CN113740538A - Method and product for detecting SARS-CoV-2 novel coronavirus IgM/IgG antibody - Google Patents
Method and product for detecting SARS-CoV-2 novel coronavirus IgM/IgG antibody Download PDFInfo
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Abstract
本发明属生物技术领域，涉及一种基于荧光免疫层析技术检测SARS‑CoV‑2新型冠状病毒IgM和IgG抗体的方法和产品。本发明的方法为一步法，其包括：将荧光微球标记的SARS‑CoV‑2的重组N蛋白抗原载于载体上；并在与微球标记抗原载体相连接的检测载体上包被大鼠抗人IgM/IgG抗体做为检测线；荧光微球标记的鸡IgY对应包被山羊抗鸡IgY抗体做为质控线；将人体血浆、血清或全血样本滴于荧光微球标记抗原的载体上，如样本中含有SARS‑CoV‑2新型冠状病毒抗体则在检测载体上能形成检测线、同时质控线为阳性；若只显示质控线不显示检测线则为阴性。本发明解决了临床对SARS‑CoV‑2新型冠状病毒感染的快速诊断问题，具有检测灵敏度高、特异性强、准确性高、操作简单快速、可选用仪器读数判定的特点。The invention belongs to the field of biotechnology, and relates to a method and product for detecting SARS-CoV-2 novel coronavirus IgM and IgG antibodies based on fluorescence immunochromatography technology. The method of the present invention is a one-step method, which comprises: loading the recombinant N protein antigen of SARS-CoV-2 labeled with fluorescent microspheres on a carrier; and coating rats on a detection carrier connected with the microsphere-labeled antigen carrier Anti-human IgM/IgG antibody is used as the detection line; chicken IgY labeled with fluorescent microspheres is correspondingly coated with goat anti-chicken IgY antibody as the quality control line; human plasma, serum or whole blood samples are dropped on the carrier of fluorescent microsphere-labeled antigen If the sample contains SARS-CoV-2 novel coronavirus antibody, a detection line can be formed on the detection carrier, and the quality control line is positive; if only the quality control line is displayed and the detection line is not displayed, it is negative. The invention solves the problem of rapid clinical diagnosis of SARS-CoV-2 novel coronavirus infection, and has the characteristics of high detection sensitivity, strong specificity, high accuracy, simple and rapid operation, and optional instrument reading judgment.
Description
Technical Field
The invention belongs to the field of biotechnology, relates to a method for detecting SRS-CoV-2 novel coronavirus antibodies in serum (plasma), and particularly relates to a method and a product for detecting SARS-CoV-2 novel coronavirus IgM and IgG antibodies based on a fluorescence immunochromatography technology.
Background
The novel coronavirus pneumonia caused by SARS-CoV-2 coronavirus is acute respiratory infectious disease, mainly manifested by fever, debilitation and dry cough, and clinically shows hypoxia-hypoxia state and dyspnea, and severe patients rapidly progress to acute respiratory distress syndrome, sepsis shock, metabolic acidosis difficult to correct and blood coagulation dysfunction. To reduce the impact and harm of new coronavirus infections on public health, those skilled in the art are working on research and development including mechanisms, diagnostics, pharmacotherapy and vaccine prevention. Wherein, the timely discovery of new coronavirus patients is the key to control the disease transmission and reduce the fatality rate of the diseases, and the establishment of a rapid and sensitive diagnosis technology has great significance.
Because the detection of antigen is limited by the screening of specific antibody and the dependence on P3 laboratory, the current rapid diagnosis method for the novel coronavirus pneumonia mainly comprises two methods: (1) fluorescent real-time quantitative PCR technology based on virus nucleic acid detection, etc.; (2) antibody detection based on ELISA and immunofluorescence techniques; practice shows that compared with nucleic acid detection, immunoassay can effectively avoid the problems of low detection rate and the like caused by a sampling mode and poor RNA nucleic acid stability; research shows that the N protein is the most stable and abundant structural protein in the SARS-CoV-2 novel coronavirus, is an ideal antigen for antibody diagnosis, and is favorable for realizing sensitive and accurate detection. At present, a kit for detecting a novel coronavirus antibody is rarely seen, and the detection rate, the detection time, the operation difficulty and the like in the prior art are urgently needed to be improved.
The fluorescence immunochromatography technology is an improved technology based on the traditional immunochromatography technology, has the advantages of high specificity, simple operation, short detection time and the like, and also has the advantages of high sensitivity and accurate quantification.
Based on the current situation of the prior art, the inventor of the application intends to provide a method for detecting SRS-CoV-2 novel coronavirus antibodies in serum (plasma), in particular to a method and a product for detecting SARS-CoV-2 novel coronavirus IgM and IgG antibodies based on a fluorescence immunochromatography technology.
Disclosure of Invention
The invention aims to provide a method for detecting SRS-CoV-2 novel coronavirus antibodies in serum (plasma) based on the current situation of the prior art, in particular to a method and a product for detecting SARS-CoV-2 novel coronavirus IgM and IgG antibodies based on a fluorescence immunochromatography technology.
The invention provides a semi-quantitative detection method of specific IgM/IgG antibody of SARS-CoV-2 novel coronavirus N protein antigen in serum and plasma based on fluorescence immunochromatographic assay technology, wherein the SARS-CoV-2 novel coronavirus N protein for antibody capture is recombinant SARS-CoV-2 novel coronavirus N protein purified by affinity column;
the method is a one-step detection method, has the characteristics of high detection sensitivity, strong specificity, high accuracy and simple and quick operation, and is suitable for various field occasions such as clinical detection, intensive care unit, bedside detection, field quarantine, epidemiological screening and the like.
The invention comprises the following steps: n protein antigen of SARS-CoV-2 loaded on the carrier as fluorescent microsphere mark; the N protein antigen of SARS-CoV-2 on the carrier is the recombinant N protein of SARS-CoV-2, which is characterized in that the protein purified by affinity column, N end has GST label, and the sequence optimized by codon is used for prokaryotic expression, and the sequence is as follows:
“ATGTCTGATAATGGTCCGCAATCAAACCAACGTAGTGCTCCGCGCATTACATTTGGTGGTCCGACAGATTCAACT GACAATAACCAGAATGGTGGTCGCAATGGTGCACGTCCAAAACAGCGCCGTCCGCAAGGTTTACCGAATAATACT GCTTCTTGGTTCACAGCTCTCACTCAGCATGGTAAGGAGGAACTTCGTTTCCCTCGTGGTCAGGGTGTTCCAATCAA CACTAATAGTGGTCCAGATGACCAAATTGGTTACTACCGTCGTGCTACTCGTCGTGTTCGTGGTGGTGACGGTAAA ATGAAAGAGCTCTCTCCGCGTTGGTACTTCTATTACCTGGGTACTGGTCCAGAAGCTTCACTTCCGTACGGTGCTAA CAAAGAAGGTATCGTATGGGTTGCAACTGAGGGTGCTTTGAATACACCGAAAGACCACATTGGTACTCGCAATCC TAATAACAATGCTGCTACTGTTCTGCAACTTCCTCAAGGTACAACATTGCCAAAAGGTTTCTACGCAGAGGGTTCTC GTGGTGGTAGTCAAGCTTCTTCTCGCTCTTCATCACGTAGTCGCGGTAATTCACGTAATTCAACTCCTGGTTCTAGT CGTGGTAATTCTCCTGCTCGTATGGCTTCTGGTGGTGGTGAAACTGCTCTCGCTCTGTTGCTGCTGGACCGTTTGAA CCAGCTTGAGTCTAAAGTTTCTGGTAAAGGTCAACAACAACAAGGTCAAACTGTTACTAAGAAATCTGCTGCTGAG GCATCTAAAAAGCCTCGCCAAAAACGTACTGCTACAAAACAGTACAACGTTACTCAAGCATTTGGTCGTCGTGGTC CAGAACAAACTCAAGGTAATTTCGGTGACCAAGACCTGATCCGTCAAGGTACTGATTACAAACATTGGCCGCAAAT TGCACAATTTGCTCCAAGTGCTTCTGCATTCTTTGGTATGTCACGCATTGGTATGGAAGTTACACCTTCTGGTACAT GGCTGACTTATCATGGTGCTATTAAATTGGATGACAAAGATCCACAATTCAAAGACAACGTTATCCTGCTGAACAA GCACATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAAAAGACTGATGAAGCTCA GCCTTTGCCGCAGCGTCAAAAGAAGCAGCCGACTGTTACTCTTCTTCCTGCTGCTGACATGGATGATTTCTCTCGTC AACTTCAAAATTCTATGAGTGGTGCTTCTGCTGATTCAACTCAGGCA”。
the detection method of the invention comprises the following steps:
(1) loading the recombinant N protein of SARS-CoV-2 marked by fluorescent microsphere on a carrier made of glass fiber; coating a detection line formed by rat anti-human IgM/IgG antibodies and a quality control line correspondingly coated with goat anti-chicken IgY antibodies by chicken IgY marked by fluorescent microspheres on a detection carrier which is a nitrocellulose membrane and is connected with a SARS-CoV-2 recombinant N protein carrier marked by the fluorescent microspheres;
(2) dripping 2 mu L of human serum, 2 mu L of plasma or 5 mu L of whole blood on a carrier coated by the recombinant N protein of SARS-CoV-2 marked by fluorescent microspheres, and dripping 80 mu L of PBS diluent; if the serum, plasma or whole blood sample to be detected contains SARS-CoV-2 novel coronavirus N protein specific IgG antibody, an IgG detection line and a quality control line can be formed on the detection carrier at the same time, and the result is IgG positive; if the serum, plasma or whole blood sample to be detected contains a SARS-CoV-2 novel coronavirus N protein specific IgM antibody, an IgM detection line and a quality control line can be simultaneously formed on the detection carrier, and the result is IgM positive; if the serum, the plasma or the whole blood sample to be detected simultaneously contains the specific IgM and IgG antibodies of the novel SARS-CoV-2 coronavirus N protein, an IgM detection line, an IgG detection line and a quality control line can be simultaneously formed on the detection carrier, and the result shows that the IgM and the IgG are simultaneously positive; if only the quality control line exists, the result is negative; and when the quality control line is not displayed, the detection is invalid.
The invention provides a fluorescence immunochromatographic rapid diagnosis kit for SARS-CoV-2 novel coronavirus antibody, which aims at the detection of SARS-CoV-2 novel coronavirus antibody and has the advantages of simple and convenient one-step method operation, short detection time, high sensitivity, strong specificity, high detectable rate, selectable instrument reading judgment and the like.
The invention relates to a fluorescence immunochromatographic rapid diagnosis kit for SARS-CoV-2 novel coronavirus antibody, which comprises an antibody detection test card and PBS sample diluent with pH7.4; the antibody detection test card comprises a test strip and a lining plate made of plastic materials and wrapping the test strip to protect the test strip, wherein the test strip is based on a plastic bottom lining, a sample pad made of glass fiber and a nitrocellulose membrane are attached to the bottom lining, two ends of the nitrocellulose membrane are respectively lapped with a marking pad and absorbent paper, the sample pad is lapped with the marking pad, the nitrocellulose membrane is provided with an IgM/IgG detection line and a quality control line, and the detection line and the quality control line are positioned between the marking pad and the absorbent paper and sequentially comprise the IgM detection line, the IgG detection line and the quality control line; the outer packaging shell comprises a sample adding slot and a result display slot.
The invention further aims to provide a preparation method of a fluorescence immunochromatographic rapid quantitative diagnostic kit for SARS-CoV-2 novel coronavirus antibody, and the method has the advantages of high stability, simplicity and good repeatability.
The preparation method of the fluorescence immunochromatographic rapid quantitative diagnostic kit mainly comprises the following steps: preparing a SARS-CoV-2 recombinant N protein band marked by fluorescent microspheres of the sample adding layer, a detection line for preparing rat anti-human IgM/IgG antibodies of the detection layer, a quality control line for correspondingly coating goat anti-chicken IgY antibodies by chicken IgY marked by the fluorescent microspheres, and then combining a fluorescence immunochromatographic rapid quantitative diagnosis test paper strip of SARS-CoV-2 novel coronavirus antibodies on a protective plastic lining plate.
The preparation method of the recombinant N protein band of SARS-CoV-2 marked by the fluorescent microsphere of the sample adding layer comprises the following steps (the specific concentration and time can be adjusted according to requirements): 3.125X 106Microspheres were activated with 250. mu.L of activation buffer (0.1M NaH)2PO4pH6.2) was washed 2 times, each time followed by 60s of sonication, and the microspheres were activated in 500. mu.L of activation buffer containing 2.5mg of N-hydroxysulfosuccinimide (sulfonHS) and 2.5mg of N- (3-dimethylaminopropyl) -N-Ethylcarbodiimide (EDC) at 20-22 ℃ for 20 min. Washing the active microspheres twice with PBS; coupling was accomplished by addition of 12.5. mu.g of recombinant N protein of SARS-CoV-2, brought to a final volume of 500. mu.L with PBS, and rotary incubated at 20-22 ℃ for 3 h; with 1mL PBS + 0.05% NaN3And 1.0% bovine serum albumin are sequentially washed with the coupling microspheres; blocking the coupled microspheres with 1mL PBS-NB for 30 min to reduce non-specific binding; microspheres were washed twice with PBS-NB and resuspended in PBS-NB to a final concentration of 2.0X 106The recombinant N protein coupled microsphere of SARS-CoV-2.
The test strip and the detection test card are assembled according to the following method:
referring to fig. 2, the test strip and the test card for detecting the novel SARS-CoV-2 coronavirus IgM/IgG antibody based on the fluorescence immunochromatography technique comprise the following contents: the test strip is composed of a sample adding layer 5, a detection layer 7 connected with the sample adding layer 5, a waste liquid water absorbing layer 8 connected with the detection layer 7, a recombinant N protein marked by fluorescent microspheres and chicken IgY 6 marked by fluorescent microspheres fixed on the sample adding layer 5, a rat anti-human IgM antibody detection line 9 fixed on the detection layer 7 and close to one end of the sample adding layer 5, a rat anti-human IgG antibody detection line 10, and a quality control line 11 fixed on the detection layer 7 and close to one end of the waste liquid water absorbing layer 8, wherein the sample adding layer 5 and the detection layer 7 can be composed of cellulose membranes which can be hydrophilic materials such as cellulose acetate membranes, cellulose nitrate membranes, non-woven fabrics, glass fiber aluminum membranes and the like and easy to uniformly distribute proteins, and the waste liquid water absorbing layer 8 can be made of water absorbing materials such as multilayer filter paper; during assembly, the sample adding layer 5, the detection layer 7 and the waste liquid water absorption layer are overlapped by about 5mm to ensure that liquid smoothly enters an adjacent layer from one layer through chromatography, materials of all parts can be assembled in sequence and then cut into strips, the cut test strip comprises the parts, and the cutting specification is 0.4 or 0.5cm wide;
referring to fig. 1 and 2, the upper part of a test strip cut into strips covers the upper half part 1 of a protective lining board, a sample adding layer 5 is correspondingly overlapped with a sample adding hole 2 of the upper half part 1 of the protective lining board, a waste liquid water absorbing layer 8 is correspondingly overlapped with a vent hole 4 of the upper half part 1 of the protective lining board, a rat antihuman IgM antibody detection line 9, a rat antihuman IgG antibody detection line 10 and a quality control line 11 which are fixed on a detection layer 7 are correspondingly overlapped with a result observation hole 3, the rat antihuman IgM antibody detection line 9, the rat antihuman IgG antibody detection line 10 and the quality control line 11 are visible through the hollowed result observation hole 3, and finally, the lower half part 12 of the protective lining board is covered on the lower half part of the test strip, and the lower half part 12 of the protective lining board is fixedly connected with the upper half part 1 of the protective lining board.
The invention provides a method and a product for detecting SARS-CoV-2 novel coronavirus IgM and IgG antibodies based on a fluorescence immunochromatography technology; the method is a one-step detection method, has the characteristics of high detection sensitivity, strong specificity, high accuracy and simple and quick operation, and is suitable for various field occasions such as clinical detection, intensive care unit, bedside detection, field quarantine, epidemic disease screening and the like.
Compared with the prior art, the invention has the obvious advantages that:
1. the invention solves the clinical problem of rapid diagnosis of SARS-CoV-2 coronavirus infection, has 100 percent positive detection rate and high specificity, improves the sensitivity and the accuracy of detection by replacing the traditional nanogold with the fluorescent microspheres, and has simple one-step operation and convenient reading.
2. The fluorescence immunochromatographic rapid quantitative diagnostic kit for the SARS-CoV-2 novel coronavirus antibody has obvious good sensitivity and detection limit and good specificity; the test strip has low manufacturing cost; does not need to contact with pathogen, and has high safety; the detection time is short, and the field test and diagnosis can be completed quickly, conveniently and effectively.
3. The preparation method of the kit is simple, and the kit has good stability and repeatability.
Drawings
Fig. 1 is a schematic view of an appearance structure of an upper surface of an external protection lining plate of a test strip, wherein 1, the upper half part of the protection lining plate, 2, a sample adding hole, 3, a result observation hole, 4 and an air hole are arranged.
Fig. 2 is a schematic diagram of the side structure of the lower surface of the test strip and the external protective lining board, wherein 5, the sample adding layer, 6, the recombinant N protein marked by the fluorescent microsphere and the chicken IgY marked by the fluorescent microsphere, 7, the detection layer, 8, the waste liquid water absorbing layer, 9, the rat anti-human IgM antibody detection line, 10, the rat anti-human IgG antibody detection line, 11, the goat anti-chicken IgY quality control line, 12, and the lower half part of the protective lining board.
Detailed Description
The invention relates to a method for detecting SARS-CoV-2 novel coronavirus IgG/IgM antibody based on fluorescence immunochromatography, a kit thereof and a preparation method of the kit, which are further described by the following specific implementation method, but the invention is not limited by the embodiment in any way.
Example 1
Materials:
1. reagent
(1) PBS buffer (ph 7.4): sterile PBS buffer solution with the components of NaCl 137mmol/L, KCl 2.7mmol/L and Na2HPO4 10mmol/L,KH2PO42 mmol/L. Subpackaging into sterile container with 20mL, storing at room temperature,
(2) buffer solution for coating protein and antibody preparation, etc., with PBS buffer (pH7.4) as in item (1),
(3) washing solutions for post-coating washing are for example PBST: PBS (pH7.4), 0.05% Tween 20,
(4) the diameter of the fluorescent microspheres can be 200nm, and the excitation light wavelength is 365nm and the emission light wavelength is 618 nm.
2. Antigen and antibody
The SARS-CoV-2 new type coronavirus recombinant N protein antigen used in the invention is a recombinant protein with GST label at the N end and prokaryotic expression by using codon optimized sequence, and is purified by affinity column;
the coated antibody used in the invention is a rat IgG anti-human IgM/IgG antibody, and the quality control line is a goat anti-chicken IgY antibody;
and (3) treating the human serum/plasma/whole blood sample solution to be detected:
2 mu L of human serum, 2 mu L of plasma or 5 mu L of whole blood are dripped on a carrier coated by the recombinant N protein of SARS-CoV-2 marked by fluorescent microspheres, and 80 mu L of PBS diluent is dripped.
And (3) judging a detection result:
the invention relates to a method for detecting SARS-CoV-2 novel coronavirus IgM/IgG antibody based on fluorescence immunochromatography technology and a product thereof, which need to be matched with a portable detector for reading fluorescence results:
c is the fluorescence intensity value of the quality control line,
t1 ═ fluorescence intensity value of IgG line
Fluorescence intensity value of T2 ═ IgM line
IgG＝T1/C；IgM＝T2/C；
IgG negative range (0-0.050) positive (>0.050)
IgM negative range (0-0.020) positive (>0.020)
The above-mentioned qualitative determination method, if there is a standard curve or a reference antibody for quantitative determination, can provide a quantitative determination method according to the format, and combine the clinical test results for comprehensive judgment.
Testing and testing of human serum/plasma/whole blood samples:
the method for detecting the SARS-CoV-2 novel coronavirus IgM/IgG antibody based on the fluorescence immunochromatography technology comprises the following operation steps:
1) taking out the test paper, horizontally placing on a table, dropwise adding 2 mu L of serum, 2 mu L of plasma or 5 mu L of whole blood into a sample adding hole, dropwise adding 80 mu L of PBS diluent, horizontally placing and standing for 10 minutes after sample adding; opening a software MD-reader, then opening a main switch behind the instrument, and pressing 'quick test'; entering an interface to be tested;
2) inserting the test card into the detection hole of the instrument with the sample adding hole facing inwards;
3) pressing a reading button to read and record a fluorescence intensity value;
in this embodiment, the method and kit for detecting the novel SARS-CoV-2 coronavirus IgM/IgG antibody based on fluorescence immunochromatography are used to detect an actual serum/plasma/whole blood sample, and the detection results are shown in the following Table 1: wherein, the sample numbers 1-15 represent 15 patients respectively, C represents quality control MFI, IgG represents MFI of IgG, and IgM represents MFI of IgM, and the result shows that the positive coincidence rate of the semi-quantitative detection method for detecting SARS-CoV-2 novel coronavirus IgM/IgG antibody by utilizing the antigen specificity of recombinant N protein is 100%; the result shows that the method of the invention can accurately and sensitively detect the novel SARS-CoV-2 coronavirus IgM/IgG antibody, and provides effective guarantee for the screening and detection of the novel SARS-CoV-2 coronavirus infection.
TABLE 1
Sequence listing
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<120> a method and product for detecting SARS-CoV-2 new type coronavirus IgM/IgG antibody
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Claims (10)
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| CN202010463796.XA CN113740538A (en) | 2020-05-27 | 2020-05-27 | Method and product for detecting SARS-CoV-2 novel coronavirus IgM/IgG antibody |
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