotic expression system;
step 2, coating the NP antigen and the S1 antigen on an enzyme linked plate, and sealing by adopting a sealing solution to obtain the enzyme linked plate coated with the NP antigen and the S1 antigen of the novel coronavirus SARS-CoV-2; the NP antigen and the S1 antigen are coupled with HRP by a sodium periodate method, after the labeled antigen is dialyzed, BSA and glycerol are added after subpackaging, and the NP antigen and the S1 antigen of the novel enzyme-labeled coronavirus SARS-CoV-2 are obtained.
8. The method of claim 7, wherein the bacterial expression plasmid is pET-28 a.
9. The method of claim 7, wherein the eukaryotic expression plasmid is pCMV 5.
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| CN111999496A (en) * | 2020-08-20 | 2020-11-27 | 必欧瀚生物技术（合肥）有限公司 | SARS-CoV-2 antigen-antibody combined detection kit and its preparation method |
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