阅读说明：本技术 A族链球菌抗原检测试纸条、试剂盒及其制备方法 (Test strip and kit for detecting group A streptococcus antigen and preparation method thereof ) 是由 杨标 金巍 刘中华 王国强 于 2019-08-21 设计创作，主要内容包括：本发明公开了一种A族链球菌抗原检测试纸条、试剂盒及其制备方法,涉及A族链球菌检测技术领域。本发明公开的A族链球菌抗原试纸条,通过将包被包括有抗A族链球菌检测抗体的硝酸纤维素膜上、包被有抗A族链球菌免疫标记抗体的免疫结合垫、吸水纸、样品垫依次贴附到聚氯乙烯底板制得；该检测卡可通过检测标记物的方法检测待检样品中是否存在A族链球菌抗原；该试纸条和包括有该试纸条的检测卡可特异快速地进行A族链球菌早期感染的检测与诊断,减低成本,快速检测,满足临床使用的需要。(The invention discloses a group A streptococcus antigen detection test strip, a kit and a preparation method thereof, and relates to the technical field of group A streptococcus detection. The invention discloses a group A streptococcus antigen test strip which is prepared by sequentially attaching an immunological combination pad, water absorption paper and a sample pad coated with an anti-group A streptococcus immunological marker antibody on a nitrocellulose membrane coated with an anti-group A streptococcus detection antibody to a polyvinyl chloride base plate; the detection card can detect whether the group A streptococcus antigen exists in a sample to be detected by a method for detecting a marker; the test strip and the test card comprising the test strip can specifically and quickly detect and diagnose the early infection of the group A streptococcus, reduce the cost, quickly detect and meet the requirement of clinical use.)

1. A preparation method of a group A streptococcus antigen test strip is characterized by comprising the following steps:

a. Adding potassium carbonate into the colloidal gold solution to adjust the pH, sequentially adding an anti-group A streptococcus antibody and a bovine serum albumin solution, centrifuging to obtain a precipitate, and resuspending the precipitate with a resuspension solution to obtain a colloidal gold-labeled anti-group A streptococcus antibody;

b. Sticking the nitrocellulose membrane on a polyvinyl chloride base plate to obtain a polyvinyl chloride base plate stuck with the nitrocellulose membrane, and placing the polyvinyl chloride base plate in room temperature, and storing the polyvinyl chloride base plate in a sealed way at room temperature for later use under the condition that the humidity is less than or equal to 30% or by adding a drying agent;

c. Diluting an anti-group A streptococcus antibody by using a coating solution to obtain a detection line working solution; diluting goat anti-mouse IgG polyclonal antibody with coating solution to obtain a control line working solution;

d. D, spraying gold on the binding pad by using the colloidal gold labeled anti-group A streptococcus antibody obtained in the step a to obtain an immune binding pad, respectively carrying out scribing treatment on the nitrocellulose membrane on the polyvinyl chloride bottom plate of the pad obtained in the step b by using the detection line working solution and the goat anti-mouse IgG multi-antibody control line working solution obtained in the step c, and drying at room temperature and under the condition that the humidity is less than or equal to 30%;

e. And sequentially assembling the sample pad, the immunological combination pad, the polyvinyl chloride base plate with the film and the absorbent paper, and cutting to obtain the A-group streptococcus antigen detection test strip.

2. the method for preparing a group A streptococcus antigen test strip according to claim 1, characterized in that: adding 0.2M potassium carbonate into the colloidal gold solution, uniformly mixing, adjusting pH, adding an anti-group A streptococcus antibody, uniformly mixing, standing for 30min, adding a bovine serum albumin solution containing 10% of bovine serum albumin, uniformly mixing, standing for 30min to obtain a mixed solution, centrifuging the mixed solution for 30min at 4 ℃ and at a rotating speed of 8000g to obtain a precipitate, and suspending the precipitate with a heavy suspension to 1/3 of the volume of the mixed solution to obtain the anti-group A streptococcus antibody solution marked by the colloidal gold.

3. The method for preparing a group A streptococcus antigen test strip according to claim 1 or 2, characterized in that: in the step a, the potassium carbonate is 0.1-0.2M potassium carbonate solution;

The concentration (W/V) of the colloidal gold solution is 0.01-0.04%

The volume ratio of the potassium carbonate solution to the colloidal gold solution is as follows: 1-10 uL: 1 ml;

The bovine serum albumin solution contains 10% of bovine serum albumin;

the volume of the bovine serum albumin solution is 1-10% of the volume of the colloidal gold solution;

The resuspension is 50mM tris solution;

The tris solution contains per 100 ml: 1g BSA, 0.5g polyvinylpyrrolidone, 2g trehalose and 4g sucrose, pH 8.5.

4. The method for preparing a group A streptococcus antigen test strip according to claim 1, characterized in that: in the step c, the coating solution is 0.01M-0.02M phosphate buffer solution, each 10mL of the coating solution contains 0.2g of trehalose, and the pH value is 7;

The concentration of the anti-group A streptococcus antibody in the detection line working solution is 0.8-1.2 mg/mL; the concentration of goat anti-mouse IgG polyclonal antibody in the working solution of the control line is 0.8-1.2 mg/mL.

5. The method for preparing a group A streptococcus antigen test strip according to claim 1, characterized in that: in the step d, the gold spraying treatment and the scribing treatment are both realized by gold spraying and film scribing equipment;

The bonding pad is a glass fiber membrane.

6. The method for preparing a group A streptococcus antigen test strip according to claim 1, characterized in that: in the step e, the sample pad is a glass fiber membrane;

The sample pad is dried after being soaked with a sample pad treatment solution before use, wherein the sample pad treatment solution is 0.05M tris solution, and the tris solution contains 0.5g casein, 0.5g surfactant S9 and 1.0mL Tween 20 per 100mL, and has a pH of 8.4-8.6.

7. A preparation method of a group A streptococcus antigen detection card is characterized by comprising the following steps: placing the group a streptococcus antigen test strip obtained in step e of claim 1 in a plastic housing, wherein the well of the plastic housing is above the sample pad and the viewing window of the plastic housing is above the test line T and the control line C.

8. A preparation method of a group A streptococcus antigen kit is characterized by comprising the following steps: the group A streptococcus toxigenic antigen detection kit obtained in claim 7 is put into an aluminum foil bag, and is packaged with a sampling straw and an instruction box to form the group A streptococcus antigen detection kit, wherein the sampling straw is optional.

9. The utility model provides a test paper strip of A clan streptococcus antigen, test paper strip top-down paste by sample pad (1), immune combination pad (2), nitrocellulose membrane (3) and water absorption pad (6) in proper order and constitute on polyvinyl chloride bottom plate (7), wherein, the A clan streptococcus antibody of mark colloidal gold that coats on the immune combination pad, nitrocellulose membrane are equipped with detection line T and control line C, and detection line has wrapped anti A clan streptococcus monoclonal antibody, and the control line has wrapped goat anti mouse IgG polyclonal antibody.

10. a group a streptococcus antigen detection card comprising a plastic housing (8) containing the strip of claim 9, wherein the plastic housing is provided with a well (9) above the sample pad and a window (10) above the test line T and the control line C.

11. A kit for detecting group A streptococcus antigen, which is obtained by boxing the detection card, the sampling pipette and the instruction in claim 10.

The technical field is as follows:

The invention relates to the technical field of group A streptococcus detection, in particular to a group A streptococcus antigen detection card and a preparation method and a detection kit thereof.

Background art:

group A streptococci (Strep-A), also known as Streptococcus pyogenes, are important pathogenic bacteria of humans worldwide. It can not only cause infection alone, but also cause mixed infection with other suppurative bacteria. Patients, especially children, are susceptible to infection-resistant treatment in short term, and are susceptible to acute pharyngitis, pyocytic disease, toxic shock syndrome, invasive fasciitis, scarlet fever, pneumonia, erysipelas, and allergic diseases, such as Acute Rheumatic Fever (ARF), Rheumatic Heart Disease (RHD), acute glomerulonephritis, etc.

it is estimated that 51.7 million people worldwide die from severe Strep-A infection and disease every year, and another 1810 million people have severe Strep-A infection, 178 million new patients every year, of which 66.3 million are invasive Strep-A infections and 16.3 million die. Strep-A in China is an extremely important pathogenic bacterium, scarlet fever induced by Strep-A is a national B-type infectious disease, about 5-6 million people are attacked every year on average, the Strep-A attracts people's extensive attention, and the Strep-A is a global hotspot subject.

At present, kits for detecting group A streptococcus are lacking in the market.

the invention content is as follows:

The invention aims to provide a group A streptococcus antigen test strip. The A group streptococcus antigen test strip can specifically and quickly detect and diagnose the early infection of A group streptococcus, reduces the cost and has high detection speed.

The invention also aims to provide a preparation method of the test strip for the group A streptococcus antigen. The group A streptococcus antigen detection card obtained by the preparation method is simple and convenient to apply, accurate in result, provides a basis for the next step of accurately treating diseases caused by group A streptococcus, and is suitable for large-scale popularization and application.

Another object of the present invention is to provide a test card and a kit comprising the above test strip for group A Streptococcus antigens. The kit can specifically and quickly detect and diagnose the early infection of the group A streptococcus, reduces the cost, quickly detects and effectively meets the requirement of clinical use.

In order to realize the purpose, the invention provides a preparation method of a group A streptococcus antigen detection test strip, which adopts the following technical scheme:

a. Adding potassium carbonate into the colloidal gold solution to adjust the pH, sequentially adding an anti-group A streptococcus antibody and a bovine serum albumin solution, centrifuging to obtain a precipitate, and resuspending the precipitate with a resuspension solution to obtain a colloidal gold-labeled anti-group A streptococcus antibody;

b. Sticking the nitrocellulose membrane on a polyvinyl chloride base plate to obtain a polyvinyl chloride base plate stuck with the membrane;

c. Diluting an anti-group A streptococcus antibody by using a coating solution to obtain a detection line working solution; diluting goat anti-mouse IgG polyclonal antibody with coating solution to obtain a control line working solution;

d. D, spraying gold on the binding pad by using the colloidal gold labeled anti-group A streptococcus antibody obtained in the step a to obtain an immune binding pad, and respectively carrying out scribing treatment on the nitrocellulose membrane on the polyvinyl chloride bottom plate of the adhesive film obtained in the step b by using the detection line working solution and the goat anti-mouse IgG multi-antibody control line working solution obtained in the step c;

e. Sequentially assembling a sample pad, an immunological combination pad, a polyvinyl chloride base plate with a film and absorbent paper as shown in figure 1, and cutting to obtain a group A streptococcus antigen detection test strip;

Preferably, the specific steps of step a in the above technical solution are: adding 0.2M potassium carbonate into the colloidal gold solution, uniformly mixing, adjusting pH, adding the anti-group A streptococcus antibody, uniformly mixing, standing for 30min, adding a bovine serum albumin solution containing 10% of bovine serum albumin, uniformly mixing, standing for 30min to obtain a mixed solution, centrifuging the mixed solution for 30min at 4 ℃ and a rotating speed of 8000g to obtain a precipitate, and resuspending the precipitate to 1/3 of the volume of the mixed solution by using a resuspension solution to obtain the colloidal gold-labeled anti-group A streptococcus antibody solution.

Preferably, in step a in the above technical scheme, the potassium carbonate is a 0.1-0.2M potassium carbonate solution.

preferably, the concentration (W/V) of the colloidal gold solution in step a in the above technical solution is 0.01% -0.04%.

preferably, in the above technical scheme, the volume ratio of the potassium carbonate solution to the colloidal gold solution in step a is: 1-10 uL: 1 ml.

preferably, the ratio of the concentration of the anti-group A streptococcus antibody in the step a to the colloidal gold solution in the technical scheme is 1-50ug:1 mL.

Preferably, the bovine serum albumin solution in step a of the above technical solution contains 8-12% of bovine serum albumin.

Preferably, in the above technical scheme, the volume of the bovine serum albumin solution added in the step a is 1-10% of the volume of the colloidal gold solution;

preferably, the resuspension in step a in the above technical scheme is 48-52mM Tris solution.

Preferably, the tris solution in step a of the above technical solution contains per 100 ml: 0.8-1.2g BSA, 0.48-0.52g polyvinylpyrrolidone, 1.8-2.2g trehalose and 3.8-4.2g sucrose, pH 8.4-8.6.

Preferably, the coating solution in step c of the above technical scheme is 0.01M-0.02M phosphate buffer solution, each 10mL of the coating solution contains 0.18-0.22g of trehalose, and the pH is 7-8.

Preferably, in the above technical solution, the concentration of the group a streptococcus antibody in the working solution of the detection line in step c is 0.8-1.2 mg/mL; the concentration of goat anti-mouse IgG polyclonal antibody in the working solution of the control line is 0.8-1.2 mg/mL.

Preferably, in the above technical solution, the gold spraying treatment and the scribing treatment in step d are both realized by a gold spraying and film scribing device.

Preferably, in the above technical solution, the bonding pad in step d is a glass fiber membrane.

Preferably, in the above technical solution, the sample pad in step e is a glass fiber membrane;

Preferably, the sample pad in step e of the above technical scheme is dried after being soaked in a sample pad treatment solution before use, wherein the sample pad treatment solution is 0.05M tris solution, and each 100mL tris solution contains 0.5g of casein, 0.5g of surfactant S9 and 1.0mL of Tween 20, and the pH is 8.5.

The invention provides a preparation method of a group A streptococcus antigen detection card, which is characterized in that the obtained group A streptococcus antigen detection test strip is placed in a plastic shell, wherein a sample adding hole of the plastic shell is arranged above a sample pad, and an observation window of the plastic shell is arranged above a detection line T and a comparison line C.

The invention provides a preparation method of a group A streptococcus antigen kit, which is characterized in that the group A streptococcus antigen detection kit obtained by the method is put into an aluminum foil bag, and is packed with a sampling straw and an instruction book to form the group A streptococcus antigen detection kit, wherein the sampling straw is selectable.

The invention provides a test strip for a group A streptococcus antigen, which is formed by sequentially sticking a sample pad, an immunological combination pad, a nitrocellulose membrane and a water absorption pad on a polyvinyl chloride bottom plate from top to bottom, wherein the immunological combination pad is coated with a group A streptococcus antibody for marking colloidal gold, the nitrocellulose membrane is provided with a detection line T and a control line C, the detection line is coated with an anti-group A streptococcus monoclonal antibody, and the control line is coated with a goat anti-mouse IgG polyclonal antibody.

The invention provides a group A streptococcus antigen detection card, which comprises a plastic shell provided with the test strip, wherein the plastic shell is provided with a sample adding hole and an observation window, the sample adding hole is arranged above a sample pad, and the observation window is arranged above a detection line T and a comparison line C.

The invention provides a group A streptococcus antigen detection kit, which is obtained by boxing the detection card, a sampling suction pipe and an instruction book.

The beneficial effects of the invention include:

(1) According to the preparation method of the A-group streptococcus antigen test strip, the treated sample pad achieves the effect of reducing the interference of other pathogens in the detection process, so that false positive is eliminated, detection is not missed, the detection result is further improved, and the test strip has good specificity.

(2) The group A streptococcus antigen detection card prepared by the method is based on an immune lateral flow chromatography technology, does not need any instrument or equipment, does not need to dilute a sample, is simple, convenient and quick, can judge whether the sample contains the group A streptococcus antigen or not through manual operation and naked eye reading, does not cause harm to the body of a detected person, and is high in detection speed and low in cost.

Description of the drawings:

FIG. 1 is a schematic structural diagram of a group A streptococcus antigen detection test strip according to an embodiment of the present invention;

FIG. 2 is a perspective view of a group A Streptococcus antigen detection card in an embodiment of the present invention;

FIG. 3 is a top view of a group A Streptococcus antigen detection test card in an embodiment of the present invention;

FIG. 4 is a graph showing the results of detecting different concentrations of group A streptococcus using the group A streptococcus antigen detection card according to the embodiment of the present invention;

FIG. 5 shows the detection results of the group A streptococcus antigen detection card in the embodiment of the present invention for detecting different pathogenic bacteria.

In the figure: 1- -sample pad; 2-an immunological binding pad; 3-nitrocellulose membrane; 4-detection line; 5-quality control line; 6-absorbent paper; 7-a polyvinyl chloride backplane; 8-a plastic housing; 9-a sample addition hole; 10-observation window.

the specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

the features and properties of the present invention are described in further detail below with reference to examples.

the immunochromatographic detection test strip shown in FIG. 1 is composed of a sample pad, an immunological combination pad, a nitrocellulose membrane and a water absorption pad which are stuck on a polyvinyl chloride base plate from top to bottom in sequence; the combination pad is coated with an immune labeled antibody, and the nitrocellulose membrane is coated with a detection antibody. During detection, the immune labeled antibody and the antibody are respectively combined with a detected substance to form a sandwich structure.

preferably, the immunological combination pad is marked with an anti-group A streptococcus immunological marker antibody, and the nitrocellulose membrane is coated with a detection antibody of anti-group A streptococcus.