WO2022134241A1 - 预测新型冠状病毒中和抗体效价的方法及其试剂盒 - Google Patents
预测新型冠状病毒中和抗体效价的方法及其试剂盒 Download PDFInfo
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- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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Definitions
- the present disclosure relates to the technical field of neutralizing antibody titer prediction, and in particular, to a method for predicting the titer of a novel coronavirus neutralizing antibody and a kit thereof.
- the human body After vaccination, the human body can produce protective antibodies through the immune response, that is, neutralizing antibodies.
- the titer of neutralizing antibody titers can be used to determine the protective effect of the vaccine.
- the detection of neutralizing antibody titers in patients with COVID-19 after cure can be used to determine whether there is a risk of secondary infection.
- PRNT detection uses live virus and needs to be completed in a BSL-3 laboratory, and the PRNT detection operation is complicated, the cycle is long, and the error is large.
- pseudovirus neutralization detection uses a relatively safe pseudovirus, it cannot simulate and replace the process of live virus infecting cells, and it also has complex operations, long cycle, and large errors, which cannot meet the requirements of rapid, throughput, and accurate detection. demand.
- the purpose of the present disclosure includes, for example, to provide a method for predicting the titer of a novel coronavirus neutralizing antibody, which can well predict the neutralizing antibody result of the novel coronavirus only through the detection of serum.
- the plaque reduction neutralization test and pseudovirus neutralization titer test have the advantages of simple operation, fast detection speed, short detection period and safe detection environment.
- the purpose of the present disclosure also includes, for example, providing a novel coronavirus neutralizing antibody titer prediction kit for the above prediction method.
- the present disclosure provides a method for predicting the titer of a novel coronavirus neutralizing antibody, the method comprising the following steps:
- step (b) correlating the detection result of step (a) with the neutralizing antibody titer.
- the biological samples to be tested are serum and plasma of cured patients with COVID-19.
- the novel coronavirus-specific protein includes at least one of novel coronavirus N protein, novel coronavirus S2-ECD protein or novel coronavirus S1-RBD protein;
- amino acid sequence of the novel coronavirus N protein is shown in Seq No.01:
- amino acid sequence of the novel coronavirus S2-ECD protein is shown in Seq No.02:
- amino acid sequence of the novel coronavirus S1-RBD protein is shown in Seq No.03.
- the affinity antibody comprises at least one of anti-human IgA, anti-human IgG, and anti-human IgM;
- the anti-human IgA includes any of goat anti-human IgA polyclonal antibody, mouse anti-human IgA monoclonal antibody, rabbit anti-human IgA monoclonal antibody, rabbit anti-human IgA polyclonal antibody A sort of;
- the anti-human IgG includes any of goat anti-human IgG polyclonal antibody, mouse anti-human IgG monoclonal antibody, rabbit anti-human IgG monoclonal antibody, rabbit anti-human IgG polyclonal antibody A sort of;
- the anti-human IgM comprises any of goat anti-human IgM polyclonal antibody, mouse anti-human IgM monoclonal antibody, rabbit anti-human IgM monoclonal antibody, rabbit anti-human IgM polyclonal antibody A sort of.
- the expression level of the novel coronavirus-specific protein combined with the affinity antibody in the step (a) is detected by quantum dot immunofluorescence method.
- the step (b) associating is performed using a software classification algorithm.
- the software classification algorithm includes at least one of logistic regression, support vector machines, and random forests.
- the present disclosure provides a novel coronavirus neutralizing antibody titer prediction kit, the kit includes: a detection reagent strip containing quantum dot-specific protein complexes;
- the quantum dot-specific protein complex is mainly obtained by coupling quantum dots and novel coronavirus-specific protein.
- the detection reagent strip adopts quantum dot immunochromatography technology, and uses the novel coronavirus N protein, novel coronavirus S2-ECD protein and novel coronavirus S1-RBD protein as detection antigens, using Quantum dot fluorescent microspheres were labeled with N, S1-RBD and S2-ECD antigen proteins to form quantum dot-specific protein complexes, and then the plasma of the new coronavirus was detected.
- the detection reagent strip includes a bottom card, a sample pad, a quantum dot label binding pad, a chromatography reaction membrane, and an absorbent paper;
- the quantum dot label binding pad is adsorbed with quantum dot-specific protein complexes
- a detection line is provided on the chromatography reaction membrane, and the detection line includes at least one of an IgA detection line, an IgG detection line, and an IgM detection line.
- the chromatography reaction membrane comprises one of NC membrane, PVDF membrane or nylon membrane, preferably nitrocellulose membrane;
- the bottom card includes one of a PVC bottom card or a PC bottom card, preferably a PC bottom card;
- the quantum dot marker binding pad comprises one of a glass fiber membrane, a polyester membrane, and a blood filter membrane, preferably a glass fiber membrane;
- the sample pad comprises one of a glass fiber membrane, a polyester membrane, and a blood filter membrane, preferably a glass fiber membrane;
- the absorbent paper comprises one of thick filter paper, glass fiber or blood filtering membrane, preferably thick filter paper.
- the chromatography reaction membrane is provided with a detection line and a quality control line;
- the detection line includes at least one of an IgA detection line, an IgG detection line, and an IgM detection line, wherein the IgA detection line contains anti-human IgA; the IgG detection line contains anti-human IgG; the IgM detection line contains anti-human IgM; and
- the quality control line contains goat anti-rabbit IgG antibody
- the kit further includes an ultraviolet irradiation device.
- the present disclosure provides a method for predicting the titer of a novel coronavirus neutralizing antibody.
- the steps of the method are: firstly providing a biological sample to be tested, and then detecting the expression of the novel coronavirus specific protein in the serum sample after binding to the affinity antibody level; the test results were then correlated with the neutralizing antibody titer to predict the neutralizing antibody titer of the novel coronavirus in the biological sample to be tested.
- the above method can well predict the neutralizing antibody result of the new coronavirus only through the detection of serum. Compared with the existing plaque reduction neutralization test and pseudovirus neutralization titer detection, it has the advantages of simple operation and fast detection speed. , The advantages of short detection period and safe detection environment can be used as an evaluation index for the second recurrence of new coronavirus vaccination or infection.
- the novel coronavirus neutralizing antibody titer prediction kit provided by the present disclosure is mainly composed of detection reagent strips containing quantum dot-specific protein complexes; the above-mentioned reagent strips use quantum dot immunochromatography technology to combine the novel coronavirus N protein, novel coronavirus
- the coronavirus S2-ECD protein and the new coronavirus S1-RBD protein were used as detection antigens, and the N, S1-RBD and S2-ECD antigen proteins were labeled with quantum dot fluorescent microspheres to form quantum dot-specific protein complexes, and then the new coronavirus was detected.
- the above-mentioned reagent strips have the advantages of high sensitivity, high specificity and quantitative detection, and avoid the missed detection of weak positive samples and false positive samples to the greatest extent.
- the quantum dot immunochromatography method used in the present disclosure is rapid in detection, and results are obtained within 10 minutes.
- the use of serum detection samples can reduce the risk of exposure of medical staff, and can be operated on-site with simple training, and each portable fluorescence detector can detect the Hundreds of samples.
- a method for predicting the titer of a novel coronavirus neutralizing antibody comprises the following steps:
- step (b) correlating the detection result of step (a) with the neutralizing antibody titer.
- the present disclosure provides a method for predicting the titer of a novel coronavirus neutralizing antibody.
- the steps of the method are: firstly providing a biological sample to be tested, and then detecting the expression of the novel coronavirus specific protein in the serum sample after binding to the affinity antibody level; the test results were then correlated with the neutralizing antibody titer to predict the neutralizing antibody titer of the novel coronavirus in the biological sample to be tested.
- the above method can well predict the neutralizing antibody result of the new coronavirus only through the detection of serum. Compared with the existing plaque reduction neutralization test and pseudovirus neutralization titer detection, it has the advantages of simple operation and fast detection speed. , The advantages of short detection period and safe detection environment can be used as an evaluation index for the second recurrence of new coronavirus vaccination or infection.
- the biological sample to be tested is serum or plasma of a cured COVID-19 patient.
- the above-mentioned biological sample to be tested is serum or plasma of a cured new crown patient, so as to predict the probability of a second recurrence of the patient after infection.
- the novel coronavirus-specific protein includes at least one of novel coronavirus N protein, novel coronavirus S2-ECD protein, or novel coronavirus S1-RBD protein;
- the novel coronavirus S2-ECD protein is the 2019-nCoV S2 subunit extra-cellular domain (ECD) protein
- the novel coronavirus S1-RBD protein is the 2019-nCoV surface spike protein S1 subunit Base receptor binding domain (receptor binding domain, RBD) protein
- the new coronavirus N protein is 2019-nCoV main protein nucleocapsid protein (Nucleocapsid Protein, N protein).
- amino acid sequence of the novel coronavirus N protein is shown in Seq No.01:
- amino acid sequence of the novel coronavirus S2-ECD protein is shown in Seq No.02:
- amino acid sequence of the novel coronavirus S1-RBD protein is shown in Seq No.03.
- the affinity antibody includes at least one of anti-human IgA, anti-human IgG and anti-human IgM;
- the anti-human IgA includes any one of goat anti-human IgA polyclonal antibody, mouse anti-human IgA monoclonal antibody, rabbit anti-human IgA monoclonal antibody, and rabbit anti-human IgA polyclonal antibody;
- the anti-human IgG includes any one of goat anti-human IgG polyclonal antibody, mouse anti-human IgG monoclonal antibody, rabbit anti-human IgG monoclonal antibody, and rabbit anti-human IgG polyclonal antibody;
- the anti-human IgM includes any one of goat anti-human IgM polyclonal antibody, mouse anti-human IgM monoclonal antibody, rabbit anti-human IgM monoclonal antibody, and rabbit anti-human IgM polyclonal antibody.
- the expression level of the novel coronavirus-specific protein combined with the affinity antibody in the step (a) is detected by quantum dot immunofluorescence method.
- the expression level of the above-mentioned novel coronavirus-specific protein combined with the affinity antibody can be detected by quantum dot immunofluorescence method, and then the fluorescence data can be obtained more intuitively, which is convenient for later data analysis. calculate.
- step (b) association is performed using a software classification algorithm.
- the software classification algorithm includes at least one of logistic regression, support vector machine and random forest.
- a novel coronavirus neutralizing antibody titer prediction kit includes: a detection reagent strip containing quantum dot-specific protein complexes;
- the quantum dot-specific protein complex is mainly obtained by coupling quantum dots and novel coronavirus-specific protein.
- the novel coronavirus neutralizing antibody titer prediction kit provided by the present disclosure is mainly composed of detection reagent strips containing quantum dot-specific protein complexes; the above-mentioned reagent strips use quantum dot immunochromatography technology to combine the novel coronavirus N protein, novel coronavirus
- the coronavirus S2-ECD protein and the new coronavirus S1-RBD protein were used as detection antigens, and the N, S1-RBD and S2-ECD antigen proteins were labeled with quantum dot fluorescent microspheres to form quantum dot-specific protein complexes, and then the new coronavirus was detected.
- the above-mentioned reagent strips have the advantages of high sensitivity, high specificity and quantitative detection, and avoid the missed detection of weak positive samples and false positive samples to the greatest extent.
- the quantum dot immunochromatography method used in the present disclosure is rapid in detection, and results are obtained within 10 minutes.
- the use of serum detection samples can reduce the risk of exposure of medical staff, and can be operated on-site with simple training, and each portable fluorescence detector can detect the Hundreds of samples.
- the detection reagent strip includes a bottom card, a sample pad, a quantum dot marker binding pad (also referred to as a release pad), a chromatography reaction film, and a absorbent paper;
- the quantum dot label binding pad is adsorbed with quantum dot-specific protein complexes
- a detection line is provided on the chromatography reaction membrane, and the detection line includes at least one of an IgA detection line, an IgG detection line, and an IgM detection line.
- the chromatography reaction membrane comprises one of NC membrane, PVDF membrane or nylon membrane, preferably nitrocellulose membrane;
- the bottom card includes one of a PVC bottom card or a PC bottom card, preferably a PC bottom card;
- the quantum dot marker binding pad comprises one of a glass fiber membrane, a polyester membrane, and a blood filter membrane, preferably a glass fiber membrane;
- the sample pad includes one of glass fiber membrane, polyester membrane, and blood filter membrane, preferably glass fiber membrane;
- the absorbent paper comprises one of thick filter paper, glass fiber or blood filtering membrane, preferably thick filter paper.
- the chromatography reaction membrane is provided with a detection line and a quality control line;
- the detection line includes at least one of an IgA detection line, an IgG detection line, and an IgM detection line, wherein the IgA detection line contains anti-human IgA; the IgG detection line contains anti-human IgG; the IgM detection line contains anti-human IgM; and
- the quality control line contains goat anti-rabbit IgG antibody
- the kit further includes an ultraviolet irradiation device.
- the N gene is transferred into E. coli for expression
- the S2-ECD gene is transferred into 293 cells for expression
- the S1-RBD protein is transferred into 293 cells for expression to obtain the new coronavirus recombinant antigen N protein and S2-ECD protein and S1-RBD protein.
- a 25cm-wide NC membrane was attached to the middle area of the PC bottom card, and then the anti-human IgA, anti-human IgG and goat anti-rabbit IgG antibodies were diluted with PBS diluent to 0.2-1 mg/ml and scratched with a gold spray. In the corresponding detection line area and quality control line area of the NC membrane. Dry in a 37°C oven for 12-24h, and seal the bag for later use.
- a 25cm-wide NC membrane was attached to the middle area of the PC bottom card, and then the anti-human IgM, anti-human IgG and goat anti-rabbit IgG antibodies were diluted with PBS diluent to 0.2-1 mg/ml and scratched with a gold-spraying film machine. In the corresponding detection line area and quality control line area of the NC membrane. Dry in a 37°C oven for 12-24h, and seal the bag for later use.
- a 25cm wide NC membrane was attached to the middle area of the PC bottom card, and then the anti-human IgA, anti-human IgM, anti-human IgG and goat anti-rabbit IgG antibodies were diluted in PBS diluent to 0.2-1 mg/ml and sprayed with gold.
- the film striper was used to mark the corresponding detection line area and quality control line area of the NC membrane. Dry in a 37°C oven for 12-24h, and seal the bag for later use.
- step (2) Reconstitute the precipitate after centrifugation in step (1) with 1000 ⁇ l of 10-50 mM MES (pH 6) buffer solution. Centrifuge the reconstituted solution under certain conditions, remove the supernatant with a pipette, and reconstitute the precipitate with 1000 ⁇ l of 10-50 mM MES (pH 6) buffer. step).
- step (3) in this example "Add 20-100 ⁇ l of the novel coronavirus S2-ECD protein that has been diluted to 1 mg/ml with 10-50 mM MES (pH 6) buffer to the reconstituted solution. 60-300min.”, the rest is the same as Example 6.
- step (3) in this example "Add 20-100 ⁇ l of the novel coronavirus S1-RBD protein that has been diluted to 1 mg/ml with 10-50 mM MES (pH 6) buffer to the reconstituted solution. 60-300min.”, the rest are the same as in Example 6.
- the quantum dot-labeled protein complex prepared in Example 6 was diluted 20-100 times with 20-100 mM Tris-HCl diluent (pH 8.0, containing 0.5% BSA, 2% trehalose) as required, and then sprayed with a film
- the gold instrument was sprayed on the treated quantum dot marker binding pad, and dried at 37°C for 12-24 hours to obtain the quantum dot marker binding pad.
- the quantum dot-labeled protein complex prepared in Example 7 is diluted 20-100 times with 20-100 mM Tris-HCl diluent (pH 8.0, containing 0.5% BSA, 2% trehalose) as required, and then pipetting The gun is sprayed on the untreated quantum dot marker binding pad, and then evenly coated with a coating rod, and finally placed in a freeze dryer for freeze drying. After the program is completed, the bag is sealed for use.
- Tris-HCl diluent pH 8.0, containing 0.5% BSA, 2% trehalose
- the quantum dot-labeled protein complex prepared in Example 7 was diluted 20-100 times with 20-100 mM Tris-HCl diluent (pH 8.0, containing 0.5% BSA, 2% trehalose) as required, and then sprayed with a film
- the gold instrument was sprayed on the treated quantum dot marker binding pad, and dried at 37°C for 12-24 hours to obtain the quantum dot marker binding pad.
- the quantum dot-labeled protein complex prepared in Example 7 is diluted 20-100 times with 20-100 mM Tris-HCl diluent (pH 8.0, containing 0.5% BSA, 2% trehalose) as required, and then pipetting The gun is sprayed on the untreated quantum dot marker binding pad, and then evenly coated with a coating rod, and finally placed in a freeze dryer for freeze drying. After the program is completed, the bag is sealed for use.
- Tris-HCl diluent pH 8.0, containing 0.5% BSA, 2% trehalose
- the quantum dot-labeled protein complex prepared in Example 8 was diluted 20-100 times with 20-100 mM Tris-HCl diluent (pH 8.0, containing 0.5% BSA, 2% trehalose) as required, and then sprayed with a film
- the gold instrument was sprayed on the treated quantum dot marker binding pad, and dried at 37°C for 12-24 hours to obtain the quantum dot marker binding pad.
- the quantum dot-labeled protein complex prepared in Example 7 is diluted 20-100 times with 20-100 mM Tris-HCl diluent (pH 8.0, containing 0.5% BSA, 2% trehalose) as required, and then pipetting The gun is sprayed on the untreated quantum dot marker binding pad, and then evenly coated with a coating rod, and finally placed in a freeze dryer for freeze drying, and the bag is sealed for use after the program is completed.
- Tris-HCl diluent pH 8.0, containing 0.5% BSA, 2% trehalose
- a novel coronavirus detection reagent strip the preparation method of the reagent strip comprises the following steps:
- the sample pad and the quantum dot marker binding pad are overlapped on one end of the chromatography reaction membrane in turn, and the absorbent paper is overlapped at the other end of the chromatography reaction membrane. Then adjust the parameters of the slitting machine to cut into strips, and cut them into 3-4mm wide. test strips.
- This example is the same as Example 12 except that the quantum dot marker binding pad prepared in Example 9 is replaced with the quantum dot marker binding pad prepared in Example 10.
- Example 12 This example is the same as Example 12 except that the quantum dot marker binding pad prepared in Example 9 is replaced with the quantum dot marker binding pad prepared in Example 11.
- Example 12 This example is the same as Example 12 except that the chromatography reaction membrane of Example 4 is replaced with the chromatography reaction membrane of Example 3.
- Example 13 This example is the same as Example 13 except that the chromatography reaction membrane of Example 4 is replaced by the chromatography reaction membrane of Example 3.
- Example 14 This example is the same as Example 14 except that the chromatography reaction membrane of Example 4 is replaced with the chromatography reaction membrane of Example 3.
- Example 12 This example is the same as Example 12 except that the chromatography reaction membrane of Example 4 is replaced by the chromatography reaction membrane of Example 2.
- Example 13 This example is the same as Example 13 except that the chromatography reaction membrane of Example 4 is replaced by the chromatography reaction membrane of Example 2.
- Example 14 This example is the same as Example 14 except that the chromatography reaction membrane of Example 4 is replaced with the chromatography reaction membrane of Example 2.
- Example 12 N protein IgA, IgM, IgG Example 13 S2-ECD protein IgA, IgM, IgG Example 14 S1-RBD protein IgA, IgM, IgG Example 15 N protein IgM, IgG Example 16 S2-ECD protein IgM, IgG Example 17 S1-RBD protein IgM, IgG Example 18 N protein IgA, IgG Example 19 S2-ECD protein IgA, IgG Example 20 S1-RBD protein IgA, IgG
- a novel coronavirus neutralizing antibody titer prediction kit which includes the detection reagent strips of Example 12 and Example 14.
- a novel coronavirus neutralizing antibody titer prediction kit includes the detection reagent strips of Example 15, Example 16 and Example 17.
- a novel coronavirus neutralizing antibody titer prediction kit includes the detection reagent strips of Example 18 and Example 20.
- a novel coronavirus neutralizing antibody titer prediction kit includes the detection reagent strips of Example 15 and Example 17.
- a novel coronavirus neutralizing antibody titer prediction kit includes the detection reagent strip of Example 14.
- the applicant used the gold standard of neutralizing titer (plaque reduction neutralization test) for the serum samples of 580 cured patients with 2019-nCoV.
- the detection of the true neutralization titer is carried out, and the specific methods are as follows:
- the antibody itself has no obvious cytotoxicity; the normal cell control is established; the virus control CPE reaches ++++.
- the lowest antibody concentration or the highest dilution of the antibody that can inhibit the cytopathic effect caused by the new coronavirus infection of 100TCID50 (protect 50% of the cells from lesions) is the antiviral effective concentration or titer of the antibody.
- the neutralization titer (whether the sample has a protective effect) of the serum samples of 580 cured patients with COVID-19 was algorithmically analyzed using the fluorescence data obtained from the kit prepared in Example 21, as follows:
- the feature vector of the fluorescence data obtained by the kit prepared in Example 21 was input into the pre-trained LR model for calculation, and the probability value of the target serum classification was obtained.
- the model is obtained by training the eigenvectors corresponding to multiple non-protective serum antibody level indicators and multiple protective serum antibody level indicators.
- the optimal combination of wavelet basis function parameters corresponding to the feature signal group and the corresponding optimal support vector machine classification model are obtained.
- the combination is optimized, and the optimal parameter combination after optimization is used to make the SVM classification conformity rate the highest.
- the classifier based on feature set achieved a comprehensive success rate of 89.66%.
- the specific detection results are:
- the classifier based on feature set achieved a comprehensive success rate of 98.10%.
- the neutralization titer (whether the sample has a protective effect) of the serum samples of 580 cured patients with new crowns was analyzed by algorithm using the fluorescence data obtained from the kit prepared in Example 22, as follows:
- the feature vector of the fluorescence data obtained by the kit prepared in Example 22 was input into the pre-trained LR model for calculation, and the probability value of the target serum classification was obtained.
- the model is obtained by training the eigenvectors corresponding to multiple non-protective serum antibody level indicators and multiple protective serum antibody level indicators.
- the optimal combination of wavelet basis function parameters corresponding to the feature signal group and the corresponding optimal support vector machine classification model are obtained.
- the combination is optimized, and the optimal parameter combination after optimization is used to make the SVM classification coincide with the highest rate.
- the classifier based on feature set achieved a comprehensive success rate of 89.48%.
- the specific detection results are:
- the classifier based on feature set achieved a comprehensive success rate of 98.62%.
- the neutralization titer (whether the sample has a protective effect) of the serum samples of 580 cured patients with new crowns was analyzed by algorithm using the fluorescence data obtained from the kit prepared in Example 23, as follows:
- the feature vector of the fluorescence data obtained by the kit prepared in Example 22 was input into the pre-trained LR model for calculation, and the probability value of the target serum classification was obtained.
- the model is obtained by training the eigenvectors corresponding to multiple non-protective serum antibody level indicators and multiple protective serum antibody level indicators.
- the optimal combination of wavelet basis function parameters corresponding to the feature signal group and the corresponding optimal support vector machine classification model are obtained.
- the combination is optimized, and the optimal parameter combination after optimization is used to make the SVM classification coincide with the highest rate.
- the classifier based on feature set achieved a comprehensive success rate of 89.83%.
- the specific detection results are:
- the classifier based on feature set achieved a comprehensive success rate of 97.93%.
- the neutralization titer (whether the sample has a protective effect) of the serum samples of 580 cured patients with COVID-19 was algorithmically analyzed using the fluorescence data obtained from the kit prepared in Example 24, as follows:
- the feature vector of the fluorescence data obtained by the kit prepared in Example 22 was input into the pre-trained LR model for calculation, and the probability value of the target serum classification was obtained.
- the model is obtained by training the eigenvectors corresponding to multiple non-protective serum antibody level indicators and multiple protective serum antibody level indicators.
- the optimal combination of wavelet basis function parameters corresponding to the feature signal group and the corresponding optimal support vector machine classification model are obtained.
- the combination is optimized, and the optimal parameter combination after optimization is used to make the SVM classification coincide with the highest rate.
- the classifier based on feature set achieved a comprehensive success rate of 88.62%.
- the specific detection results are:
- the classifier based on feature set achieved a comprehensive success rate of 92.93%.
- the present disclosure provides a method for predicting the titer of a novel coronavirus neutralizing antibody.
- the steps of the method are: firstly providing a biological sample to be tested, and then detecting the expression of the novel coronavirus specific protein in the serum sample after binding to the affinity antibody level; and then correlate the test results with the neutralizing antibody titer to predict the neutralizing antibody titer of the new coronavirus in the biological sample to be tested.
- the above method can well predict the neutralizing antibody result of the new coronavirus only through the detection of serum. Compared with the existing plaque reduction neutralization test and pseudovirus neutralization titer detection, it has the advantages of simple operation and fast detection speed. , The advantages of short detection period and safe detection environment can be used as an evaluation index for the second recurrence of new coronavirus vaccination or infection.
- the novel coronavirus neutralizing antibody titer prediction kit provided by the present disclosure is mainly composed of detection reagent strips containing quantum dot-specific protein complexes; the above-mentioned reagent strips use quantum dot immunochromatography technology to combine the novel coronavirus N protein, novel coronavirus
- the coronavirus S2-ECD protein and the new coronavirus S1-RBD protein were used as detection antigens, and the N, S1-RBD and S2-ECD antigen proteins were labeled with quantum dot fluorescent microspheres to form quantum dot-specific protein complexes, and then the new coronavirus was detected.
- the above-mentioned reagent strips have the advantages of high sensitivity, high specificity and quantitative detection, and avoid the missed detection of weak positive samples and false positive samples to the greatest extent.
- the quantum dot immunochromatography method used in the present disclosure is fast in detection, and the results are obtained within 10 minutes.
- the use of serum detection samples can reduce the exposure risk of medical staff, and it can be operated on-site with simple training, and each portable fluorescence detector can detect the upper limit in 1 hour. Hundreds of samples.
Abstract
提供一种预测新型冠状病毒中和抗体效价的方法及其试剂盒，属于中和抗体效价预测技术领域，该预测新型冠状病毒中和抗体效价的方法为：首先提供待检生物样品，然后检测血清样品中的新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平；随后将检测结果与中和抗体效价相关联，以预测待检生物样品中新型冠状病毒的中和抗体效价。上述方法仅通过血清的检测即可很好的预测得到新型冠状病毒的中和抗体结果，相较于现有的空斑减少中和试验和假病毒中和效价检测具有操作简单、检测速度快、检测周期短，检测环境安全的优势，可作为新型冠状病毒疫苗接种或感染后二次复发的评估指标。
Description
相关申请的交叉引用
本申请要求于2020年12月21日提交中国专利局的申请号为2020115238569、名称为“预测新型冠状病毒中和抗体效价的方法及其试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
本公开涉及中和抗体效价预测技术领域，尤其是涉及一种预测新型冠状病毒中和抗体效价的方法及其试剂盒。
人体接种疫苗后可通过免疫应答产生保护性抗体，即中和抗体。对中和抗体效价进行滴度测定，可以判断疫苗的保护效果。治愈后新冠患者中和抗体效价检测，可用于判断是否存在二次感染的风险。
目前已经建立的冠状病毒中和抗体效价检测方法包括空斑减少中和试验(Plaque Reduction Neutralization Test，PRNT)和假病毒中和效价检测(Pseudovirus Based Neutralization Assay，PBNA)等。PRNT检测使用活病毒，需在BSL-3级实验室完成，且PRNT检测操作复杂、周期长、误差大。假病毒中和检测虽使用安全性较高的假病毒，但无法模拟和替代活病毒侵染细胞的过程，且其同样存在操作复杂、周期长、误差大，无法满足快速、通量、精准检测的需求。
因此，研究开发一种新型冠状病毒中和抗体效价的预测方法和试剂盒，以用于快速评价人体的中和效价水平，尤为重要。
有鉴于此，特提出本公开。
发明内容
本公开的目的包括，例如提供一种预测新型冠状病毒中和抗体效价的方法，该方法仅通过血清的检测即可很好的预测得到新型冠状病毒的中和抗体结果，相较于现有的空斑减少中和试验和假病毒中和效价检测具有操作简单、检测速度快、检测周期短，检测环境安全的优势。
本公开的目的还包括，例如提供一种用于上述预测方法的新型冠状病毒中和抗体效价预测试剂盒。
为了实现本公开的上述目的，特采用以下技术方案：
本公开提供的一种预测新型冠状病毒中和抗体效价的方法，所述方法包括以下步骤：
(a)、提供待检生物样品，检测血清样品中的新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平；
(b)、将步骤(a)的检测结果与中和抗体效价相关联。
在一种或多种实施方式中，所述待检生物样品为治愈后新冠患者的血清和血浆。
在一种或多种实施方式中，所述新型冠状病毒特异性蛋白包括新型冠状病毒N蛋白、新型冠状病毒S2-ECD蛋白或新型冠状病毒S1-RBD蛋白中的至少一种；
在一种或多种实施方式中，所述新型冠状病毒N蛋白的氨基酸序列如Seq No.01所示:
在一种或多种实施方式中，所述新型冠状病毒S2-ECD蛋白的氨基酸序列如Seq No.02所示:
在一种或多种实施方式中，所述新型冠状病毒S1-RBD蛋白的氨基酸序列如Seq No.03所示。
在一种或多种实施方式中，所述亲和抗体包括抗人IgA、抗人IgG和抗人IgM中的至少一种；
在一种或多种实施方式中，所述抗人IgA包括羊抗人IgA多克隆抗体、鼠抗人IgA单克隆抗体、兔抗人IgA单克隆抗体、兔抗人IgA多克隆抗体中的任意一种；
在一种或多种实施方式中，所述抗人IgG包括羊抗人IgG多克隆抗体、鼠抗人IgG单克隆抗体、兔抗人IgG单克隆抗体、兔抗人IgG多克隆抗体中的任意一种；
在一种或多种实施方式中，所述抗人IgM包括羊抗人IgM多克隆抗体、鼠抗人IgM单克隆抗体、兔抗人IgM单克隆抗体、兔抗人IgM多克隆抗体中的任意一种。
在一种或多种实施方式中，所述步骤(a)新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平采用量子点免疫荧光法进行检测。
在一种或多种实施方式中，所述步骤(b)关联采用软件分类算法进行。
在一种或多种实施方式中，所述软件分类算法包括逻辑斯蒂回归、支持向量机和随机森林中的至少一种。
本公开提供的一种新型冠状病毒中和抗体效价预测试剂盒，所述试剂盒包括：含有量子点特异性蛋白复合物的检测试剂条；
所述量子点特异性蛋白复合物主要由量子点和新型冠状病毒特异性蛋白偶联得到。
在一种或多种实施方式中，所述检测试剂条采用量子点免疫层析技术，将新型冠状病毒N蛋白、新型冠状病毒S2-ECD蛋白和新型冠状病毒S1-RBD蛋白作为检测抗原，使用量子点荧光微球标记N、S1-RBD和S2-ECD抗原蛋白形成量子点特异性蛋白复合物，随后对新型冠状病毒的血浆进行检测。
在一种或多种实施方式中，所述检测试剂条包括底卡、样品垫、量子点标记物结合垫、层析反应膜和吸水纸；
其中，所述量子点标记物结合垫吸附有量子点特异性蛋白复合物；
所述层析反应膜上设置有检测线，所述检测线包括IgA检测线、IgG检测线、IgM检测线中的至少一条。
在一种或多种实施方式中，所述层析反应膜包括NC膜、PVDF膜或尼龙膜中的一种，优选为硝酸纤维素膜；
在一种或多种实施方式中，所述底卡包括PVC底卡或PC底卡中的一种，优选为PC底卡；
在一种或多种实施方式中，所述量子点标记物结合垫包括玻璃纤维膜、聚酯膜、滤血膜中的一种，优选为玻璃纤维膜；
在一种或多种实施方式中，所述样品垫包括玻璃纤维膜、聚酯膜、滤血膜中的一种，优选为玻璃纤维膜；
在一种或多种实施方式中，所述吸水纸包括厚滤纸、玻璃纤维或滤血膜中的一种，优选厚滤纸。
在一种或多种实施方式中，所述层析反应膜上设置有检测线和质控线；
所述检测线包括IgA检测线、IgG检测线、IgM检测线中的至少一条，其中，IgA检测线含有抗人IgA；IgG检测线含有抗人IgG；IgM检测线含有抗人IgM；并且
所述质控线含有羊抗兔IgG抗体；
在一种或多种实施方式中，所述试剂盒还包括紫外照射装置。
与现有技术相比，本公开的有益效果为：
本公开提供的预测新型冠状病毒中和抗体效价的方法，所述方法的步骤为：首先提供待检生物样品，然后检测血清样品中的新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平；随后将检测结果与中和抗体效价相关联，以预测待检生物样品中新型冠状病毒的中和抗体效价。上述方法仅通过血清的检测即可很好的预测得到新型冠状病毒的中和抗体结果，相较于现有的空斑减少中和试验和假病毒中和效价检测具有操作简单、检测速度快、检测周期短，检测环境安全的优势，可作为新型冠状病毒疫苗接种或感染后二次复发的评估指标。
本公开提供的新型冠状病毒中和抗体效价预测试剂盒主要由含有量子点特异性蛋白复合物的检测试剂条组成；上述试剂条采用量子点免疫层析技术，将新型冠状病毒N蛋白、新型冠状病毒S2-ECD蛋白和新型冠状病毒S1-RBD蛋白作为检测抗原，使用量子点荧光微球标记N、S1-RBD和S2-ECD抗原蛋白形成量子点特异性蛋白复合物，随后对新型冠状病毒的血浆进行检测，上述试剂条具有灵敏度高、特异度高以及可定量检测的优势，最大程度避免了弱阳性样本漏检和假阳性样本误检。同时本公开使用的量子点免疫层析法检测迅速，10分钟内出结果，使用血清检测样本可降低医护人员暴露风险，简单培训即可现场操作，而且每台便携式荧光检测仪1小时能检测上百个样本。
下面将结合实施例对本公开的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
根据本公开的一个方面，一种预测新型冠状病毒中和抗体效价的方法，所述方法包括以下步骤：
(a)、提供待检生物样品，检测血清样品中的新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平；
(b)、将步骤(a)的检测结果与中和抗体效价相关联。
本公开提供的预测新型冠状病毒中和抗体效价的方法，所述方法的步骤为：首先提供待检生物样品，然后检测血清样品中的新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平；随后将检测结果与中和抗体效价相关联，以预测待检生物样品中新型冠状病毒的中和抗体效价。上述方法仅通过血清的检测即可很好的预测得到新型冠状病毒的中和抗体结果，相较于现有的空斑减少中和试验和假病毒中和效价检测具有操作简单、检测速度快、检测周期短，检测环境安全的优势，可作为新型冠状病毒疫苗接种或感染后二次复发的评估指标。
在本公开的一种优选实施方式中，所述待检生物样品为治愈后新冠患者的血清或血浆。
作为一种优选的实施方式，上述待检生物样品为治愈后新冠患者的血清或血浆，进而预测患者感染后二次复发的概率。
在本公开的一种优选实施方式中，所述新型冠状病毒特异性蛋白包括新型冠状病毒N蛋白、新型冠状病毒S2-ECD蛋白或新型冠状病毒S1-RBD蛋白中的至少一种；
注：所述新型冠状病毒S2-ECD蛋白为2019-nCoVS2亚基胞外结构域(extra-cellular domain,ECD)蛋白；所述新型冠状病毒S1-RBD蛋白为2019-nCoV表面刺突蛋白S1亚基受体结合域(receptor binding domain,RBD)蛋白；所述新型冠状病毒N蛋白为2019-nCoV主要蛋白核壳体蛋白(Nucleocapsid Protein,N蛋白)。
在上述优选实施方式中，所述新型冠状病毒N蛋白的氨基酸序列如Seq No.01所示:
在上述优选实施方式中，所述新型冠状病毒S2-ECD蛋白的氨基酸序列如Seq No.02所示:
在上述优选实施方式中，所述新型冠状病毒S1-RBD蛋白的氨基酸序列如Seq No.03所示。
在本公开的一种优选实施方式中，所述亲和抗体包括抗人IgA、抗人IgG和抗人IgM中的至少一种；
在上述优选实施方式中，所述抗人IgA包括羊抗人IgA多克隆抗体、鼠抗人IgA单克隆抗体、兔抗人IgA单克隆抗体、兔抗人IgA多克隆抗体中的任意一种；
在上述优选实施方式中，所述抗人IgG包括羊抗人IgG多克隆抗体、鼠抗人IgG单克隆抗体、兔抗人IgG单克隆抗体、兔抗人IgG多克隆抗体中的任意一种；
在上述优选实施方式中，所述抗人IgM包括羊抗人IgM多克隆抗体、鼠抗人IgM单克隆抗体、兔抗人IgM单克隆抗体、兔抗人IgM多克隆抗体中的任意一种。
在本公开的一种优选实施方式中，所述步骤(a)新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平采用量子点免疫荧光法进行检测。
作为一种优选的实施方式，上述新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平可以采用量子点免疫荧光法进行检测，进而可以更为直观的得到荧光数据，以便于后期的数据分析计算。
在本公开的一种优选实施方式中，所述步骤(b)关联采用软件分类算法进行。
在上述优选实施方式中，所述软件分类算法包括逻辑斯蒂回归、支持向量机和随机森林中的至少一种。
根据本公开的一个方面，一种新型冠状病毒中和抗体效价预测试剂盒，所述试剂盒包括：含有量子点特异性蛋白复合物的检测试剂条；
所述量子点特异性蛋白复合物主要由量子点和新型冠状病毒特异性蛋白偶联得到。
本公开提供的新型冠状病毒中和抗体效价预测试剂盒主要由含有量子点特异性蛋白复合物的检测试剂条组成；上述试剂条采用量子点免疫层析技术，将新型冠状病毒N蛋白、新型冠状病毒S2-ECD蛋白和新型冠状病毒S1-RBD蛋白作为检测抗原，使用量子点荧光微球标记N、S1-RBD和S2-ECD抗原蛋白形成量子点特异性蛋白复合物，随后对新型冠状病毒的血浆进行检测，上述试剂条具有灵敏度高、特异度高以及可定量检测的优势，最大程度避免了弱阳性样本漏检和假阳性样本误检。同时本公开使用的量子点免疫层析法检测迅速，10分钟内出结果，使用血清检测样本可降低医护人员暴露风险，简单培训即可现场操作，而且每台便携式荧光检测仪1小时能检测上百个样本。
在本公开的一种优选实施方式中，所述检测试剂条包括底卡、样品垫、量子点标记物结合垫(也可称为释放垫)、层析反应膜和吸水纸；
其中，所述量子点标记物结合垫吸附有量子点特异性蛋白复合物；
所述层析反应膜上设置有检测线，所述检测线包括IgA检测线、IgG检测线、IgM检测线中的至少一条。
在上述优选实施方式中，所述层析反应膜包括NC膜、PVDF膜或尼龙膜中的一种，优选为硝酸纤维素膜；
在上述优选实施方式中，所述底卡包括PVC底卡或PC底卡中的一种，优选为PC底卡；
在上述优选实施方式中，所述量子点标记物结合垫包括玻璃纤维膜、聚酯膜、滤血膜中的一种，优选为玻璃纤维膜；
在上述优选实施方式中，所述样品垫包括玻璃纤维膜、聚酯膜、滤血膜中的一种，优选为玻璃纤维膜；
在上述优选实施方式中，所述吸水纸包括厚滤纸、玻璃纤维或滤血膜中的一种，优选厚滤纸。
在上述优选实施方式中，所述层析反应膜上设置有检测线和质控线；
所述检测线包括IgA检测线、IgG检测线、IgM检测线中的至少一条，其中，IgA检测线含有抗人IgA；IgG检测线含有抗人IgG；IgM检测线含有抗人IgM；并且
所述质控线含有羊抗兔IgG抗体；
在本公开的一种优选实施方式中，所述试剂盒还包括紫外照射装置。
下面将结合实施例对本公开的技术方案进行进一步地说明。
实施例1
制备新型冠状病毒重组抗原N蛋白、S1-RBD蛋白和S2-ECD蛋白：
(1)、采用基因克隆技术，PCR扩增编码新型冠状病毒抗原的N基因和S2-ECD基因、S1-RBD蛋白；
(2)、N基因转入大肠杆菌表达，S2-ECD基因转入293细胞中使其表达，S1-RBD蛋白转入293 细胞中表达，获得新型冠状病毒重组抗原N蛋白和S2-ECD蛋白和S1-RBD蛋白。
实施例2
制备层析反应膜(IgA、IgG两条检测线)：
将25cm宽的NC膜有贴在PC底卡中间区域，之后将抗人IgA、抗人IgG以及羊抗兔IgG抗体分别用PBS稀释液中稀释成0.2～1mg/ml并用喷金划膜仪划在NC膜相应的检测线区和质控线区。37℃烘箱12-24h烘干，封袋备用。
实施例3
制备层析反应膜(IgM、IgG两条检测线)：
将25cm宽的NC膜有贴在PC底卡中间区域，之后将抗人IgM、抗人IgG以及羊抗兔IgG抗体分别用PBS稀释液中稀释成0.2～1mg/ml并用喷金划膜仪划在NC膜相应的检测线区和质控线区。37℃烘箱12-24h烘干，封袋备用。
实施例4
制备层析反应膜(IgA、IgM、IgG三条检测线)：
将25cm宽的NC膜有贴在PC底卡中间区域，之后将抗人IgA、抗人IgM、抗人IgG以及羊抗兔IgG抗体分别用PBS稀释液中稀释成0.2～1mg/ml并用喷金划膜仪划在NC膜相应的检测线区和质控线区。37℃烘箱12-24h烘干，封袋备用。
实施例5
制备样品垫：
用移液枪和涂布棒将20～100mMTris-HCl稀释液(pH8.0，含0.5％BSA,0.5％PVP,0.5％TWEEN-20)均匀涂布在未处理量子点标记物结合垫上，37℃12～24h烘干，封袋备用。
实施例6
制备量子点-标记蛋白复合物(特异性蛋白为新型冠状病毒N蛋白)：
(1)、取200μl的qds加入800μl的MES(pH 6)缓冲液中，加入40～160μg EDC和10～40μg NHS活化，37℃活化15-60min。将活化后的溶液在一定条件下离心，用移液枪去除上清。
(2)、将步骤(1)离心后沉淀用1000μl的10～50mM MES(pH 6)缓冲液复溶沉淀，若复溶不彻底可用超声协助复溶。将复溶后的溶液在一定条件下离心，用移液枪去除上清，1000μl的10～50mM MES(pH 6)缓冲液复溶沉淀，若复溶不彻底可用超声协助复溶(可省略此步骤)。
(3)、向复溶溶液中加入20～100μl的已用10～50mM MES(pH 6)缓冲液稀释成1mg/ml的新型冠状病毒N蛋白，37℃偶联60-300min。
(4)、偶联结束后加入100μl的20～100mM Tris-HCl稀释液(pH8.0,含100～500mM甘氨酸+10％BSA)进行封闭，37℃封闭30～120min。
(5)、将封闭好后的溶液在一定条件下离心，用移液枪去除上清。500-1000μl Tris-HCl稀释液复溶沉淀，若复溶不彻底可用超声协助复溶，继续在一定条件下离心，用移液枪去除上清(可省略此步骤)。500-1000μl的20～100mM Tris-HCl稀释液(pH8.0)复溶沉淀，若复溶不彻底可用超声协助复溶，4℃保存，得到量子点-标记蛋白复合物。
实施例7
制备量子点-标记蛋白复合物(特异性蛋白为新型冠状病毒S2-ECD蛋白)：
本实施例除步骤(3)为：“向复溶溶液中加入20～100μl的已用10～50mM MES(pH 6)缓冲液稀释成1mg/ml的新型冠状病毒S2-ECD蛋白，37℃偶联60-300min。”外，其余同实施例6。
实施例8
制备量子点-标记蛋白复合物(特异性蛋白为新型冠状病毒S1-RBD蛋白)：
本实施例除步骤(3)为：“向复溶溶液中加入20～100μl的已用10～50mM MES(pH 6)缓冲液稀释成1mg/ml的新型冠状病毒S1-RBD蛋白，37℃偶联60-300min。”外，其余同实施例6。
实施例9
制备量子点标记物结合垫(特异性蛋白为新型冠状病毒N蛋白)：
将实施例6制备好的量子点-标记蛋白复合物用20～100mM Tris-HCl稀释液(pH8.0，含0.5％BSA,2％海藻糖)根据需求稀释20-100倍后用划膜喷金仪喷点于已处理量子点标记物结合垫上，37℃12～24h烘干，得到量子点标记物结合垫。或者将实施例7制备好的量子点-标记蛋白复合物用20～100mM Tris-HCl稀释液(pH8.0，含0.5％BSA,2％海藻糖)根据需求稀释20-100倍后用移液枪喷点在未处理的量子点标记物结合垫上，再用涂布棒涂布均匀，最后放入冷冻干燥机冷冻干燥，待程序运行完毕后封袋备用。
实施例10
制备量子点标记物结合垫(特异性蛋白为新型冠状病毒S2-ECD蛋白)：
将实施例7制备好的量子点-标记蛋白复合物用20～100mM Tris-HCl稀释液(pH8.0，含0.5％BSA,2％ 海藻糖)根据需求稀释20-100倍后用划膜喷金仪喷点于已处理量子点标记物结合垫上，37℃12～24h烘干，得到量子点标记物结合垫。或者将实施例7制备好的量子点-标记蛋白复合物用20～100mM Tris-HCl稀释液(pH8.0，含0.5％BSA,2％海藻糖)根据需求稀释20-100倍后用移液枪喷点在未处理的量子点标记物结合垫上，再用涂布棒涂布均匀，最后放入冷冻干燥机冷冻干燥，待程序运行完毕后封袋备用。
实施例11
制备量子点标记物结合垫(特异性蛋白为新型冠状病毒S1-RBD蛋白)：
将实施例8制备好的量子点-标记蛋白复合物用20～100mM Tris-HCl稀释液(pH8.0，含0.5％BSA,2％海藻糖)根据需求稀释20-100倍后用划膜喷金仪喷点于已处理量子点标记物结合垫上，37℃12～24h烘干，得到量子点标记物结合垫。或者将实施例7制备好的量子点-标记蛋白复合物用20～100mM Tris-HCl稀释液(pH8.0，含0.5％BSA,2％海藻糖)根据需求稀释20-100倍后用移液枪喷点在未处理的量子点标记物结合垫上，再用涂布棒涂布均匀，最后放入冷冻干燥机冷冻干燥，待程序运行完毕后封袋备用。
实施例12
一种新型冠状病毒检测试剂条，所述试剂条的制备方法包括以下步骤：
将实施例4制得的层析反应膜、实施例9制得的量子点标记物结合垫、实施例5制得的样品垫以及吸水纸搭接粘附在PVC背板上；
其中样品垫和量子点标记物结合垫依次搭接在层析反应膜的一端，吸水纸搭接在层析反应膜的另一端，然后调整切条机参数进行切条，切成宽3-4mm的试纸条。
实施例13
本实施例除将实施例9制得的量子点标记物结合垫替换为实施例10制得的量子点标记物结合垫外，其余同实施例12。
实施例14
本实施例除将实施例9制得的量子点标记物结合垫替换为实施例11制得的量子点标记物结合垫外，其余同实施例12。
实施例15
本实施例除将实施例4的层析反应膜替换为实施例3的层析反应膜外，其余同实施例12。
实施例16
本实施例除将实施例4的层析反应膜替换为实施例3的层析反应膜外，其余同实施例13。
实施例17
本实施例除将实施例4的层析反应膜替换为实施例3的层析反应膜外，其余同实施例14。
实施例18
本实施例除将实施例4的层析反应膜替换为实施例2的层析反应膜外，其余同实施例12。
实施例19
本实施例除将实施例4的层析反应膜替换为实施例2的层析反应膜外，其余同实施例13。
实施例20
本实施例除将实施例4的层析反应膜替换为实施例2的层析反应膜外，其余同实施例14。
上述实施例12～19中试剂条的特异性蛋白和检测线的设置如下：
| 组别 | 特异性蛋白 | 检测线 |
| 实施例12 | N蛋白 | IgA、IgM、IgG |
| 实施例13 | S2-ECD蛋白 | IgA、IgM、IgG |
| 实施例14 | S1-RBD蛋白 | IgA、IgM、IgG |
| 实施例15 | N蛋白 | IgM、IgG |
| 实施例16 | S2-ECD蛋白 | IgM、IgG |
| 实施例17 | S1-RBD蛋白 | IgM、IgG |
| 实施例18 | N蛋白 | IgA、IgG |
| 实施例19 | S2-ECD蛋白 | IgA、IgG |
| 实施例20 | S1-RBD蛋白 | IgA、IgG |
实施例21
一种新型冠状病毒中和抗体效价预测试剂盒，所述试剂盒包括实施例12和实施例14的检测试剂 条。
实施例22
一种新型冠状病毒中和抗体效价预测试剂盒，所述试剂盒包括实施例15、实施例16和实施例17的检测试剂条。
实施例23
一种新型冠状病毒中和抗体效价预测试剂盒，所述试剂盒包括实施例18和实施例20的检测试剂条。
实施例24
一种新型冠状病毒中和抗体效价预测试剂盒，所述试剂盒包括实施例15和实施例17的检测试剂条。
实施例25
一种新型冠状病毒中和抗体效价预测试剂盒，所述试剂盒包括实施例14的检测试剂条。
实验例1真实效价检测
为了更好为验证本申请预测新型冠状病毒中和抗体效价的方法的准确性，申请人将580例治愈后新冠患者的血清样本采用中和效价的金标准(空斑减少中和试验)进行了真实中和效价的检测，具体方法如下：
1、取一块新的96孔板，1-10列每孔加50μl待检血清原液，再用8道孔排枪加每孔100TCID50/50μl病毒液50μl，稍微吹打使血清与病毒液混均。11列先每孔加50μl维持液，再于11列的第一孔加50μl标准阳性血清，将标准阳性血清往下作1:2、1:22至1:28倍稀释。12列第1孔加标准阴性血清原液50μl，再加100TCID50/50μl病毒液50μl。用维持液将100TCID50/50μl的病毒液作4次连续10倍稀释，稀释成10TCID50/50μl、1TCID50/50μl、0.1TCID50/50μl,依次加到第12列的3到6孔，每孔加50μl，再每孔加50μl维持液。将培养板置37℃CO
2培养箱中和1h。
2、将细胞长至单层的培养板中的维持液倒掉，然后将上一步已中和1h的血清病毒液转移到长了单层细胞的培养板中，转移的时候要小心，以免加的液体把细胞吹掉了。
3、再每孔加150μl维持液，微量振荡器上震荡30s，混均。将培养板置37℃CO
2培养箱中，96h后开始观察记录结果。
判定结果是需满足：抗体本身无明显细胞毒性；正常细胞对照成立；病毒对照CPE达++++。以能抑制细胞不受100TCID50的新型冠状病毒感染(保护50％细胞不产生病变)所致的细胞病变效应的最低抗体浓度或抗体最高稀释度，为该抗体的抗病毒有效浓度或效价。
其中，当中和效价低于40对应的抗体水平样本时，无保护作用；当中和效价高于40对应的抗体水平样本时，表示有保护作用。
实施例26
利用上述实施例21～25的新型冠状病毒中和抗体效价预测试剂盒对实验例1中的580例治愈后新冠患者的血清样本进行检测，并测定各样本检测后的IgA检测线、IgG检测线、IgM检测线上的荧光数值，具体检测结果如下：
实验例2
利用上述实施例21制得的试剂盒得到的荧光数据对580例治愈后新冠患者的血清样本的中和效价(样本是否具有保护作用)进行算法分析，具体如下：
(1)基于LR的中和效价水平预测方法：
将实施例21制得的试剂盒得到的荧光数据的特征向量输入预先训练好的LR模型中进行计算，得到目标血清分类的概率值。模型通过多个无保护作用的血清中抗体水平指标和多个有保护作用的血清中抗体水平指标对应的特征向量训练得到。
其优选的函数值为：y＝2.09686313(S1-IgM)+3.05375507(S1-IgG)+0.91398949(S1-IgA)+0.2027609(S2-IgA)+4.79033777(S2-IgG)+1.13682287(S2-IgM)-0.382805366具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率：
| 真阳性 | 97.07％ | 真阴性 | 67.65％ |
| 假阳性 | 32.35％ | 假阴性 | 2.93％ |
| 准确率 | 90.17％ |
(2)、基于支持SVM的中和效价水平预测方法：
将实施例21制得的试剂盒得到的荧光数据的归一化后得其特征向量，随机选取80％的数据集作为训练集，以剩余20％的数据集作为测试集，训练集用于训练SVM模型，得到最优分类超平面；
利用训练集对SVM模型进行训练，得到特征信号组对应的小波基函数参数的最佳组合，以及相对应的最佳支持向量机分类模型；过程中，采用自适应遗传算法对小波基函数的参数组合进行优化，利用优化后的最佳参数组合使得支持向量机分类符合率最高。
具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率，基于特征集的分类器取得了89.66％的综合成功率。
| 真阳性 | 92.79％ | 真阴性 | 79.41％ |
| 假阳性 | 20.59％ | 假阴性 | 7.21％ |
| 准确率 | 89.66％ |
(3)、基于RF的中和效价水平预测方法：
预处理后随机选取80％的数据集作为训练集，以剩余20％的数据集作为测试集，训练集用于训练RF模型，具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率，基于特征集的分类器取得了98.10％的综合成功率。
| 真阳性 | 98.65％ | 真阴性 | 96.32％ |
| 假阳性 | 3.68％ | 假阴性 | 1.35％ |
| 准确率 | 98.10％ |
实验例3
利用上述实施例22制得的试剂盒得到的荧光数据对580例治愈后新冠患者的血清样本的中和效价(样本是否具有保护作用)进行算法分析，具体如下：
(1)基于LR的中和效价水平预测方法：
将实施例22制得的试剂盒得到的荧光数据的特征向量输入预先训练好的LR模型中进行计算，得到目标血清分类的概率值。模型通过多个无保护作用的血清中抗体水平指标和多个有保护作用的血清中抗体水平指标对应的特征向量训练得到。
其优选的函数值为：y＝3.18895222(S1-IgG)+0.94068715(S1-IgA)+0.34774695(S2-IgA)+4.96019302(S2-IgG)-0.297491254具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率：
| 真阳性 | 97.07％ | 真阴性 | 66.18％ |
| 假阳性 | 33.82％ | 假阴性 | 2.93％ |
| 准确率 | 89.83％ |
(2)、基于支持SVM的中和效价水平预测方法：
将实施例21制得的试剂盒得到的荧光数据的归一化后得其特征向量，随机选取80％的数据集作为训练集，以剩余20％的数据集作为测试集，训练集用于训练SVM模型，得到最优分类超平面；
利用训练集对SVM模型进行训练，得到特征信号组对应的小波基函数参数的最佳组合，以及相对应的最佳支持向量机分类模型；过程中，采用自适应遗传算法对小波基函数的参数组合进行优化，利用优化后的最佳参数组合使得支持向量机分类符合率最高。
具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率，基于特征集的分类器取得了89.48％的综合成功率。
| 真阳性 | 92.79％ | 真阴性 | 78.68％ |
| 假阳性 | 21.32％ | 假阴性 | 7.21％ |
| 准确率 | 89.48％ |
(3)、基于RF的中和效价水平预测方法：
预处理后随机选取80％的数据集作为训练集，以剩余20％的数据集作为测试集，训练集用于训练RF模型，具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率，基于特征集的分类器取得了98.62％的综合成功率。
| 真阳性 | 99.32％ | 真阴性 | 96.32％ |
| 假阳性 | 3.68％ | 假阴性 | 0.68％ |
| 准确率 | 98.62％ |
实验例4
利用上述实施例23制得的试剂盒得到的荧光数据对580例治愈后新冠患者的血清样本的中和效价(样本是否具有保护作用)进行算法分析，具体如下：
(1)基于LR的中和效价水平预测方法：
将实施例22制得的试剂盒得到的荧光数据的特征向量输入预先训练好的LR模型中进行计算，得到目标血清分类的概率值。模型通过多个无保护作用的血清中抗体水平指标和多个有保护作用的血清中抗体水平指标对应的特征向量训练得到。
其优选的函数值为：y＝2.12011703(S1-IgM)+3.06835539(S1-IgG)+4.78491836(S2-IgG)+1.14629294(S2-IgM)-0.354555657
具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率：
| 真阳性 | 97.30％ | 真阴性 | 67.65％ |
| 假阳性 | 32.35％ | 假阴性 | 2.70％ |
| 准确率 | 90.34％ |
(2)、基于支持SVM的中和效价水平预测方法：
将实施例21制得的试剂盒得到的荧光数据的归一化后得其特征向量，随机选取80％的数据集作为训练集，以剩余20％的数据集作为测试集，训练集用于训练SVM模型，得到最优分类超平面；
利用训练集对SVM模型进行训练，得到特征信号组对应的小波基函数参数的最佳组合，以及相对应的最佳支持向量机分类模型；过程中，采用自适应遗传算法对小波基函数的参数组合进行优化，利用优化后的最佳参数组合使得支持向量机分类符合率最高。
具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率，基于特征集的分类器取得了89.83％的综合成功率。
| 真阳性 | 92.34％ | 真阴性 | 81.62％ |
| 假阳性 | 18.38％ | 假阴性 | 7.66％ |
| 准确率 | 89.83％ |
(3)、基于RF的中和效价水平预测方法：
预处理后随机选取80％的数据集作为训练集，以剩余20％的数据集作为测试集，训练集用于训练RF模型，具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率，基于特征集的分类器取得了97.93％的综合成功率。
| 真阳性 | 98.65％ | 真阴性 | 95.59％ |
| 假阳性 | 4.41％ | 假阴性 | 1.35％ |
| 准确率 | 97.93％ |
实验例5
利用上述实施例24制得的试剂盒得到的荧光数据对580例治愈后新冠患者的血清样本的中和效价(样本是否具有保护作用)进行算法分析，具体如下：
(1)基于LR的中和效价水平预测方法：
将实施例22制得的试剂盒得到的荧光数据的特征向量输入预先训练好的LR模型中进行计算，得到目标血清分类的概率值。模型通过多个无保护作用的血清中抗体水平指标和多个有保护作用的血清中抗体水平指标对应的特征向量训练得到。
其优选的函数值为：y＝5.63203179(S2-IgG)-0.183104242
具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率：
| 真阳性 | 96.85％ | 真阴性 | 68.38％ |
| 假阳性 | 31.62％ | 假阴性 | 3.15％ |
| 准确率 | 90.17％ |
(2)、基于支持SVM的中和效价水平预测方法：
将实施例21制得的试剂盒得到的荧光数据的归一化后得其特征向量，随机选取80％的数据集作为训练集，以剩余20％的数据集作为测试集，训练集用于训练SVM模型，得到最优分类超平面；
利用训练集对SVM模型进行训练，得到特征信号组对应的小波基函数参数的最佳组合，以及相对应的最佳支持向量机分类模型；过程中，采用自适应遗传算法对小波基函数的参数组合进行优化，利用优化后的最佳参数组合使得支持向量机分类符合率最高。
具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率，基于特征集的分类器取得了88.62％的综合成功率。
| 真阳性 | 93.47％ | 真阴性 | 72.79％ |
| 假阳性 | 27.21％ | 假阴性 | 6.53％ |
| 准确率 | 88.62％ |
(3)、基于RF的中和效价水平预测方法：
预处理后随机选取80％的数据集作为训练集，以剩余20％的数据集作为测试集，训练集用于训练RF模型，具体检测结果为：
并计算出该模型下的真阳性率、假阳性率、真阴性率、假阴性率和预测准确率，基于特征集的分类器取得了92.93％的综合成功率。
| 真阳性 | 94.37％ | 真阴性 | 88.24％ |
| 假阳性 | 11.76％ | 假阴性 | 5.63％ |
| 准确率 | 92.93％ |
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
本公开提供的预测新型冠状病毒中和抗体效价的方法，所述方法的步骤为：首先提供待检生物样品，然后检测血清样品中的新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平；随后将检测结果与中和抗体效价相关联，以预测待检生物样品中新型冠状病毒的中和抗体效价。上述方法仅通过血清的检测即可很好的预测得到新型冠状病毒的中和抗体结果，相较于现有的空斑减少中和试验和假病毒中和效价检测具有操作简单、检测速度快、检测周期短，检测环境安全的优势，可作为新型冠状病毒疫苗接种或感染后二次复发的评估指标。
本公开提供的新型冠状病毒中和抗体效价预测试剂盒主要由含有量子点特异性蛋白复合物的检测试剂条组成；上述试剂条采用量子点免疫层析技术，将新型冠状病毒N蛋白、新型冠状病毒S2-ECD蛋白和新型冠状病毒S1-RBD蛋白作为检测抗原，使用量子点荧光微球标记N、S1-RBD和S2-ECD抗原蛋白形成量子点特异性蛋白复合物，随后对新型冠状病毒的血浆进行检测，上述试剂条具有灵敏度高、特异度高以及可定量检测的优势，最大程度避免了弱阳性样本漏检和假阳性样本误检。同时本公开使用的量子点免疫层析法检测迅速，10分钟内出结果，使用血清检测样本可降低医护人员暴露风险，简单培训即可现场操作，而且每台便携式荧光检测仪1小时能检测上百个样本。
Claims (17)
- 一种预测新型冠状病毒中和抗体效价的方法，其特征在于，所述方法包括以下步骤：(a)、提供待检生物样品，检测血清样品中的新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平；(b)、将步骤(a)的检测结果与中和抗体效价相关联。
- 根据权利要求1所述的预测新型冠状病毒中和抗体效价的方法，其特征在于，所述待检生物样品为治愈后新冠患者的血清或血浆。
- 根据权利要求1或2所述的预测新型冠状病毒中和抗体效价的方法，其特征在于，所述新型冠状病毒特异性蛋白包括新型冠状病毒N蛋白、新型冠状病毒S2-ECD蛋白或新型冠状病毒S1-RBD蛋白中的至少一种；优选地，所述新型冠状病毒N蛋白的氨基酸序列如Seq No.01所示:优选地，所述新型冠状病毒S2-ECD蛋白的氨基酸序列如Seq No.02所示:优选地，所述新型冠状病毒S1-RBD蛋白的氨基酸序列如Seq No.03所示。
- 根据权利要求1-3中任一项所述的预测新型冠状病毒中和抗体效价的方法，其特征在于，所述亲和抗体包括抗人IgA、抗人IgG和抗人IgM中的至少一种；优选地，所述抗人IgA包括羊抗人IgA多克隆抗体、鼠抗人IgA单克隆抗体、兔抗人IgA单克隆抗体、兔抗人IgA多克隆抗体中的任意一种；优选地，所述抗人IgG包括羊抗人IgG多克隆抗体、鼠抗人IgG单克隆抗体、兔抗人IgG单克隆抗体、兔抗人IgG多克隆抗体中的任意一种；优选地，所述抗人IgM包括羊抗人IgM多克隆抗体、鼠抗人IgM单克隆抗体、兔抗人IgM单克隆抗体、兔抗人IgM多克隆抗体中的任意一种。
- 根据权利要求1-4中任一项所述的预测新型冠状病毒中和抗体效价的方法，其特征在于，所述步骤(a)新型冠状病毒特异性蛋白与亲和抗体结合后的表达水平采用量子点免疫荧光法进行检测。
- 根据权利要求1-5中任一项所述的预测新型冠状病毒中和抗体效价的方法，其特征在于，所述步骤(b)关联采用软件分类算法进行。
- 根据权利要求6所述的预测新型冠状病毒中和抗体效价的方法，其特征在于，所述软件分类算法包括逻辑斯蒂回归、支持向量机和随机森林中的至少一种。
- 一种新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述试剂盒包括：含有量子点特异性蛋白复合物的检测试剂条；所述量子点特异性蛋白复合物主要由量子点和新型冠状病毒特异性蛋白偶联得到。
- 根据权利要求8所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述检测试剂条采用量子点免疫层析技术，将所述新型冠状病毒N蛋白、所述新型冠状病毒S2-ECD蛋白和所述新型冠状病毒S1-RBD蛋白作为检测抗原，使用量子点荧光微球标记N、S1-RBD和S2-ECD抗原蛋白形成量子点特异性蛋白复合物，随后对新型冠状病毒的血浆进行检测。
- 根据权利要求8或9所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述检测试剂条包括底卡、样品垫、量子点标记物结合垫、层析反应膜和吸水纸；其中，所述量子点标记物结合垫吸附有量子点特异性蛋白复合物；所述层析反应膜上设置有检测线，所述检测线包括IgA检测线、IgG检测线、IgM检测线中的至少一条。
- 根据权利要求8-10中任一项所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述层析反应膜包括NC膜、PVDF膜或尼龙膜中的一种，优选为硝酸纤维素膜。
- 根据权利要求8-11中任一项所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述底卡包括PVC底卡或PC底卡中的一种，优选为PC底卡。
- 根据权利要求8-12中任一项所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述量子点标记物结合垫包括玻璃纤维膜、聚酯膜、滤血膜中的一种，优选为玻璃纤维膜。
- 根据权利要求8-13中任一项所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述样品垫包括玻璃纤维膜、聚酯膜、滤血膜中的一种，优选为玻璃纤维膜。
- 根据权利要求8-14中任一项所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述吸水纸包括厚滤纸、玻璃纤维或滤血膜中的一种，优选厚滤纸。
- 根据权利要求8或9所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述层析反应膜上设置有检测线和质控线；所述检测线包括IgA检测线、IgG检测线、IgM检测线中的至少一条，其中，所述IgA检测线含有 抗人IgA；所述IgG检测线含有抗人IgG；所述IgM检测线含有抗人IgM；并且所述质控线含有羊抗兔IgG抗体。
- 根据权利要求8-16中任一项所述的新型冠状病毒中和抗体效价预测试剂盒，其特征在于，所述试剂盒还包括紫外照射装置。
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| CN113267634B (zh) * | 2021-07-20 | 2021-10-08 | 南京申基医药科技有限公司 | 一种联合检测IgG、IgM和中和抗体的试纸条及其制备方法、试剂盒 |
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