CN112526139A - Indirect ELISA detection method for representing plasma neutralizing antibody activity of COVID-19 rehabilitation patient - Google Patents

Indirect ELISA detection method for representing plasma neutralizing antibody activity of COVID-19 rehabilitation patient Download PDF

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CN112526139A
CN112526139A CN202011241648.XA CN202011241648A CN112526139A CN 112526139 A CN112526139 A CN 112526139A CN 202011241648 A CN202011241648 A CN 202011241648A CN 112526139 A CN112526139 A CN 112526139A
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antigen
plasma
covid
indirect elisa
neutralizing antibody
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孔庆明
吕杭军
袁亚杰
陆绍红
高孟
卓洵辉
金子
丁豪杰
丁建祖
楼涤
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Hangzhou Medical College
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Hangzhou Medical College
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Abstract

The invention discloses an indirect ELISA detection method for detecting the activity of a plasma neutralizing antibody of a patient recovering COVID-19, which comprises the following steps: (1) the enzyme label plate is coated by using the combination of the RBD antigen and the N antigen of the SARS-CoV-2 virus, and is sealed by using sealing liquid after being coated; (2) diluting blood plasma of a patient to be detected with COVID-19 rehabilitation, and adding the diluted blood plasma into the ELISA plate processed in the step (1) for incubation; (3) after washing the plate, adding a secondary antibody for incubation; (4) and (3) washing the plate after incubation, adding a color development liquid for color development, and detecting the absorbance value, wherein the higher the absorbance value is, the higher the activity of the plasma neutralizing antibody of the patient to be detected the COVID-19 rehabilitation is. The indirect ELISA detection method established by the application can be used for carrying out new crown IgG antibody detection and new crown epidemiological investigation of large-scale crowds, and can be used for preliminarily screening potential new crown plasma donors with high neutralizing antibody titer, so that the method is safer, more convenient and quicker than a neutralization experiment in a BSL3 laboratory.

Description

Indirect ELISA detection method for representing plasma neutralizing antibody activity of COVID-19 rehabilitation patient
Technical Field
The invention relates to the technical field of biological detection, in particular to an indirect ELISA detection method for representing the activity of a plasma neutralizing antibody of a COVID-19 rehabilitation patient.
Background
SARS-CoV-2 is a pandemic due to its rapid spread and high infectivity. Until now, there is still a lack of effective therapeutic measures in the clinic. Historical experience has demonstrated that Convalescent Plasma (CP) therapy, also known as passive immunotherapy, can be a treatment option in the absence of drugs and vaccines and has been successfully applied to both SARS-CoV in 2003 and MERS-CoV in 2012 for diseases caused by coronaviruses. CP contains specific Neutralizing Antibody (Neutralizing Antibody) of corresponding pathogenic microorganism, and can bind with antigen on microorganism surface, thereby preventing pathogenic microorganism from adhering to target cell receptor and invading cell.
SARS-CoV-2 is a single-stranded positive-strand RNA virus of the genus Beta of the family Coronaviridae, having a genome size of about 29.9kb, encoding 4 structural proteins spike glycoprotein (Spick, S), Membrane protein (M), Envelope protein (E) and nucleoprotein (N). The receptor binding domain RBD on the spike protein S mediates virus invasion into host cells by binding with Angiotensin-converting enzyme 2 (ACE 2) receptor, and is the core antigen that stimulates the body to produce neutralizing antibodies.
CP donor information, active ingredient and quality control, and recipient indication are key factors in determining the efficacy of a treatment. Prior to infusion, the level of neutralizing antibodies in CP against SARS-CoV-2 is especially important. At present, virus neutralization experiments are the only effective and accurate method for evaluating the titer of plasma neutralizing antibodies, but the method uses true viruses and has certain danger. In addition, the neutralization experiment needs to be operated in a BSL-3 level biological safety laboratory, the requirements on equipment and operators are high, and most units cannot achieve the neutralization experiment. Although studies report that the pseudovirus neutralization experiment can avoid the risk of live virus operation, the pseudovirus neutralization experiment needs a series of complicated experimental operations such as cell culture, preparation of an ACE2 overexpression cell line, cell transfection and the like, and is long in time consumption. When detecting clinical plasma neutralizing antibodies, large-scale and rapid detection is difficult to realize. The indirect ELISA has the advantages of rapidness, simplicity and high sensitivity, and is suitable for large-scale screening of basic units and hospitals. In view of the deficiencies of the neutralization assay, we attempted to establish an indirect ELISA method for the characterization of neutralizing antibodies against SARS-CoV-2 in plasma.
The invention patent application with the publication number of CN111337689A discloses a novel coronavirus detection kit, which comprises a bottom plate, a colloidal gold pad, an NC membrane, a water absorption pad and a sample pad, wherein the colloidal gold pad is fixed on the bottom plate and coated with an antigen, the NC membrane is provided with a quality control line and a detection line, the water absorption pad and the sample pad are fixed at the detection line end of the NC membrane, the water absorption pad is fixed at the quality control line end, the novel coronavirus detection kit is characterized in that the antigen coated on the colloidal gold pad is a mixed antigen protein formed by mixing a colloidal gold-labeled N protein and a colloidal gold-labeled S-RBD protein, and the detection line arranged on the NC membrane is a mouse anti-human IgM antibody and mouse anti-human IgG antibody detection line. However, the prior art kit is used for detecting whether the novel coronavirus is infected, is a qualitative detection method, cannot be used for representing the plasma neutralizing antibody activity of the COVID-19 rehabilitation patients, and cannot realize quantitative detection of the IgG antibody level in the CP.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an indirect ELISA detection method for representing the plasma neutralizing antibody activity of a COVID-19 rehabilitation patient.
An indirect ELISA detection method for characterizing the plasma neutralizing antibody activity of a COVID-19 convalescent patient, comprising the following steps:
(1) the enzyme label plate is coated by using the combination of the RBD antigen and the N antigen of the SARS-CoV-2 virus, and is sealed by using sealing liquid after being coated;
(2) diluting blood plasma of a patient to be detected with COVID-19 rehabilitation, and adding the diluted blood plasma into the ELISA plate processed in the step (1) for incubation;
(3) after washing the plate, adding a secondary antibody for incubation;
(4) and (3) washing the plate after incubation, adding a color development liquid for color development, and detecting the absorbance value, wherein the higher the absorbance value is, the higher the activity of the plasma neutralizing antibody of the patient to be detected the COVID-19 rehabilitation is.
Preferably, the combination ratio of the RBD antigen and the N antigen in the step (1) is 1: 1/7-7. More preferably, the combination ratio of the RBD antigen and the N antigen in the step (1) is 1: 3-5.
Preferably, in step (2), the plasma of the convalescent patient to be tested for COVID-19 is first inactivated and then diluted. More preferably, the inactivation is carried out by inactivating the plasma of the patient to be tested for COVID-19 recovery in a 56 ℃ water bath for 30 min. More preferably, the dilution is performed by initial dilution 1: 100 with blocking solution and then 2-fold gradient dilution for 8 gradients.
The invention also provides a kit for indirect ELISA detection for representing the activity of the plasma neutralizing antibody of the COVID-19 rehabilitation patient, which comprises the RBD antigen and the N antigen of SARS-CoV-2 virus. Preferably, the combination ratio of the RBD antigen and the N antigen in the kit is 1: 1/7-7. More preferably, the combination ratio of the RBD antigen and the N antigen in the kit is 1: 3-5.
The invention also provides application of the kit in the fields of new crown IgG antibody detection and new crown epidemiological investigation of large-scale population, preliminary evaluation of new crown convalescent plasma neutralizing antibody titer and the like.
The invention discloses an indirect ELISA detection method for representing the plasma neutralizing antibody activity of a COVID-19 rehabilitation patient, which takes recombinant RBD and N protein as the sensitivity and specificity of detection antigenPreferably, the sensitivity is 94.7 percent and 100 percent respectively, and the specificity is 96.2 percent and 88.5 percent respectively; s1 is poor in sensitivity (86.0%) and specificity (73.1%) and is not suitable for being used as a detection antigen; within batch repeatability (CV)<2%) and good batch-to-batch reproducibility (F: 4.578, p: 0.123, p)>0.05). IgG antibody levels against recombinant RBD correlate with virus neutralizing antibody titers (R)2＝0.2564，p<0.0001, pseudovirus; r20.2933, p 0.0017, euvirus), whereas IgG antibody levels against recombinant S1 and N antigens were not correlated with neutralizing antibody titers (R1)2＝0.0210，p＝0.2827；R20.0659, p 0.0539). Further mixing RBD and N antigen at a certain ratio (1: 3-1: 5) improves detection sensitivity (100%) and specificity (96.2%) compared with RBD and N antigen alone.
Potential novel corona plasma donors with high neutralizing antibody titers can be initially screened using the indirect ELISA assay established herein. The method is safer than the classical neutralization test, is free of virus operation, and is used for inactivating the blood plasma of patients with COVID-19. In addition, the operation is simple, the operation in a BSL-3 grade laboratory is not needed, and the cell culture and the like are not needed. The method can help clinical institutions realize large-scale rapid preliminary assessment of the anti-SARS-CoV-2 neutralizing antibody level in plasma.
The detection kit takes the N protein and the S-RBD protein as antigens, quantitatively detects the IgG antibody level in the CP, evaluates the plasma neutralizing antibody level of the new coronary rehabilitation patients through correlation analysis, has the advantages of high sensitivity, high safety, accurate quantification, time saving and labor saving, and is suitable for new coronary IgG antibody detection and new coronary epidemiological investigation of large-scale crowds and preliminary evaluation of the plasma neutralizing antibody titer of the new coronary rehabilitation patients.
Drawings
FIG. 1 is a graph showing the results of ELISA detection of IgG antibody OD values in plasma of normal human and SARS-CoV-2 convalescent patient, wherein graphs A-C are OD values of plasma of healthy human and plasma of new coronary convalescent patient, graph A is RBD, graph B is S1, and graph C is N; graphs D-F are t-tests of the healthy human plasma OD value and the new coronary rehabilitation patient plasma OD value corresponding to A-C, graph D is RBD, graph E is S1, and graph F is N.
FIG. 2 is a graph showing the results of detecting the anti-RBD, S1 and N antigen IgG antibody levels in the plasma of SARS-CoV-2 convalescent patient.
FIG. 3 is a graph showing the correlation between the anti-SARS-CoV-2 IgG antibody level and the neutralizing antibody titer, wherein graphs A to C are graphs showing the correlation between the IgG antibody level and the neutralizing antibody titer against pseudoviruses, graph A is RBD, graph B is S1, and graph C is N; panel D is a graph of an assay correlating IgG antibody levels to neutralizing antibody titers from a euvirus strain.
FIG. 4 is a graph showing the results of plate coating with different ratios of recombinant RBD and N antigen.
Detailed Description
Main experimental reagents and consumables:
polystyrene 96-well plate, 5% skim milk powder, PBST (1 XPBS + 0.05% tween 20), pH 9.6 carbonate buffer, goat anti-human IgG-HRP secondary antibody (Solarbio), TMB developing solution (Byunyan), 2% H2SO4SARS-CoV-2 convalescent patients and normal human plasma (Zhejiang province' S blood center, ethical examination batch No. 2020-IIT-18) HEK293-ACE2 cells, SARS-CoV-2-S pseudovirus, DMEM medium (Gibico), fetal bovine serum (Gibico), Luciferase detection kit (Biovision).
Example 1
Pseudovirus neutralization experiments:
HEK293-ACE2 cells were plated in 96 well cell culture plates at a density of 30,000 cells/well and cultured at 37 ℃ for 12 hours; 50 mu L of new crown Spike pseudovirus (provided by Beijing Yiqian Shenzhou Co., Ltd.) and 50 mu L of serially diluted plasma to be detected are incubated for 1 hour, and then 293T-ACE2 cells are infected, and after 12 hours, the culture medium is replaced by a fresh medium for continuous culture for 48 to 72 hours; the method is characterized in that a chemiluminescence method is adopted to detect the luminescence value RLU of Luciferase, the pseudovirus inhibition rate of the blood plasma to be detected is calculated according to the reading value of the RLU, and the neutralization effect of the blood plasma to be detected is evaluated (completed by Beijing Yinqiao Shenzhou science and technology Co., Ltd.).
Indirect ELISA detection of plasma anti-SARS-CoV-2 IgG antibodies:
coating detection antigens RBD, S1 and N protein (donated by Hangzhou medical college virus) with the concentration of 5 μ g/mL in a 96-well plate, 100 μ L/well, and standing overnight at 4 ℃; inactivating the blood plasma to be detected in a 56 ℃ water bath for 30 min; then using 5% skimmed milk powder to carry out 1: 100 initial dilution, 2 times serial dilution, and 8 gradients in total; PBST plate washing 5 times, 1 min/time, patting dry; adding 250 mu L of 5% skimmed milk powder into each hole, and sealing in water bath at 37 ℃ for 2 hours; spin-drying the sealing liquid in the holes, washing the plates for 5 times (1 min/time) by PBST, and drying by beating; adding 100 mu L of blood plasma diluent to be detected into each hole, and sealing in water bath at 37 ℃ for 1 hour; spin-drying the liquid in the holes, washing the plate for 5 times (1 min/time) by PBST, and drying by beating; adding goat anti-human IgG-HRP secondary antibody diluted by 1: 5000, 100 mu L/hole, and incubating in water bath at 37 ℃ for 30 min; spin-drying the liquid in the holes, washing the plate for 5 times (1 min/time) by PBST, and drying by beating; adding 50 mu LTMB developing solution into each hole, and incubating in water bath at 37 ℃ for 10-15 min; the absorbance value per well was determined at OD450 nm.
Cut-off value: in indirect ELISA detection, the cut-off value of a positive result is determined as the average OD value of negative plasma antibodies plus 0.15; when a new crown plasma donor with neutralizing antibody activity is screened, the plasma IgG antibody titer is more than or equal to 200 and is judged to be positive by referring to FDA standard, and a pseudovirus neutralizing antibody IC50More than or equal to 30 is positive.
And (3) repeatability verification: coating 100 mu L/hole recombinant N antigen on the same enzyme label plate, setting 4 repeats, randomly taking a positive serum to dilute 200 times, detecting the OD value of each hole, and analyzing the repeatability in batches. Three ELISA plates were used to coat the same amount of N antigen, 100. mu.L/well, each ELISA plate was repeated 2 times, OD was measured, and the batch-to-batch reproducibility was analyzed.
RBD in combination with N antigen: a single RBD plate has higher specificity and lower sensitivity than N antigen, so that RBD and N antigen are coated in different ratios of 1: 1, 1: 3, 1: 5, 1: 7, 3: 1, 5: 1 and 7: 1, a positive sample is randomly selected, diluted in 1: 200, 1: 800 and 1: 3200, and the OD value is used for plotting, and the optimal ratio is analyzed.
Statistical treatment: the data were statistically analyzed and plotted using IBM SPSS 26.0 software and Graphad prism 8.0. Respectively and randomly selecting 10 healthy human plasma and positive sample plasma as line graphs; scattergrams of IgG antibody levels in plasma of 57 recovered SARS-CoV-2 patients were plotted. Calculating the sensitivity, specificity and repeatability (batch-to-batch and batch-to-batch) of indirect ELISA for detecting different antigens; and analyzing the consistency and correlation of the IgG antibody titer against the recombinant RBD and the N antigen in the plasma of the SARS-CoV-2 convalescent patient and the neutralizing antibody titer.
As a result:
sensitivity and specificity: the sensitivity of RBD, S1 and N antigens as detection antigens is 94.7% (54/57), 86.0% (49/57) and 100% (57/57), and the specificity is 96.2% (25/26), 73.1% (19/26) and 88.5% (23/26); the specificity and sensitivity of the RBD antigen and the N antigen are higher than those of the S1 antigen, the cross-reactivity of the RBD antigen is the weakest, and the cross-reactivity of the S1 antigen is the strongest (figure 1). The anti-N antigen IgG antibody levels were higher than those of anti-S1 and RBD antigens (fig. 2).
And (3) repeatability verification: the repeatability in batches is good, and CV is less than 2%; good reproducibility between batches (F-4.578, p-0.123, p > 0.05).
And (3) consistency analysis: the detection rate of IgG antibodies against recombinant RBD, S1 and N antigen by indirect ELISA was 0.048 (p 0.452, p 0.05), 0.026 (p 0.788, p 0.05), 0.00 (p 1.0, p 0.05) according to the results of the analysis of the consistency with the results of the pseudovirus neutralization test using the statistical method of the four-lattice Charcot square test; the results of ELISA detection by recombinant RBD, S1 and N antigen are inconsistent with the results of the pseudovirus neutralization experiment.
And (3) correlation analysis: performing correlation analysis on the anti-recombinant RBD, S1 and N antigen IgG antibody levels and the titer of the pseudovirus neutralizing antibodies in the plasma of 57 positive COVID-19 convalescent patients; plasma IgG antibody titers against recombinant RBD were correlated with neutralizing antibody titers (R)2＝0.2564，p<0.0001, pseudovirus; r20.2933, p 0.0017, euvirus); plasma IgG antibody titers against recombinant S1 and N antigens were not related to neutralizing antibody titers (R2＝0.0210，p＝0.2827；R20.0659, p 0.0539) (fig. 3).
RBD in combination with N antigen: the RBD and N are wrapped in different proportions, preferably in a ratio of 1: 3 to 1: 5 (FIG. 4).

Claims (10)

1. An indirect ELISA method for characterizing the plasma neutralizing antibody activity of a COVID-19 convalescent patient, comprising the steps of:
(1) the enzyme label plate is coated by using the combination of the RBD antigen and the N antigen of the SARS-CoV-2 virus, and is sealed by using sealing liquid after being coated;
(2) diluting blood plasma of a patient to be detected with COVID-19 rehabilitation, and adding the diluted blood plasma into the ELISA plate processed in the step (1) for incubation;
(3) after washing the plate, adding a secondary antibody for incubation;
(4) and (3) washing the plate after incubation, adding a color development liquid for color development, and detecting the absorbance value, wherein the higher the absorbance value is, the higher the activity of the plasma neutralizing antibody of the patient to be detected the COVID-19 rehabilitation is.
2. The indirect ELISA detection method of claim 1 wherein the combination ratio of RBD antigen to N antigen in step (1) is 1: 1/7-7.
3. The indirect ELISA detection method of claim 2 wherein the combination ratio of RBD antigen to N antigen in step (1) is 1: 3 to 5.
4. The indirect ELISA assay of claim 1 wherein in step (2) the plasma of the convalescent patient to be assayed for COVID-19 is inactivated and then diluted.
5. The indirect ELISA assay of claim 4 wherein the inactivation is performed by subjecting plasma from a patient whose COVID-19 recovery is to be assayed to inactivation in a 56 ℃ water bath for 30 min.
6. The indirect ELISA assay of claim 4 wherein the dilution is performed by a 1: 100 initial dilution with blocking solution followed by a 2-fold gradient of 8 gradients.
7. A kit for indirect ELISA detection for characterizing the plasma neutralizing antibody activity of a COVID-19 convalescent patient is characterized by comprising an RBD antigen and an N antigen of SARS-CoV-2 virus.
8. The kit of claim 7, wherein the RBD antigen and N antigen are combined in a ratio of 1: 1/7-7.
9. The kit of claim 8, wherein the RBD antigen and N antigen are combined in a ratio of 1: 3 to 5.
10. Use of the kit according to claims 7 to 9 for screening plasma for neutralizing antibody activity against neocorona plasma.
CN202011241648.XA 2020-11-09 2020-11-09 Indirect ELISA detection method for representing plasma neutralizing antibody activity of COVID-19 rehabilitation patient Pending CN112526139A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115219726A (en) * 2022-09-06 2022-10-21 昭衍（苏州）新药研究中心有限公司 Method for detecting serum neutralizing antibody titer by SARS-CoV-2 pseudovirus

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CN111337689A (en) * 2020-04-03 2020-06-26 山西医科大学 Novel coronavirus detection kit
CN111474348A (en) * 2020-04-20 2020-07-31 广州市丰华生物工程有限公司 Novel detection kit and detection method for coronavirus
CN111505277A (en) * 2020-03-10 2020-08-07 四川省人民医院 2019 novel coronavirus IgG antibody detection kit
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CN111505277A (en) * 2020-03-10 2020-08-07 四川省人民医院 2019 novel coronavirus IgG antibody detection kit
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115219726A (en) * 2022-09-06 2022-10-21 昭衍（苏州）新药研究中心有限公司 Method for detecting serum neutralizing antibody titer by SARS-CoV-2 pseudovirus
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