CN113834943A - Test strip for realizing synergistic detection of neocorolla total antibody and neutralizing antibody and preparation method thereof - Google Patents
Test strip for realizing synergistic detection of neocorolla total antibody and neutralizing antibody and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a test strip for synergistically detecting a neocorona total antibody and a neutralizing antibody, which comprises the following components in part by weight: sample pad, nitrocellulose membrane, absorbent pad and end liner, sample pad, nitrocellulose membrane and absorbent pad overlap joint in proper order paste on the end liner, the last sample pad end of nitrocellulose membrane to absorbent pad end have set gradually release line, T2 detection line, T1 competition line and C matter accuse line, the parcel tracer particle-marker conjugate on the release line, T1 competition package is ACE2 protein, the package of T2 detection line is by SARS-CoV-2 antigen. According to the invention, through the detection of the total anti-cooperative neutralizing antibody, the detection pertinence and specificity are improved under the condition that the original detection sensitivity of the test strip is not reduced, and the HOOK problem caused by overhigh concentration of a substance to be detected is solved on the premise of ensuring the detection accuracy of the kit.
Description
Technical Field
The invention relates to the field of medical inspection, in particular to a method for realizing cooperative detection of a new crown total antibody and a neutralizing antibody.
Background
2019 the novel coronavirus (SARS-CoV-2) is a new strain of coronavirus found in human in 2019. The symptoms of the virus are fever, hypodynamia, dry cough and gradual dyspnea, and severe patients show acute respiratory distress syndrome, septic shock, metabolic acidosis and blood coagulation dysfunction which are difficult to correct. Because the novel coronavirus is confirmed to have a phenomenon of human transmission and has infectivity in a latent period, and a specific treatment method for diseases caused by the novel coronavirus is not available at present, the identification of virus-infected patients and the timely isolation and treatment are particularly urgent and important.
At present, the novel coronavirus detection reagents on the market mainly comprise two types: one type is nucleic acid detection reagent, which comprises detection means such as a gene sequencing method (DNA sequencing), reverse transcription-polymerase chain reaction (RT-PCR) and the like; the other is an antibody detection reagent, and mainly comprises methods such as chemiluminescence, immunochromatography and the like. The nucleic acid detection reagent has the characteristics of strong specificity, high detection sensitivity and the like, but the average detection time is 2-3h due to the steps of sample treatment, nucleic acid extraction, PCR reaction and the like, the time consumption is long, specialized technical personnel are required to be matched with instruments and equipment, reagent consumables, and the requirement on the use environment is high. The chemiluminescence detection time of the magnetic particles generally needs 30-60min, the requirement on the use environment is high, and the cost of matched detection instruments and reagent consumables is high, so that the magnetic particles are difficult to be used on a large scale in primary hospitals. The average detection time of immunochromatography is about 15min, and the detection kit is small in size and convenient to carry, can be used in any medical environment as a rapid screening means, and can play a great screening advantage in the period of large-area personnel flow and aggregation.
The products related to the immunochromatography mainly comprise: the test strip for detecting total coronavirus and the test strip for detecting neutralizing antibody, wherein the total anti-immunochromatographic test strip is generally applied to a double-antigen sandwich method, and the detection result is mainly used as a supplementary detection index for a novel coronavirus nucleic acid detection negative suspected case or used in cooperation with nucleic acid detection in the diagnosis of the suspected case; the test strip mainly adopts a competitive inhibition method, and the new coronavirus neutralizing antibody has important significance for determining infection rate, group immunity and prediction along with the continuous prevalence of the novel coronavirus and the wide development of vaccine research. The detection object of the new crown total antibody comprises all antibodies which can be aggregated with the new crown S protein or a Receptor Binding Domain (RBD) positioned at S1, namely the sum of the antibodies comprising the neutralizing antibody and the rest of non-neutralizing antibodies, has high sensitivity, but has low pertinence and specificity, so that the detection result cannot be traced, and the accuracy of the result is difficult to guarantee. Meanwhile, because the content of the new crown total antibody in the human body is often higher, in order to ensure that the sample still has reliable detection sensitivity when the content of the total antibody is lower, the problem of HOOK is easily caused when the content of the total antibody of the sample is high, and then a false negative value report result is caused.
Disclosure of Invention
According to the invention, by coordinating the detection principle of the neutralizing antibody on the basis of total antibody detection, the detection pertinence and specificity are obviously improved under the condition of not reducing the original sensitivity of the total antibody detection, the HOOK effect when the concentration of a sample to be detected is too high is avoided, and the detection accuracy is ensured; meanwhile, the invention avoids the noise factor fluctuation of the drying and dissolving release of the label compound coated on the glass fiber by the way that the SARS-CoV-2 antigen and/or RBD antigen combined with the tracer label is coated on the NC membrane release line in a concentration way, and can ensure the uniform release of the label after the sample is added by matching with the special stationary liquid components, effectively reduce the residue of the label compound on the NC membrane and obviously improve the detection precision of the reagent.
In order to achieve the purpose, the invention adopts the following technical means: a test strip for detecting the synergy of a corona total antibody and a neutralizing antibody is characterized by comprising: sample pad, nitrocellulose membrane, absorbent pad and end liner, sample pad, nitrocellulose membrane and absorbent pad overlap joint paste in proper order on the end liner, the last sample pad end of nitrocellulose membrane to absorbent pad end have set gradually release line, T2 detection line, T1 competition line and C matter accuse line, the trace particle-marker conjugate of peridium on the release line, at least one in T2 detection line peridium SARS-CoV-2 antigen or RBD antigen T1 competition line peridium ACE2 protein.
Preferably, in the tracer particle-marker conjugate, the tracer particle is at least one selected from fluorescent microspheres, colloidal gold, latex microspheres, magnetic particles or nanocarbon, preferably fluorescent microspheres, and the marker is a combination of at least one of SARS-CoV-2 antigen or RBD antigen and IgY antibody.
Preferably, the amount of coating of the tracer particle-label conjugate on the release line is between 0.5 and 1.0ul/cm, preferably between 0.7 and 1.0 ul/cm.
Preferably, the coating amount of the ACE2 protein on the T1 competitive line is 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm; the coating amount of the SARS-CoV-2 antigen on the T2 detection line is 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm.
A preparation method of a test strip for detecting the synergy of a neocorona total antibody and a neutralizing antibody is characterized by comprising the following steps:
(1) treatment of nitrocellulose membrane:
coating a release line, a T2 detection line, a T1 competition line and a C quality control line on a nitrocellulose membrane, and then drying:
a. coating of release thread:
coating the tracer particle-marker conjugate on the nitrocellulose membrane at a position 1-2mm close to the end of the sample pad;
preparing a T2 detection line:
coating at least one of RBD antigen solution or SARS-CoV-2 antigen solution at a position 4-6mm away from the release line and far away from the sample pad end;
c, preparing a T1 competitive line:
coating an ACE2 protein solution at a position 1-3mm away from a T2 detection line and far away from the end of a sample pad;
d.C position of the control line:
coating the IgY antibody solution at a position 1-3mm away from a T1 competitive line and far away from the end of a sample pad;
(2) assembling:
sequentially adhering a sample pad, a nitrocellulose membrane and a water absorption pad on a bottom lining, and cutting into test strips with the width of 3-5 mm.
Preferably, in the preparation of the release line in step (1), the marker is a combination of at least one of RBD antigen or SARS-CoV-2 antigen and IgY antibody, and the tracer particle is at least one selected from fluorescent microsphere, colloidal gold, latex microsphere, magnetic microparticle or nanocarbon, preferably fluorescent microsphere;
the preparation method of the tracer particle-marker conjugate comprises the following steps:
a, uniformly mixing 300-500ug of tracer particles and 60-100ug of at least one of RBD antigen or SARS-CoV-2 antigen for 30-40min, adding 5-10% BSA, stirring for 10-30min, centrifuging at 12000-14000rpm for 20-30min, removing supernatant, and dissolving precipitate with fluorescent labeling redissolution;
b. uniformly stirring and mixing 300-500ug of tracer particles and 60-100ug of IgY antibody for 30-40min, adding 5-10% BSA, stirring for 10-30min, centrifuging at 12000-14000rpm for 20-30min, removing the supernatant, and dissolving the precipitate with a fluorescent labeling redissolution;
c. and mixing the RBD antigen or SARS-CoV-2 antigen marked microsphere and IgY antibody marked microsphere according to the ratio of 1: (0.2-0.5), and then suspending in a fluorescence labeling complex solution to obtain the tracer particle-marker conjugate.
Preferably, in step (1), the tracer particle-label conjugate is coated at the release line in an amount of 0.5-1ul/cm, preferably 0.7-1.0 ul/cm; the ACE2 protein solution is coated at the T1 competitive line in an amount of 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm; the SARS-CoV-2 antigen solution or RBD antigen solution is coated at the T2 detection line in the amount of 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm; the IgY antibody solution is coated at the C quality control line in an amount of 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm. .
Preferably, the concentration of the tracer particle-label conjugate is 0.5-1.0mg/ml, preferably 0.8-1.0 mg/ml; the concentration of the ACE2 protein solution is 0.5-1.0mg/ml, preferably 0.8-1.0 mg/ml; the concentration of the SARS-CoV-2 antigen solution or RBD antigen solution is 0.5-1.0mg/ml, preferably 0.8-1.0mg/ml.
Preferably, the components of the fluorescent labeling complex solution comprise: 10-50mmol/L buffer solution, 5-10g/L surfactant and 40-50g/L protective agent; the buffer is selected from at least one of Tris, borate, phosphate or PBS buffer; the surfactant is selected from at least one of Tween-20, Tween-80 or Triton-X100; the protective agent is at least one of sucrose or trehalose.
Preferably, the fluorescent labeling complex solution also comprises 10-30g/L, PEG 1-10g/L of protein and 1-5g/L of preservative, wherein the protein is at least one selected from casein or BSA; the preservative is selected from at least one of sodium azide or proclin-300.
The invention has the beneficial effects that:
(1) according to the invention, through the detection of the total anti-cooperative neutralizing antibody, the detection pertinence and specificity are improved under the condition that the original detection sensitivity of the test strip is not reduced, and the HOOK problem caused by overhigh concentration of a substance to be detected is solved on the premise of ensuring the detection accuracy of the kit.
(2) The test strip of the invention adopts SARS-CoV-2 antigen or/and RBD antigen marked by trace particles to coat the release line of a nitrocellulose membrane (NC membrane) in a concentration mode, avoids the noise factor fluctuation released by drying and dissolving fixed glass fiber, and the marker is fixed on the NC membrane and adopts proper fluorescence-marked redissolution components, thereby reducing the residue of the marker on the NC membrane, simultaneously ensuring the uniform release of the marker after the sample is added, and effectively improving the precision of the reagent.
Drawings
FIG. 1 is a diagram showing the structure of a test strip prepared in example 1 of the present invention (1-release line, 2-T2 detection line; 3-T1 competition line, 4-quality control line C, 5-sample pad, 6-nitrocellulose membrane, 7-absorbent pad, 8-backing)
FIG. 2 is a graph showing the linear relationship between the concentration of the calibrator and the signal value of the group A test strip T2/T1 in example 5;
FIG. 3 is a graph showing the linear relationship between the concentration of the calibrator and the signal value of the test strip T2/T1 in group B in example 5 of the present invention;
FIG. 4 is a graph showing the linear relationship between the concentration of the calibrator and the T/C signal value detected by the test strip group C in example 5 of the present invention;
FIG. 5 is a linear relationship graph of the concentration of the calibrator and the T/C signal value detected by the group D test strips in embodiment 5 of the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are included to more clearly and clearly illustrate the technical solutions of the present invention by way of illustration. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit the invention in any way.
Example 1 preparation of fluorescent test strip 1
1. Preparation of fluorescent labeling complex solution
The preparation is carried out according to the following formula, fully stirred and uniformly mixed, and stored at 2-8 ℃.
2. Preparation of tracer particle-label conjugates
(1) Adding 60 mu g of RBD antigen into 300 mu g of fluorescent microspheres, stirring at room temperature for reaction for 40min, adding 5% BSA, and sealing and stirring for 20 min;
(2)14000rpm, centrifuging for 20min, discarding the supernatant, recovering the volume of the precipitate with a fluorescent labeling compound solution, and storing at 4 ℃;
(3) adding 60 mu g of chicken IgY into 300 mu g of fluorescent microspheres, stirring and reacting for 40min at room temperature, adding 5% BSA, and sealing and stirring for 20 min;
(4)14000rpm, centrifuging for 20min, discarding the supernatant, recovering the volume of the precipitate with a fluorescent labeling compound solution, and storing at 4 ℃;
(5) and (3) mixing the RBD antigen marked microspheres and the chicken IgY marked microspheres according to the ratio of 5: mixing at a ratio of 1, and then suspending the mixture in a fluorescent labeling complex solution to obtain the tracer particle-marker conjugate.
3. Assembly of test strips
(1) Treatment of nitrocellulose membranes
Coating a release line, a T2 detection line, a T1 competition line and a C quality control line on the nitrocellulose membrane, and then drying:
a. position of release line:
coating the tracer particle-marker conjugate on the nitrocellulose membrane at a position 2mm close to the end of the sample pad in an amount of 0.7 ul/cm;
position of T2 detection line:
coating the RBD antigen solution with the concentration of 0.8mg/ml on the nitrocellulose membrane at a position 6mm away from the release line and far away from the end of the sample pad in an amount of 0.7 ul/cm;
position of T1 contention line:
coating an ACE2 protein solution with the concentration of 0.8mg/ml on a nitrocellulose membrane at a position 3mm away from a T2 detection line and far away from the end of a sample pad in an amount of 0.7 ul/cm;
d.C position of the control line:
an IgY antibody solution with a concentration of 0.8mg/ml was coated on the nitrocellulose membrane at a distance of 3mm from the T1 competition line and away from the end of the sample pad in an amount of 0.7 ul/cm.
The plastic bottom lining, the sample pad and the water absorption pad are general materials in the field, the sample pad, the nitrocellulose membrane and the water absorption pad are sequentially and tightly overlapped on the plastic bottom lining, the adhered intermediate is cut into test strips with preset width by a cutting machine, and only the test strip card box is assembled, so that the finished test strip (the test strip structure is shown in figure 1) can be obtained.
Example 2 preparation of fluorescent test strip 2
The difference between the method for preparing the test strip in this example and that in example 1 is that: the preparation process of the tracing particle-marker conjugate adopts SARS-CoV-2 antigen marked fluorescent microsphere, and the T2 detection line is coated with SARS-CoV-2 antigen solution.
Example 3 preparation of fluorescent test strip 3
The difference between the method for preparing the test strip in this example and that in example 1 is that: the contention line T1 is missing.
Example 4 preparation of fluorescent test strip 4
The difference between the method for preparing the test strip in this example and that in example 1 is that: detection line T2 is absent.
Example 5 test strip Performance validation
In order to verify the detection performance of the test strip of embodiment 1 of the present invention, 4 groups of test strips are provided for verification:
group A: the test strip prepared in embodiment 1 of the invention;
group B: the test strip prepared in embodiment 2 of the invention;
group C: the test strip prepared in embodiment 3 of the invention;
group D: the test strip prepared in embodiment 4 of the invention.
(1) Detection result of novel crown neutralizing antibody standard substance
The four groups of test strips are used for respectively carrying out accuracy test on the calibrator, 2 times of the test are repeated, signals are read by an immune quantitative analyzer (Q8 pro, Yuanhui-Gi Biotechnology Co., Ltd. in Chongqing), and the relative deviation between the measured mean value and the target value is calculated for carrying out accuracy verification. The results are shown in the following table:
TABLE 1 test results for standards
From the above experimental results, the relative deviations of the test value 1 and the target value 1 of the four test strips are respectively 0.46%, -0.97%, -4.38%, and-1.51%, and the relative deviations of the test value 2 and the target value 2 are respectively-0.46%, -0.81%, -18.24%, and-3.95%. The test strip (group A) prepared in the embodiment 1 of the invention has better detection accuracy than the test strips of the groups B, C and D.
(2) Determination of precision
Preparing low-value, medium-value and high-value calibrator solutions, repeatedly measuring the calibrator solutions with different concentrations for 10 times by using three groups of test strips, respectively calculating a measurement mean value and a standard deviation, calculating a variation coefficient, and performing repeated investigation, wherein the detection results are shown in the following table:
TABLE 2 test paper strip precision
From the above experimental results, the coefficients of variation of the four test strips in the low value sample detection are 0.95%, 1.35%, 2.99% and 5.96%, the coefficients of variation in the median sample detection are 1.08%, 1.53%, 2.71% and 3.98%, and the coefficients of variation in the high value sample detection are 0.99%, 1.75%, 8.02% and 4.07%, respectively. The experimental results show that the test strips prepared in example 1 (group a) and example 2 (group B) of the present invention have better precision in detecting low-value, medium-value and high-value samples than the test strips in groups C and D.
(3) Clinical evaluation
Selecting 53 positive samples for completing injection of the new corona vaccine and 119 negative samples for not inoculating the new corona vaccine, and detecting the positive samples and the negative samples by using the three groups of test strips respectively, wherein the detection results are shown in the following table:
TABLE 3 clinical Properties of test strips
Injecting: the positive sample is a sample for completing vaccination; the negative sample is a sample without vaccination; clinical sensitivity (number of positive tests/number of positive samples)%; clinical specificity ═ (number of negative tests/number of negative samples)%; the total coincidence rate is (number of correct detections/total samples)%.
From the above experimental results, the clinical sensitivities of the four test strips are respectively 98.11%, 96.22%, 54.71% and 67.92%, the clinical specificities are respectively 100%, 98.32%, 96.64% and 92.44%, and the total coincidence rates are respectively 99.42%, 97.67%, 83.72% and 84.88%. The experimental results show that the clinical compliance of the test strip prepared in example 1 (group a) and the test strip prepared in example 2 (group B) is significantly better than that of the test strips in groups C and D.
(4) HOOK RESISTANCE VERIFICATION
Preparing a calibrator solution with ultrahigh concentration (the concentration is 10000ng/ml), diluting with ultrapure water in proportion to prepare various concentration gradient solutions, testing the calibrator solution by using three groups of test strips, respectively repeating the determination for three times for each sample, reading signals by an immune quantitative analyzer, and obtaining the detection results shown in the following table:
TABLE 4 HOOK RESISTANCE TEST
The detection results of the four groups of test strips are compared and analyzed with the concentration value of a calibrator (as shown in the attached figures 2-5), the experimental results show that the detection defects of the test strips of the group C are based on the sandwich principle of a detection line, the high linear end of the test strips is particularly above 2000ng/ml, HOOK causes false negative, the detection defects of the test strips of the group D are based on the fact that the sensitivity of the low end of a competition method is not distinguished obviously particularly near the reference range of 300ng/ml, the test strips of the group A and the test strips of the group B are combined with the advantages of the sandwich method and the competition method at the same time, the test strips of the group A and the test strips of the group B have good detection accuracy at the low linear end and the high linear end, and have good HOOK resistance in the whole linear range.
According to the test results, the test strip improves the detection pertinence and specificity through the detection of the total anti-cooperative neutralizing antibody under the condition of not reducing the original detection sensitivity of the test strip, and effectively overcomes the problem of HOOK when the concentration of the substance to be detected is too high on the premise of ensuring the detection accuracy of the kit.
Example 6 Effect of Release line set on NC films on detection Performance
In order to verify that the technical scheme that a release line is arranged on the NC membrane of the test strip to replace a conventional combination pad can effectively improve the detection precision of the test strip, the test strip is provided with the following two groups of test strips:
group A: the test strip of embodiment 1 of the invention;
group B: the preparation method of the test strip is different from that of the embodiment 1 in that: the release line is absent, and the test strip is provided with a binding pad on which the tracer particle-marker conjugate is coated.
Preparing low-value, medium-value and high-value calibrator solutions, repeatedly measuring the calibrator solutions with different concentrations for 10 times by using three groups of test strips, respectively calculating a measurement mean value and a standard deviation, calculating a variation coefficient, and performing repeated investigation, wherein the detection results are shown in the following table:
TABLE 5 precision verification
From the above experimental results, the coefficients of variation of the two test strips in the low value sample detection are 0.88% and 7.48%, the coefficients of variation in the median sample detection are 0.65% and 6.03%, and the coefficients of variation in the high value sample detection are 0.71% and 6.90%. The experimental results show that the test strip (group A) prepared in the embodiment 1 of the invention has better precision than the test strips in the group B when detecting low-value, medium-value and high-value samples.
Example 7 Effect of concentration of labeled microspheres on Release line on assay Performance
In order to verify the influence of the concentration of the marked microspheres on the release line on the detection linearity of the test strip, the following 5 groups of experiments were set up:
group A: the preparation method of the test strip is different from that of the embodiment 1 in that: the concentration of the marked microspheres on the release line is 0.1 mg/ml;
group B: the preparation method of the test strip is different from that of the embodiment 1 in that: the concentration of the marked microspheres on the release line is 0.5 mg/ml;
group C: the preparation method of the test strip is different from that of the embodiment 1 in that: the concentration of the marked microspheres on the release line is 1 mg/ml;
group D: the preparation method of the test strip is different from that of the embodiment 1 in that: the concentration of the marked microspheres on the release line is 1.5 mg/ml;
group E: the preparation method of the test strip is different from that of the embodiment 1 in that: the concentration of the marked microspheres on the release line is 5 mg/ml;
preparing a median calibrator solution, repeatedly measuring the median calibrator solution for 10 times by using five groups of test strips, respectively calculating a measurement mean value and a standard deviation, calculating a variation coefficient, and carrying out repeated investigation, wherein the detection results are shown in the following table:
TABLE 6 verification of precision
| Sample(s) | A | B | C | D | E |
| Release line marking microsphere concentration | 0.1mg/ml | 0.5mg/ml | 1.0mg/ml | 1.5mg/ml | 5mg/ml |
| Precision (CV) | 5.26％ | 0.85％ | 0.65％ | 1.58％ | 4.21％ |
The test result shows that when the concentration range of the marked microspheres on the release line is 0.5-1.0mg/ml, the detection precision of the test strip is good, the CV values are all less than 2%, and when the concentration is 1.0mg/ml, the precision is best and can reach 0.65%.
According to the experimental results, the test strip adopts the technical scheme that SARS-CoV-2 antigen or RBD antigen marked by the tracer particles is coated on a nitrocellulose membrane (NC membrane) release line in a concentration mode to replace the conventional tracer particle marker coated on a bonding pad, so that on one hand, the noise factor fluctuation caused by the drying and dissolution release of the test strip fixed on a glass cellulose membrane is avoided. On the other hand, the NC membrane stationary liquid component with special components is adopted to help the tracer particle markers to coat on the release line, so that the residue of the markers on the NC membrane in the chromatography process is reduced, the markers are uniformly released after the sample is added, and the detection precision of the reagent is effectively improved.
Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments without departing from the principles and spirit of the present invention.
Claims (10)
1. A test strip for detecting the synergy of a corona total antibody and a neutralizing antibody is characterized by comprising: sample pad, nitrocellulose membrane, absorbent pad and end liner, sample pad, nitrocellulose membrane and absorbent pad overlap joint paste in proper order on the end liner, the last sample pad end of nitrocellulose membrane to absorbent pad end have set gradually release line, T2 detection line, T1 competition line and C matter accuse line, the trace particle-marker conjugate of peridium on the release line, at least one in T2 detection line peridium SARS-CoV-2 antigen or RBD antigen T1 competition line peridium ACE2 protein.
2. The test strip for the cooperative detection of the corona total antibody and the neutralizing antibody according to claim 1, wherein in the tracer particle-label conjugate, the tracer particle is at least one selected from fluorescent microsphere, colloidal gold, latex microsphere, magnetic particle or nano carbon, preferably fluorescent microsphere, and the label is a combination of at least one selected from SARS-CoV-2 antigen or RBD antigen and IgY antibody.
3. The test strip for the synergistic detection of the novel crown-like total antibody and the neutralizing antibody according to claim 2, wherein the coating amount of the tracer particle-marker conjugate on the release line is 0.5 to 1.0ul/cm, preferably 0.7 to 1.0 ul/cm.
4. The test strip for the synergistic detection of the total antibodies and the neutralizing antibodies of the neocorolla as claimed in claim 3, wherein the coating amount of ACE2 protein on the competitive line of T1 is 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm; the coating amount of the SARS-CoV-2 antigen on the T2 detection line is 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm.
5. The method for preparing the test strip for the cooperative detection of the corona total antibody and the neutralizing antibody according to any one of claims 1 to 4, wherein the method comprises the following steps:
(1) treatment of nitrocellulose membrane:
coating a release line, a T2 detection line, a T1 competition line and a C quality control line on a nitrocellulose membrane, and then drying:
a. coating of release thread:
coating the tracer particle-marker conjugate on the nitrocellulose membrane at a position 1-2mm close to the end of the sample pad;
preparing a T2 detection line:
coating at least one of RBD antigen solution or SARS-CoV-2 antigen solution at a position 4-6mm away from the release line and far away from the sample pad end;
c, preparing a T1 competitive line:
coating an ACE2 protein solution at a position 1-3mm away from a T2 detection line and far away from the end of a sample pad;
d.C position of the control line:
coating the IgY antibody solution at a position 1-3mm away from a T1 competitive line and far away from the end of a sample pad;
(2) assembling:
sequentially adhering a sample pad, a nitrocellulose membrane and a water absorption pad on a bottom lining, and cutting into test strips with the width of 3-5 mm.
6. The method for preparing the test strip for the cooperative detection of the corona total antibody and the neutralizing antibody according to claim 5, wherein the method comprises the following steps: in the step (1) preparing the release line, the marker is a composition of at least one of RBD antigen or SARS-CoV-2 antigen and IgY antibody, and the tracer particle is at least one of fluorescent microsphere, colloidal gold, latex microsphere, magnetic particle or nano carbon, preferably fluorescent microsphere;
the preparation method of the tracer particle-marker conjugate comprises the following steps:
a, uniformly mixing 300-500ug of tracer particles and 60-100ug of at least one of RBD antigen or SARS-CoV-2 antigen for 30-40min, adding 5-10% BSA, stirring for 10-30min, centrifuging at 12000-14000rpm for 20-30min, removing supernatant, and dissolving precipitate with fluorescent labeling redissolution;
b. uniformly stirring and mixing 300-500ug of tracer particles and 60-100ug of IgY antibody for 30-40min, adding 5-10% BSA, stirring for 10-30min, centrifuging at 12000-14000rpm for 20-30min, removing the supernatant, and dissolving the precipitate with a fluorescent labeling redissolution;
c. mixing the RBD antigen or SARS-CoV-2 antigen marked microsphere and IgY antibody marked microsphere in the ratio of 1 to (0.2-0.5), and then suspending in fluorescent labeling complex solution to obtain the tracer particle-marker conjugate.
7. The method for preparing the test strip for the cooperative detection of the corona total antibody and the neutralizing antibody according to claim 6, wherein the method comprises the following steps: in step (1), the tracer particle-label conjugate is coated at the release line in an amount of 0.5-1ul/cm, preferably 0.7-1.0 ul/cm; the ACE2 protein solution is coated at the T1 competitive line in an amount of 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm; the SARS-CoV-2 antigen solution or RBD antigen solution is coated at the T2 detection line in the amount of 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm; the IgY antibody solution is coated at the C quality control line in an amount of 0.5-1.0ul/cm, preferably 0.7-1.0 ul/cm. .
8. The method for preparing the test strip for the cooperative detection of the corona total antibody and the neutralizing antibody according to any one of claims 6 to 7, wherein the method comprises the following steps: the concentration of the tracer particle-marker conjugate is 0.5-1.0mg/ml, preferably 0.8-1.0 mg/ml; the concentration of the ACE2 protein solution is 0.5-1.0mg/ml, preferably 0.8-1.0 mg/ml; the concentration of the SARS-CoV-2 antigen solution or RBD antigen solution is 0.5-1.0mg/ml, preferably 0.8-1.0mg/ml.
9. The method for preparing the test strip for the synergistic detection of the neocorona total antibody and the neutralizing antibody according to claim 8, wherein the fluorescent labeling complex solution comprises the following components: 10-50mmol/L buffer solution, 5-10g/L surfactant and 40-50g/L protective agent; the buffer is selected from at least one of Tris, borate, phosphate or PBS buffer; the surfactant is selected from at least one of Tween-20, Tween-80 or Triton-X100; the protective agent is at least one of sucrose or trehalose.
10. The method for preparing the test paper strip for the synergistic detection of the neo-corona total antibodies and the neutralizing antibodies according to claim 9, wherein the fluorescent labeling compound solution further comprises 10-30g/L, PEG 1-10g/L of protein and 1-5g/L of preservative, wherein the protein is at least one selected from casein and BSA; the preservative is selected from at least one of sodium azide or proclin-300.
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