CN114324879B

CN114324879B - Novel qualitative detection kit for coronavirus neutralizing antibody based on colloidal gold double-antibody sandwich method

Info

Publication number: CN114324879B
Application number: CN202111653477.6A
Authority: CN
Inventors: 卜令杰; 李扬霞; 陈超
Original assignee: Zhejiang Mole Biotechnology Co ltd
Current assignee: Jiangsu Mole Bioscience Co ltd; Zhejiang Mole Biotechnology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-09-27
Anticipated expiration: 2041-12-30
Also published as: CN114324879A

Abstract

The invention relates to a novel coronavirus neutralizing antibody qualitative detection kit based on a colloidal gold double-antibody sandwich method, which comprises a test strip and a sample diluent, wherein the test strip comprises a sheet, a novel coronavirus S-RBD protein marked by nano colloidal gold, a sample pad, a detection line and a quality control line, and is characterized in that the novel coronavirus S-RBD protein marked by nano colloidal gold contains BSA (bovine serum albumin) and PEG (polyethylene glycol), the sample diluent comprises a surfactant, a Tris buffer solution and bovine serum albumin, and the surfactant is selected from one or more of laurinol polyethylene oxide (9) ether, laurinol polyoxyethylene ether, fatty alcohol epoxy ether and oleyl alcohol polyoxyethylene (10) ether. By adopting the technical scheme of the invention, the stability of the S-RBD colloidal gold marker can be improved, and the sensitivity and specificity of the kit can be improved.

Description

Novel qualitative detection kit for coronavirus neutralizing antibody based on colloidal gold double-antibody sandwich method

Technical Field

The invention relates to the field of disease diagnosis, in particular to a novel qualitative detection kit for a coronavirus neutralizing antibody based on a colloidal gold double-antibody sandwich method and application thereof.

Background

The colloidal gold labeling technology is a labeling method developed in the last 80 th century after three major labeling technologies of fluorescein, radioisotope and enzyme. In 1971, Faulk and Taylor introduced colloidal gold into immunochemistry, and the labeling method is widely applied to the fields of drug detection, biomedical detection, pesticide and veterinary drug detection, clinical disease detection and the like. At present, there are hundreds of commercial colloidal gold immunochromatographic kits at home and abroad, for example, the following are common: early pregnancy kit, drug detection kit, fecal occult blood detection kit, infectious disease detection kit and the like.

The colloidal gold labeling technology is to use colloidal gold as a labeling tracer and apply the labeled tracer to an immunoelectron microscope at first; with the development of immunology, the antibody is gradually applied to animal agglutination experiments, immunoblotting, optical lens staining, immunospot and immunochromatography. Due to the high electron density on the surface of the colloidal gold, the colloidal gold can be firmly combined with most proteins without destroying the activity of the proteins.

The colloidal gold is applied to the immunodetection, and the colloidal gold is mainly used for marking an antigen or an antibody; an antibody or antigen immunologically reactive with the labeled antigen or antibody is then immobilized on the substrate, whereby binding of the antigen or antibody is revealed as a color change visible to the naked eye. The binding degree of colloidal gold and protein, the pH of the reaction system, the concentration of the reacted protein, etc., all affect the binding of the protein and the colloidal gold.

In 2019, outbreaks of new coronaviruses have caused global pandemics, and the epidemics have been of long duration and repetitiveness. This virus is formally named SARS-CoV-2 by the International Committee for Classification of viruses, and the disease caused by this virus is named COVID-19 by WHO. SARS-CoV-2 belongs to the family of coronaviruses, β to coronaviruses; is elliptical or circular, and has a diameter of 60-140 nm. The virus consists of a single-stranded positive-strand RNA, a protein capsid, and an envelope (with spikes outside) 3 partial structure. Its sgRNA is responsible for encoding 4 structural proteins and accessory proteins, spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The present research shows that SARS-CoV-2 completes the infection process mainly through the RBD structure domain of S protein combining with the corresponding receptor ACE2 (angiotensin receptor protein) on the host target cell and entering the cell. The neutralizing antibody of the novel coronavirus can be combined with SARS-CoV-2S protein RBD, thereby blocking the combination of S-RBD and ACE2 receptor. Vaccines for the novel coronaviruses are also prepared based on this principle. The detection method based on the principle can be used for detecting the neutralizing antibody in the human body.

At present, the main detection method is colloidal gold chromatography detection based on a competitive method of S-RBD and ACE2 combination. The main principle is to mark S-RBD or ACE2, detect line coating ACE2 or S-RBD, judge whether the sample contains neutralizing antibody by whether color is developed.

For example, chinese patent application CN112051400A discloses an immunochromatographic kit for detecting novel neutralizing antibodies against coronavirus and a detection method, wherein the kit comprises a detection card and a sample diluent, the detection card comprises a PVC base plate, a sample pad, a blood filter pad, a conjugate pad, a nitrocellulose membrane and absorbent paper, the sample pad, the blood filter pad, the conjugate pad, the nitrocellulose membrane and the absorbent paper are sequentially overlapped and staggered with each other and then adhered to the PVC base plate to form a test paper plate, and the detection card is obtained by cutting. The immunochromatography kit for detecting the novel coronavirus neutralizing antibody, which is prepared by the invention, can quickly detect the novel coronavirus neutralizing antibody, is simple to operate, intuitive in result, safe and cheap, and is suitable for on-site quick detection; secondly, the method for detecting the content of the neutralizing antibody of the novel coronavirus by using the kit prepared by the invention can accurately determine the content of the substance to be detected by combining with a colloidal gold immunoassay reading instrument, and the result is obtained within 15 min.

However, how to improve the stability of the S-RBD colloidal gold label and improve the sensitivity and specificity of the kit still remains a technical problem to be further solved by those skilled in the art.

Disclosure of Invention

The invention aims to provide a novel qualitative detection kit for coronavirus neutralizing antibodies based on a colloidal gold double-antibody sandwich method. In order to realize the purpose of the invention, the following technical scheme is adopted:

the invention relates to a novel coronavirus neutralizing antibody qualitative detection kit based on a colloidal gold double-antibody sandwich method, which comprises a test strip and a sample diluent, wherein the test strip comprises a sheet, a novel coronavirus S-RBD protein marked by nano colloidal gold, a sample pad, a detection line and a quality control line, and is characterized in that the novel coronavirus S-RBD protein marked by nano colloidal gold contains BSA (bovine serum albumin) and PEG (polyethylene glycol), the sample diluent comprises a surface active agent, a Tris buffer solution and bovine serum albumin, and the surface active agent is selected from one or more of laurinol polyethylene oxide (9) ether, laurinol polyoxyethylene ether, fatty alcohol epoxy ether and oleyl polyoxyethylene (10) ether. The kit of the invention can refer to a schematic diagram shown in figure 1 and a physical diagram shown in figure 2.

In a preferred embodiment of the present invention, the average particle size of the nano colloidal gold is 30 to 50 nm.

In a preferred embodiment of the present invention, the sample pad contains a surface active material propylene oxide-ethylene oxide-vinyl diamine copolymer. By containing the surface active substance propylene oxide-ethylene oxide-vinyl diamine copolymer on the sample pad, the sensitivity and the specificity of the product are effectively improved. Meanwhile, the product can be used for detecting three samples of whole blood, serum and plasma.

In a preferred embodiment of the present invention, the average molecular weight of the PEG is 18000-25000. By controlling the molecular weight of PEG within the above preferred range, it is helpful to further improve the stability of the nano colloidal gold labeled S-RBD protein of the novel coronavirus.

In a preferred embodiment of the invention, the sample diluent further comprises a preservative. Such preservatives include, but are not limited to, Proclin300, sodium azide, krovin 300, and the like.

In a preferred embodiment of the invention, the detection line is coated with S protein trimer of SARS-CoV-2, and the quality control line is coated with ACE2 protein. Through the detection line and the quality control line, the effectiveness of the product can be effectively monitored.

The invention also relates to the application of the detection kit in preparing a diagnostic reagent for detecting the novel coronavirus neutralizing antibody.

In a preferred embodiment of the invention, the novel coronavirus neutralizing antibody is derived from whole blood, serum and/or plasma.

By adopting the technical scheme, the invention has the following beneficial effects:

firstly, the invention adopts a double-antigen sandwich colloidal gold immunochromatography, and the prepared colloidal gold kit can qualitatively detect 1ng/ml neutralizing antibody in blood, plasma or serum. Compared with the commercial product, the product has high sensitivity. The marked protein uses SARS-CoV-2S-RBD protein, which can be combined with neutralizing antibody specifically, therefore, the product has higher sensitivity; the coating protein uses trimer of S protein, and can be specifically combined with neutralizing antibody, thereby avoiding cross-linking with protein analogue with similar structure. When in marking, BSA and PEG are added, so that the marking yield is effectively improved, and the stability of the gold-labeled solution after marking is greatly improved.

Secondly, the buffer system of the kit is added with a surface active substance of propylene oxide-ethylene oxide-vinyl diamine copolymer, so that the sensitivity and specificity of the product are effectively improved. Meanwhile, the product can be used for detecting three samples of whole blood, serum and plasma.

In addition, in the sample diluent of the kit, lauryl alcohol polyoxyethylene (9) ether is added, so that the color development of the product is obviously improved.

Finally, the kit prepared by the method avoids the phenomenon that the product fails to be monitored due to independent quality control lines. The quality control line of the method can be specifically combined with the gold label. If the detection gold marks a problem, the quality control line cannot be developed, and the quality of the product can be effectively monitored. However, in the case of detection of a sample in which a very high concentration of neutralizing antibody exists, the color development of the quality control line may be lighter; most of the gold labels are gathered at the position of the detection line, so that the residual gold labels are less, the color development of the quality control line is lighter, but the condition that the quality control line cannot be observed cannot occur under the condition that the product is effective. When the product prepared by the method is detected, the red line at the detection line position is positive, the red line is negative when no red line exists, and the red line is always displayed at the quality control line position. The result observation is convenient and visual.

Drawings

FIG. 1 is a schematic view of a test strip of the present invention;

FIG. 2 is a diagram showing an example of the kit of the present invention

FIG. 3 shows the color development without lauryl alcohol polyethylene oxide (9) ether;

FIG. 4 shows the color development of laurinol polyoxyethylene (9) ether.

Detailed Description

The present invention is further illustrated by the following examples which are intended to be purely exemplary of the invention and are not intended to be limiting, since all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Example 1

1 preparation of colloidal gold

The prepared colloidal gold is colloidal gold sol prepared by a trisodium citrate reduction method, and the particle diameter is 40 nm. Has uniform size, and can effectively improve the combination of the colloidal gold and the SARS-CoV-2S-RBD protein.

(1) 2ml of chloroauric acid aqueous solution with the concentration of 0.1g/ml is prepared and stored away from light for later use.

(2) 10ml of trisodium citrate aqueous solution with the concentration of 0.1g/ml is prepared and stored away from light for standby.

(3) Adding water with the volume of the colloidal gold solution to be prepared into the beaker, wherein the water comprises the following components: 1L of water; the beaker was then placed on a magnetic stirrer and heated to boiling.

(4) Adding 1.53ml of chloroauric acid aqueous solution into a beaker while heating; then 0.24ml of trisodium citrate solution was added to the beaker containing aqueous chloroauric acid solution and stirring was continued for 5 minutes to obtain a 40nm colloidal gold solution.

(5) Stopping heating, then under the condition of stirring, cooling the reacted colloidal gold solution to room temperature of 2-8 ℃, and storing in dark place for later use.

2 novel coronavirus S-RBD protein marker

The technology adopts a direct marking method. Namely, the novel coronavirus S-RBD protein which is sold in the market is directly reacted with a colloidal gold solution at room temperature, and then the reaction product is centrifuged to obtain the required gold marker. The specific method comprises the following steps:

2.1 determination of the pH of the label:

(1) 1ml of each of the gold colloidal solutions prepared in 1 was put into an EP tube, and 10 tubes in total were counted, and then the gold colloidal solutions were adjusted to pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 with 0.1M potassium carbonate, respectively, and labeled. Then, SARS-CoV-2S-RBD protein was added in an amount of 10. mu.g/ml, and vortexed to mix well.

(2) Each solution was allowed to stand for 10 minutes, and 10% sodium chloride solution was added to each EP tube in an amount of 100. mu.l/tube, followed by vortex mixing.

(3) After standing for 15 minutes, the color change was observed.

(4) Selecting the pH of the tube of the colloidal gold solution which is not precipitated, changed into purple or black as the optimal labeling pH; i.e. the pH of the solution just after the color change and still wine-red is the optimum pH. I.e. pH 7.5

2.2 determination of the amount of marker

(1) 1ml of each of the gold colloidal solutions prepared in step 1 was put into an EP tube, 10 tubes in total, and then the pH of the gold colloidal solutions was adjusted to 7.5 with 0.1M potassium carbonate, respectively, and labeled. Then, SARS-CoV-2S-RBD protein was added in an amount of 2. mu.g/ml, 4. mu.g/ml, 6. mu.g/ml, 8. mu.g/ml, 10. mu.g/ml, 12. mu.g/ml, 14. mu.g/ml, 16. mu.g/ml, 18. mu.g/ml, 20. mu.g/ml, respectively, and vortexed to mix well.

(2) After standing for 10 minutes, an aqueous solution of BSA (0.85 parts BSA per ten parts water) was added to each tube in an amount of 24. mu.l/ml, and vortexed to mix.

(3) PEG20000 aqueous solution (0.2 parts PEG2000 per ten parts water) was added in an amount of 20. mu.l/ml and vortexed to mix.

(4) After standing for 15 minutes, an aqueous sodium chloride solution (1.5 parts of sodium chloride per ten parts of water) was added to each tube in an amount of 100. mu.l/ml, and vortexed to mix them.

(5) After standing for 2 hours, the tubes were observed for sedimentation and color change. Selecting the amount of protein in the tube of the colloidal gold solution which is not precipitated, changed into purple or black as the optimal labeling amount; i.e., the protein content of the solution that just did not undergo a color change and still appeared wine-red was the optimal labeling amount. I.e., 8. mu.g/ml.

2.3 colloidal gold labeling of SARS-CoV-2S-RBD protein

(1) The colloidal gold solution is weighed into a beaker according to the preparation amount, such as: 100ml of the colloidal gold solution was put into a beaker.

(2) The pH of the gold colloid solution was adjusted to 7.5 with 0.1M potassium carbonate solution. And placed on a magnetic stirrer for stirring.

(3) The concentration of SARS-CoV-2 protein was diluted to 1mg/ml with 2mM Tris buffer, pH7.4, and then 0.8ml of the diluted protein solution was added to the colloidal gold solution. The solution was added with stirring.

Preparation of Tris buffer solution: weighing Tris according to the amount of 2.2 parts of Tris per hundred parts of water, placing the Tris in a beaker, adding 9 parts of water, adjusting the pH value to 7.4 by using 6M hydrochloric acid solution, and then fixing the volume to the required volume by using purified water to obtain the finished product.

(4) Stirring for 25 minutes on a magnetic stirrer;

(5) to 100ml of the above solution, an aqueous solution of 8.5% BSA was added in an amount of 24. mu.l/ml, and the stirring was continued for 45 minutes.

(6) To the above solution was added 2% PEG20000 in an amount of 20. mu.l/ml, and the stirring was continued for 1 hour.

(7) Transferring the reacted solution into a centrifugal tube;

(8) centrifugation at 8000-15000 rpm/min at 4 ℃, for example: centrifuging at 12000rpm/min for 35 min; and removing the supernatant.

(9) And (3) re-dissolving the precipitate by using 1 part per hundred of 2mM Tris buffer solution with the pH value of 7.4 to obtain the gold-labeled S-RBD protein.

2.4 preparation of buffer systems for Whole blood, serum and plasma Loading

The method prepares a buffer system simultaneously suitable for whole blood, serum and plasma samples, and prepares the buffer system into a sample pad.

2.4.1 preparation of the buffer System

(1) Weighing borax according to the amount of 4 parts per hundred of borax, putting the borax into a beaker, adding 80 parts of water into the beaker, and putting the beaker on a magnetic stirrer for stirring.

(2) Adding 0.3 part of ethylenediamine tetraacetate into the beaker, and continuously stirring;

(3) adding 0.1 part of casein salt into the solution, and continuously stirring;

(4) 1 part of propylene oxide-ethylene oxide-vinyl diamine copolymer (commercially available) is added to the solution, and stirring is continued;

(5) adding 2 parts of Proclin300 per ten thousand parts into the solution, and continuing stirring;

(6) to the above solution, 0.5 parts per ten thousand of Tween20 was added and stirring was continued.

(7) After the solution is completely dissolved and clarified, the pH of the solution is adjusted to 8.2 by using hydrochloric acid or sodium hydroxide, and then purified water is used for fixing the volume to the required volume.

(8) And (5) keeping the temperature at room temperature for later use.

2.4.2 preparation of sample pad

(1) A commercially available glass fiber mat (length: width: 300 mm: 254mm) was prepared;

(2) soaking the glass fiber mat according to the amount of 1 sheet per hundred parts; the glass fiber mat was first soaked in the solution for 10 minutes, then removed and excess water was gently filtered off with a glass rod. Turn over, soak the glass fiber pad into the solution again for 10 minutes, filter off the excess water gently with a glass rod. And obtaining the sample pad.

(3) The resulting sample pad was placed in an oven at 37 ℃ and dried overnight (time 17-20 hours).

(4) After drying, the sample pad is stored in a sealed state and placed in a dry environment.

2.5 preparation of the sheet

(1) Sticking the nitrocellulose membrane on a PVC bottom plate;

(2) preparation of coating liquid:

and (3) detection line: SARS-CoV-2 was diluted to 1mg/ml with 0.01M PB, and the amount of the detection line was prepared in an amount of 50. mu.l per patch.

Quality control line: SARS-CoV-2 was diluted to 0.5mg/ml with 0.01M PB, and the amount of the control line was prepared in an amount of 50. mu.l per tablet.

Coating: the solution was coated on a nitrocellulose membrane with a scratch-off agent.

(3) The coated sheet was placed in a 37 ℃ oven and dried overnight.

(4) And (5) storing the dried sheet in a sealed manner and storing the sheet in a dry place.

2.6 preparation of gold-labeled strip

(1) The gold standard solution prepared at 2.3 was diluted with 0.01M PB buffer containing 1 part bovine serum albumin and 20 parts sucrose per hundred. The dilution ratio was 46 parts of the gold-labeled solution and 54 parts of the above-mentioned 0.01M PB solution.

(2) The diluted solution was sprayed with gold labels in an amount of 45. mu.l per strip using a gold labeler. The sprayed substrate mat was a glass fiber mat (length: width: 300 mm: 84 mm).

(3) And after spraying, putting the gold label strip into a 37 ℃ oven, and drying overnight.

(4) And sealing the dried gold label strips in a self-sealing bag, and then storing in a dry environment in a dark place.

2.7 Assembly of reagent cards

(1) Cutting the prepared gold label strip into small strips with the length of 300mm and the width of 8mm, sticking the small strips on the lower edge of the nitrocellulose membrane of the sheet, and pressing the small strips by 1-2mm through the nitrocellulose membrane;

(2) cutting the sample pad into small strips with the length of 300mm and the width of 17mm, and sticking the small strips to the lower end of the sheet; the lower edge of the sample pad was aligned with the lower edge of the sheet and the upper edge of the sample pad was finished with the white edge of the gold stripe.

(3) The absorbent filter paper was cut into small strips of length 300mm and width 17mm, with the upper edge of the absorbent filter paper aligned with the upper edge of the sheet and the lower edge pressed through the nitrocellulose membrane by about 2 mm.

(4) And pressing the pasted reagent card. Then cut into small strips 3.5mm wide by a cutter.

(5)2.8 Assembly of the kit: and (3) placing the cut small strips with the diameter of 3.5mm into a card shell of the kit to obtain the SARS-CoV-2 neutralizing antibody detection kit.

3 preparation of sample dilution

A solution was prepared by adding 0.6 part of Tris, 0.7 part of bovine serum albumin, 8 parts of sodium chloride, 1.5 parts of laurylpolyoxyethylene (9) ether and 2 parts of Proclin300 per ten thousand parts of water per hundred parts of water, and the pH of the solution was adjusted to 8.0.

4 test

1 drop (about 20-25 mul) of whole blood/serum/plasma is added into the sample adding hole of the kit, then 2 drops (about 75-80 mul) of sample diluent is added into the sample adding hole, the time is kept for 15 minutes, and the detection result can be observed. The detection sensitivity of the kit prepared by the method is 1ng/ml (novel coronavirus neutralizing antibody).

Example 2

In order to further explain the technical effects of the present invention, the following comparative experiments were also conducted.

Comparative experiments with respect to labeling:

1. labeling method with PEG 20000:

1. the colloidal gold solution is weighed into a beaker according to the preparation amount, such as: 100ml of the colloidal gold solution was put into a beaker.

The pH of the gold colloid solution was adjusted to 7.5 with 0.1M potassium carbonate solution. And placed on a magnetic stirrer for stirring.

The concentration of SARS-CoV-2 protein was diluted to 1mg/ml with 2mM Tris buffer, pH7.4, and then 0.8ml of the diluted protein solution was added to the colloidal gold solution. The solution was added with stirring.

Stirring for 25 minutes on a magnetic stirrer;

to 100ml of the above solution was added an 8.5% BSA aqueous solution in an amount of 24. mu.l/ml, and the stirring was continued for 45 minutes.

To the above solution was added 2% PEG20000 in an amount of 20. mu.l/ml, and the stirring was continued for 1 hour.

Transferring the reacted solution into a centrifugal tube;

centrifuging at 4 deg.C at 10000rpm/min for 35 min; and removing the supernatant.

And (3) re-dissolving the precipitate by using 1 part of 2mM Tris buffer solution with the pH value of 7.4 per hundred parts to obtain the gold-labeled S-RBD protein. The gold label prepared in this way was denoted as a gold label.

Labeling method without PEG 20000:

The pH of the gold colloid solution was adjusted to 7.5 with 0.1M potassium carbonate solution. And placed on a magnetic stirrer to stir.

Stirring for 25 minutes on a magnetic stirrer;

to 100ml of the above solution, an aqueous solution of 8.5% BSA was added in an amount of 24. mu.l/ml, and the stirring was continued for 45 minutes.

Transferring the reacted solution into a centrifugal tube;

centrifuging at 12000rpm/min for 35 min at 4 deg.C; and removing the supernatant.

Reconstituting the pellet with 1 part per ten 2mM Tris buffer pH 7.4;

continuously centrifuging the re-dissolved solution at the temperature of 4 ℃ at the centrifugal speed of 12000rpm/min for 40 minutes; and removing the supernatant.

And (3) re-dissolving the precipitate by using 1 part of 2mM Tris buffer solution with the pH value of 7.2 per hundred parts to obtain the gold-labeled S-RBD protein. The markers prepared in this way were labeled as B gold.

3 test

3.1 the prepared gold-labeled S-RBD protein was diluted in 10mM phosphate buffer in percentage and then tested. The two colloidal gold markers are diluted by the same concentration (45 percent and 55 percent); and coating the gold label solution on the polyester films respectively by using a gold spraying machine to prepare the gold label strip.

3.2 the prepared A gold label and B gold label are put into an environment of 45 ℃ for accelerated aging test, and the observation is carried out every 2 weeks. When observing, the concentration of the coating protein used for forming the kit, the NC membrane and the sample pad are the same.

3.3 testing: and respectively adding a negative solution and a positive solution (neutralizing antibody solution) into the sample adding hole of the kit, and testing. Each test was repeated 2 times.

Negative solution: 0.05M Borax buffer pH8.0, containing 0.7 parts BSA, 0.5 parts Tween20 per hundred parts.

The concentrations of the positive solutions (solutions of neutralizing antibodies against SARS-CoV-2) were: 0.1ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 100 ng/ml.

4, test results:

45% concentration gold mark test result statistical table

55% concentration gold standard test result statistical table

Note: in the table, "+" indicates positive, "-" indicates negative, and "+" indicates weak positive.

The test result shows that the positive detection result of the kit prepared by adding the PEG 20000A gold label is always positive along with the progress of the accelerated experiment, and the negative detection result is unchanged. And the positive detectable threshold of the kit prepared by the B gold mark is gradually increased along with the increase of the accelerated aging time, and the kit cannot detect low concentration, so that the stability of the kit is lower than that of the kit prepared by the A gold mark. Namely: the gold-labeled stability of the added PEG20000 is better than that of the non-added PEG.

Comparative experiments with buffer systems:

the buffer system was used to prepare sample pads for processing glass fiber membranes. The specific experiment is as follows:

1. preparing glass fiber treating fluid:

weighing borax according to the amount of 4 parts per hundred of borax, putting the borax into a beaker, adding 80 parts of water into the beaker, and putting the beaker on a magnetic stirrer for stirring.

Adding 0.3 part of ethylenediamine tetraacetate into the beaker, and continuously stirring;

adding 0.1 part of casein salt into the solution, and continuously stirring;

0 part (that is, not containing) of 0.1 part, 0.5 part, 1 part and 1.5 parts of propylene oxide-ethylene oxide-vinyl diamine copolymer (commercially available) are respectively added into the solution and continuously stirred;

adding 2 parts of Proclin300 per ten thousand parts into the solution, and continuing stirring;

to the above solution, 0.5 parts per ten thousand of Tween20 was added and stirring was continued.

After the solution is completely dissolved and clarified, the pH of the solution is adjusted to 8.2 by using hydrochloric acid or sodium hydroxide, and then purified water is used for fixing the volume to the required volume.

The glass fiber treatment solutions containing propylene oxide-ethylene oxide-vinyl diamine copolymers (commercially available) with different concentrations were respectively designated as a0 solution, B0.1 solution, C0.5 solution, D1 solution and E1.5 solution, and were kept at room temperature.

2. Preparation of sample pad

A commercially available glass fiber mat (length: width: 300 mm: 254mm) was prepared;

soaking the glass fiber mat according to the amount of 1 sheet per hundred parts; first, the glass fiber mat was soaked in the solution for 10 minutes, then taken out, and excess water was gently filtered off with a glass rod. Turn over, soak the glass fiber pad into the solution again for 10 minutes, filter off the excess water gently with a glass rod. And obtaining the sample pad.

The resulting sample pad was placed in an oven at 37 ℃ and dried overnight (time 17-20 hours).

After drying, an A0 sample pad, a B0.1 sample pad, a C0.5 sample pad, a D1 sample pad and an E1.5 sample pad were obtained, stored in a closed state and placed in a dry environment.

Testing

(1) Cutting the prepared A-E sample pad into small strips of 17mm x 300mm, assembling the small strips with the same coated sheet, gold labels and absorbent paper to form test strips, and putting the test strips into a reagent box for later use. The kits prepared for the A-E sample pads were designated as A0 kit, B0.1 kit, C0.5 kit, D1 kit, and E1.5 kit.

(2) And respectively adding a negative solution and a positive solution (neutralizing antibody solution) into the sample adding hole of the kit, and testing. One for each test.

Test results

Experimental data show that 0.5-1.5 parts of propylene oxide-ethylene oxide-vinyl diamine copolymer is added into each hundred parts of glass fiber treatment fluid, so that the sensitivity of the product can be effectively improved. Compared with the kit without the sample pad, the kit prepared by adding more than 0.5 parts of sample pad has the advantage that the concentration of the newly detectable coronavirus neutralizing antibody is increased by ten times. The lowest detectable concentration was 1 ng/ml.

Comparative experiments with sample dilutions:

1. preparation of sample diluent

Solutions containing no lauryl polyoxyethylene (9) ether and lauryl polyoxyethylene (9) ether were prepared and compared.

2. Preparation method

2.1 preparation of a solution not containing lauryl Polyoxyethylene (9) Ether

A solution was prepared by adding 0.6 part of Tris, 0.7 part of bovine serum albumin, 8 parts of sodium chloride and 2 parts of Proclin300 per ten thousand parts of water per hundred parts of water, and the pH of the solution was adjusted to 8.0. Is marked as F liquid

2.2 preparation of a solution containing lauryl Polyoxyethylene (9) Ether

Adding 0.6 part of Tris, 0.7 part of bovine serum albumin, 8 parts of sodium chloride and 2 parts of Proclin300 in ten thousand parts of water into each hundred parts of water, and respectively preparing solutions containing 0.5 part of lauryl alcohol polyethylene oxide (9) ether, 1 part of lauryl alcohol polyethylene oxide (1.5) ether, 2 parts of lauryl alcohol polyethylene oxide (9) ether, wherein the solutions are respectively marked as J solution, K solution, L solution and M solution; and the pH of the solution was adjusted to 8.0.

3. And (3) testing: and (3) preparing a positive solution containing a neutralizing antibody by using the solution, adding 3 drops of the solution and the prepared positive solution into a sample adding hole of the kit, testing, timing for 15 minutes, and observing the result.

4. And (3) testing results:

the results show (as shown in fig. 3 and 4) that the test results for the solution containing laurylpolyethylene oxide (9) ether are deep red, superior to those without and that the coloration of the lines is clearly clearer and more aesthetic than those without.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims

1. The novel coronavirus neutralizing antibody qualitative detection kit based on the colloidal gold double-antibody sandwich method comprises a test strip and a sample diluent, wherein the test strip comprises a sheet, a novel coronavirus S-RBD protein marked by nano-colloidal gold, a sample pad, a detection line and a quality control line, and is characterized in that the novel coronavirus S-RBD protein marked by nano-colloidal gold contains BSA (bovine serum albumin) and PEG (polyethylene glycol), the sample diluent comprises a surfactant, a Tris buffer solution and bovine serum albumin, the surfactant is lauryl alcohol polyethylene oxide (9) ether, and the sample pad contains a surfactant epoxypropane-ethylene oxide-vinyl diamine copolymer.

2. The detection kit according to claim 1, wherein the average particle size of the nano-colloidal gold is 30-50 nm.

3. The detection kit according to claim 1, wherein the average molecular weight of PEG is 18000-25000.

4. The test kit of claim 1, wherein the sample diluent further comprises a preservative.

5. The test kit of claim 1, wherein the test line is coated with S protein trimer of SARS-CoV-2, and the quality control line is coated with ACE2 protein.

6. Use of the test kit according to any one of claims 1 to 5 for the preparation of a diagnostic reagent for the detection of neutralizing antibodies against novel coronaviruses.

7. The use according to claim 6, wherein the novel coronavirus neutralizing antibody is derived from whole blood, serum and/or plasma.