CN114252601A - New corona total antibody and neutralizing antibody latex rapid detection test paper and preparation method thereof - Google Patents
New corona total antibody and neutralizing antibody latex rapid detection test paper and preparation method thereof Download PDFInfo
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- 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
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Abstract
The invention provides novel coronavirus total antibody and neutralizing antibody latex rapid detection test paper and a preparation method and application thereof. The test paper is prepared by sequentially pasting a chromatographic membrane comprising a solid phase detection line T1 with a mouse anti-human IgG/IgM antibody, a detection line T2 with a gene recombinant novel coronavirus ACE2 protein and a quality control line, a combination pad which is pre-sprayed with microspheres coupled with different colors and is used for gene recombinant novel coronavirus spike protein RBD-mFC and rabbit IgG antibody, and other auxiliary materials. The invention adopts the capture method and sandwich method principle, is used for in vitro qualitative detection of novel coronavirus total antibodies and neutralizing antibodies in human blood samples, can effectively eliminate false positives, and improves the detection accuracy.
Description
Technical Field
The invention belongs to the technical field of in-vitro detection of inspection medicine, and particularly relates to novel coronavirus total antibody and neutralizing antibody rapid detection test paper by an emulsion method, and a preparation method and application thereof.
Background
When new coronavirus is immersed in human body, the human body generates antibody capable of specifically neutralizing virus at the later stage of resisting virus, the antibody belongs to the class of IgG (other classes such as IgM antibody are antibodies which are generated at the early stage and used for emergency, and have weak specificity), and the specific is RBD protein on the surface of the virus surface, so the antibody is called RBD antibody.
The RBD is a region on the receptor binding domain of the spike protein (spike) on the surface of the outer shell of the novel coronavirus particle, and is the specific site of the virus. Neutralizing antibody detection is typically used for total neutralizing antibodies, including IgA, IgM, and IgG, in samples from convalescent patients with new coronavirus infections, and patients immunized with new coronavirus vaccines.
Disclosure of Invention
The invention mainly aims to provide a combined detection kit for total antibodies and neutralizing antibodies. The technical problem to be solved is that a user needs to detect the self neutralizing antibody after vaccination, and a non-specific result appears.
In order to achieve the above objects, the present invention provides a method for determining whether a sample contains IgG/IgM antibodies against a novel coronavirus by a capture method, and determining whether the sample contains a neutralizing antibody against the novel coronavirus by a sandwich method
In one aspect, the present application provides a novel rapid test strip for detecting total and neutralizing antibodies of coronavirus latex, which comprises a chromatographic membrane, a buffer pad and a binding pad; the binding pad is pre-adsorbed with diluted protein conjugate coupled with colored microspheres, the chromatographic membrane is sequentially provided with a detection line T and a quality control line C along the liquid chromatography direction, and a buffer pad is arranged between the chromatographic membrane 2 and the binding pad 4.
Furthermore, the detection lines are T1 and T2, wherein the T1 line is a mixed coating line of mouse anti-human IgG and IgM, and the T2 line is a coating line of the gene recombinant novel coronavirus SARS-COV 2-RBD-HIS.
Furthermore, the coating of the quality control line is goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody.
Furthermore, one end of the combination pad is sprayed with a gene recombination novel coronavirus SARS-COV2-RBD-mFC coupled with blue microspheres.
Further, the cushion pad 3 is a blood filter membrane or glass fiber.
Furthermore, the color of the color microspheres corresponding to the detection line and the quality control line is different, and can be blue, red or black.
Further, the coating material of the T1 line is diluted to a concentration value of 0.5-1mg/mL by using a phosphate buffer solution containing 1-5% of sucrose; coating material of T2 line was diluted with 0.05M MES buffer containing 0.3-0.6% bovine serum albumin; the coating material of the control line C was diluted with a buffer solution of Tris-HCl pH8.0 containing Bovine Serum Albumin (BSA) at 0.3-0.6%.
Further, the coupling step of the colored microspheres comprises:
washing the microspheres with 0.05M MES solution with pH of 6.0-6.5 as reaction buffer solution, and diluting to 1%; activating by conventional EDC-NHS two-step method, adding labeling material at a ratio of 80-160 micrograms per milligram of microsphere, reacting for 3-4 hours, adding ethanolamine to terminate, centrifuging at 10000rpm, discarding supernatant, and diluting with 0.1M Tris-hydrochloric acid buffer solution with pH8.0 containing 0.5-1% norrin, 0.1-1% PVP K30, and 0.5-1% PEG6000 to desired concentration.
On the other hand, the application provides the application of the rapid detection test paper in preparing a new crown antibody detection kit or a new crown vaccine effect detection kit.
Along the flowing direction of the sample, the chromatographic membrane 2 is sequentially coated with mouse anti-human IgG antibody/mouse anti-human IgM antibody (T1 line), SARS-COV2-RBD-HIS (T2 line) and goat anti-rabbit IgG antibody (quality control line).
The conjugate pad 4 was sprayed with blue latex conjugated SARS-COV2-RBD-mFC and red latex conjugated rabbit IgG antibodies.
The mFc tag can be other gene recombination tags with amino groups.
The quality control line system used by the chromatographic membrane 2 and the combined pad 4 can be used without influencing a detection line system and without non-specificity, and comprises rabbit serum and goat anti-rabbit serum, mouse serum and goat anti-mouse serum, and other DNP-BSA and rabbit anti-DNP antibodies.
The cushion pad 3 can gather the sample, reduce the time for the sample to pass through the chromatographic membrane 2 and ensure the stability of the result within the result judging time.
When the product is tested, a blood sample to be tested and sample diluent are sequentially added on the sample pad. If the sample contains the novel coronavirus neutralizing antibody, the sample is combined with SARS-COV2-RBD-mFC coupled with blue microspheres on the combination pad respectively to form a neutralizing antibody-RBD protein complex. The complex moves forward along the strip, sequentially passing through line T1, line T2, and the control line. Wherein, the RBD marked by the neutralizing antibody-blue microsphere is captured by the mouse anti-human IgG and IgM antibodies coated by the T1 line to form a blue T1 line, and when the rest of the neutralizing antibody-RBD complex passes through the T2 line, the complex is captured by the RBD of the T2 line solid phase and forms a blue line. The rabbit IgG antibody marked by the red latex is combined by the goat anti-rabbit IgG antibody coated on the quality control line to form a red line.
Drawings
Fig. 1 is a schematic structural diagram of a test strip provided in embodiment 1 of the present invention; wherein 1 is an absorption pad, 2 is a chromatographic membrane (2 a-quality control line, 2 b-detection line T2, 2 c-detection line T1), 3 is a blocking pad, 4 is a combination pad, 5 is a sample pad, and 6 is an adhesive plate (the surface is provided with adhesive, and release paper is normally used for covering).
Detailed Description
EXAMPLE 1 preparation of test strips
The test strip with the structure shown in figure 1 is prepared according to the following method
1. Chromatographic membrane
1.1 Nitrocellulose Membrane
The product uses YN-80 backing film produced by Shantou Yineng film industry Co. The film width should be chosen to be 2.2 cm.
2.2 Main raw materials
Respectively diluting the coating raw materials of the detection line and the quality control line by using different diluents, wherein T1 lines are mouse anti-human IgG and IgM, and the concentration value is diluted by using a phosphate buffer solution containing 4% of sucrose to be 0.60 mg/mL; test line T2 is RBD-HIS, diluted with MES buffer containing 0.50% bovine serum albumin (0.05M) to a concentration of 0.80 mg/mL; the control line was goat anti-rabbit IgG antibody, and the concentration was 1.0mg/mL diluted with Tris-HCl (pH8.0) buffer containing 0.50% bovine serum albumin.
2.3 coating operation
The amount of the liquid sprayed by the coating machine is generally set to 0.085 to 0.120. mu.l/mm, and the rail speed is 60mm/s, so that coating is performed.
Transferring the coated cellulose nitrate reaction membrane to a drying room to be dried overnight for not less than 20 hours, controlling the temperature to be 35-40 ℃ and the humidity to be not more than 30%.
And (3) adding a drying agent into the dried chromatographic membrane, filling the chromatographic membrane into an aluminum foil bag, sealing, and storing at room temperature, wherein the validity period is 1 year.
2. Combined pad
2.1 materials and reagents
The blue microspheres and the red microspheres with the particle size of 300nm are prepared by the Shantou biological medicine technology company Limited.
The novel coronavirus SARS-COV2-RBD-mFC protein is from Jiangsu Dongzhi anti-biological medicine science and technology limited.
2.2 coupling of microspheres
Included in this reagent are blue latex conjugated SARS-COV2-RBD-mFC (suitable for detection lines 2b, 2c) and red latex conjugated rabbit IgG antibody (suitable for quality control line 2 a).
Taking appropriate amounts of blue microspheres and red microspheres respectively, diluting with ultrapure water until the solid content is 0.5%, centrifuging for 5 minutes at 2500 r, discarding the precipitate, centrifuging the supernatant again for 12000r for 5 minutes, discarding the supernatant, precipitating with Liu, diluting the microspheres to a concentration of 1% by using 0.05M PH6.0MES solution as reaction buffer solution.
Activating by a conventional EDC-NHS two-step method, and respectively adding marking raw materials according to the proportion of 80-160 micrograms per milligram of microspheres, wherein the marking raw materials comprise: 60 micrograms of SARS-COV2-RBD-mFC and 80 micrograms of rabbit IgG antibody, after the rotational reaction is carried out for 3 to 4 hours, 3 microliters of ethanolamine is added into each milliliter of microspheres for termination cost for 30 minutes, the microspheres are centrifuged for 5 minutes at 12000rpm, the supernatant is discarded, and the precipitate is remained; blocking was performed using a blocking solution of 0.05M Tris-hydrochloric acid (pH8.0) containing 1% sodium caseinate for 30 minutes, followed by centrifugation at 12000rpm for 10 minutes, and the supernatant was discarded to leave a precipitate.
The microsphere conjugate was diluted to a certain concentration using 0.1M Tris-HCl working solution pH8.0 containing 1% sodium caseinate, 1% PVP K30, 0.5% PEG 6000.
2.3 conjugate pad preparation
The coating machine is used for cleaning the pipeline, the liquid spraying amount is set to be 0.8-1.2 mu l/mm, and the air pump is controlled to be 1.2-2 psi. Spraying on glass fiber pad, and drying overnight at humidity of less than or equal to 30% for at least 20 hr.
Drying the bonding pad, adding desiccant, packaging into aluminum foil bag, sealing, and storing at room temperature.
3. Sample pad
Glass fibers were treated with 0.05M phosphate buffer (pH7.6) containing 1% anti-cell antibody, 0.5% sodium caseinate, 2% Tween 20. Drying overnight for not less than 20 hours.
4. Inspection method
4.1 Assembly slitting
The components of the product, such as the absorption pad, the chromatographic membrane, the combination pad, the sample pad and the like, are sequentially stuck on the plastic substrate and are cut according to different widths for later use.
Firstly, tearing off the adhesive tape release paper at one end of the sticking plate (6), and sticking the position of the quality control line (2a) of the chromatographic film (2) downwards along the small double-sided adhesive; then, the absorbent pad (1) and the chromatographic carrier (2) are laminated at a position of about 1 mm near the control line (2a) for attachment.
Reversely, tearing off the adhesive tape release paper at the other end, laminating the blocking pad (3) by about 1 mm, and sticking the chromatographic film (2) at one end close to the detection line T2(2 c); then laminating the colloidal gold-antibody combined pad (4) with the blocking pad (3) and pasting under the pressure of about 1 mm; finally, the sample pad (5) was also laminated to the conjugate pad (4) and adhered to the adhesive plate (6) at about 1 mm pressure. All the components and auxiliary materials must be adhered in place.
And installing a card shell outside the test strip, namely the card type reagent.
4.2 inspection operation
4.1 sample Collection
The blood of the user is collected according to a conventional method, or the blood of the tip of the user's finger is directly used.
4.2 sample detection
Dropping 2 drops of sample to be detected on the sample pad of the detection reagent, and then dropping 1 drop of sample diluent, thereby avoiding the addition of bubbles. The timing is started, and the result judgment is carried out within 10-20 minutes.
4.3 interpretation of test results
(1) Invalid result: the control line (2a in the drawing) did not develop color. Indicating that the reagent is invalid or improperly operated and needs to be retested.
(2) Positive results: the detection line T1 (2c in the figure) is colored, and the detection line T2(2 b in the figure) is weakly colored. And prompting that the novel coronavirus IgG antibody and/or IgM antibody is detected in the sample to be detected, and meanwhile, the novel coronavirus neutralizing antibody is positive.
(3) Suspected results are: the detection line T1 is colored, and the detection (T2) is not colored, which indicates that the novel coronavirus IgG antibody and/or IgM antibody is detected in the sample to be detected, but the novel coronavirus neutralizing antibody is not detected, and the detection is suggested to occur after 7 days.
(4) Negative results: neither detection line T1 nor T2 developed color. Suggesting that no novel coronavirus antibody was detected in the sample.
5. Evaluation of Performance
The population inoculated with the novel inactivated coronavirus vaccine was selected, and their blood samples were collected for a total of 250 portions. The novel coronavirus IgG antibody/IgM antibody is tested by using a reagent of the same variety, the reagent is a product of a medical instrument registration certificate approved by the State drug administration, and possible combinations of the positive results displayed by the detection results are as follows: one or 2 of the IgM antibody and the IgG antibody were positive.
Summary of table test results
Including 17 samples that were suspect.
The inventive reagents described above were tested as positive (170 parts) and negative (63 parts) samples, using a reagent that has obtained U.S. FDA EUA as a comparison reagent.
Statistics of the underlying data
| Numbering | Numerical ratio | Percentage (95% confidence interval) |
| Positive rate of agreement | 166/169 | 98.22％(94.90％-99.63％) |
| Negative rate of agreement | 60/64 | 93.75％(84.76％-98.27％) |
| Positive predictive value | 166/170 | 97.65％(94.09％-99.36％) |
| Negative predictive value | 60/63 | 95.24％(86.71％-99.01％) |
| Total rate of agreement | 226/233 | 97.00％(93.91％-98.78％) |
Kappa consistency test
| Numbering | Statistical value |
| Kappa number | 0.9242, better consistency |
| Notation mistake Se (K) | 0.0282 |
| 95% confidence interval | 0.8690-0.9795 |
| Marking error Se0(K) | 0.066 |
| Test value Z | Z is 14.1087, and probability value P is 0.0000 |
| Test results | P<0.05, reject HO, Kappa value from totality other than 0 |
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A novel coronavirus total antibody and neutralizing antibody latex rapid detection test paper is characterized in that: the test paper comprises a chromatographic membrane, a buffer pad and a combination pad; the binding pad is pre-adsorbed with diluted protein conjugate coupled with colored microspheres, the chromatographic membrane is sequentially provided with a detection line T and a quality control line C along the liquid chromatography direction, and a buffer pad is arranged between the chromatographic membrane 2 and the binding pad 4.
2. The rapid test strip of claim 1, wherein the detection lines are T1 and T2, wherein the T1 line is a mixed coating line of mouse anti-human IgG and IgM, and the T2 line is a coating line of the genetically modified novel coronavirus SARS-COV 2-RBD-HIS.
3. The rapid test strip according to claim 1 or 2, wherein the coating material of the quality control line is goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody.
4. The rapid test strip according to any one of claims 1 to 3, wherein one end of the conjugate pad is coated with a blue microsphere coupled recombinant coronavirus SARS-COV 2-RBD-mFC.
5. The rapid test strip of claim 4, wherein the other end of the conjugate pad is sprayed with a red latex-conjugated rabbit IgG antibody.
6. The rapid test strip according to any one of claims 1 to 5, wherein the buffer pad is a blood filter or a glass fiber.
7. The rapid test strip according to any one of claims 1 to 6, wherein the color of the colored microspheres corresponding to the detection line and the quality control line is different, and the color of the microspheres can be selected from blue, red and black.
8. The rapid test strip according to any one of claims 2 to 7, wherein the coating material of the T1 line is diluted to a concentration value of 0.5 to 1mg/mL using a phosphate buffer solution containing 1 to 5% sucrose; coating material of T2 line was diluted with 0.05M MES buffer containing 0.3-0.6% bovine serum albumin; the coating material of the control line C was diluted with a buffer solution of Tris-HCl pH8.0 containing Bovine Serum Albumin (BSA) at 0.3-0.6%.
9. The rapid test strip according to any one of claims 1 to 8, wherein the colored microsphere coupling step comprises:
washing the microspheres with 0.05M MES solution with pH of 6.0-6.5 as reaction buffer solution, and diluting to 1%; activating by conventional two-step method of EDC-NHS, adding labeling raw material according to the proportion of 80-160 micrograms per milligram of microsphere, reacting for 3-4 hours, adding ethanolamine for stopping, centrifuging at 12000rpm, discarding supernatant, leaving precipitate, and diluting to required concentration by using 0.1M Tris-hydrochloric acid buffer solution of pH8.0 containing 0.5-1% norrin, 0.1-1% PVP K30 and 0.5-1% PEG 6000.
10. Use of the rapid test strip according to any one of claims 1 to 9 in the preparation of a new corona antibody test kit or a new corona vaccine effect test kit.
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