CN116183904A - Simian pox antigen, HIV1/2 antibody and P24 antigen joint detection test strip and preparation method thereof - Google Patents

Abstract

The invention relates to the field of antigen-antibody detection, in particular to a joint detection test strip for monkey pox antigen, HIV1/2 antibody and P24 antigen for oral effusion and a preparation method thereof. According to the invention, the detection of the monkey pox antigen, the HIV1/2 antibody and the P24 antigen is introduced into the same colloidal gold test strip for the first time, so that triple detection is realized, the detection of the HIV is carried out simultaneously when the monkey pox is detected, the HIV potentially infected people can be detected in time, and the invisible transmission risk of the HIV is reduced.

Description

Combined test strip for monkeypox antigen, HIV1/2 antibody and P24 antigen and preparation method thereof

Technical Field

The present invention relates to the field of antigen-antibody detection, and in particular to a joint detection test strip of monkeypox antigen, HIV1/2 antibody and P24 antigen and a preparation method thereof. The test strip is particularly suitable for realizing sensitive detection of oral exudate.

Background Art

On July 21, 2022, Geneva time, the World Health Organization held its second meeting on the monkeypox outbreak in multiple countries, and subsequently announced that the monkeypox epidemic constituted a "Public Health Emergency of International Concern" (PHEIC), which is the highest level of alert that WHO can issue.

Monkeypox is a zoonosis caused by the monkeypox virus (MPXV) and was first discovered in the primeval forests of West and Central Africa. The incubation period of monkeypox is usually 6 to 13 days, but can also be 5 to 21 days. The typical clinical manifestation is a short prodromal period of fever, followed by a gradual development of a typical rash with lesions such as induration and central depression; the rash starts on the head or face, develops to the limbs and trunk, and may affect the eyes and genital area. The rash period undergoes the evolution of macules, papules, vesicles, and pustules, which dry up and scab off after 2 to 4 weeks. The number of rashes varies from person to person, and can bleed or merge into large blisters. When the rash appears, the patient is contagious. Monkeypox can be transmitted through contaminants such as broken skin or mucous membranes, blood, body fluids, respiratory droplets (requires prolonged face-to-face contact), and bedding of infected people, and close contact during sexual intercourse, and can also be transmitted through the placenta. Although monkeypox is not as deadly as smallpox, the mortality rate of Central African monkeypox can be as high as 10% (in contrast, the mortality rate of West African monkeypox is 1%), which is still a serious threat to human social security. Although the mortality rate of monkeypox is much lower than that of smallpox, the scars left by infection are comparable to disfigurement.

Acquired Immunodeficiency Syndrome (AIDS), also known as AIDS, is a serious infectious disease caused by human immunodeficiency virus (HIV) infection. It is currently incurable and is a chronic progressive disease with a high mortality rate. Therefore, in terms of AIDS control, early diagnosis, timely detection of infected persons and control of further transmission have become the top priorities of AIDS prevention and control. The transmission routes of HIV mainly include blood transmission, sexual contact, drug injection and mother-to-child transmission. According to the different clinical manifestations of AIDS, it can be divided into acute phase, asymptomatic phase and AIDS phase. The most common clinical manifestation is fever, which may be accompanied by sore throat, night sweats, nausea, vomiting, diarrhea, rash, joint pain, lymphadenopathy and neurological symptoms. Most patients have mild clinical symptoms, which last for 1 to 3 weeks and then resolve on their own. HIV RNA and p24 antigen can be detected in the blood during this period. HIV antibodies can appear in the serum of most patients 2 to 6 weeks after HIV1/2 infection, which can be used as an important indicator for early diagnosis and disease screening. At present, the fourth-generation HIV detection reagents that simultaneously detect HIV-1 p24 antigen and HIV1/2 antibodies are gradually replacing the third-generation reagents that only detect HIV1/2 antibodies, which also shortens the detection window period from 21 days to 14 days.

Since May 2022, there have been many atypical cases of monkeypox around the world. According to data survey statistics, since the monkeypox epidemic is mainly prevalent among gay men, a large number of patients have rashes starting from the genitals and perianal areas, and then spread to other parts of the body. The main epidemic groups of the monkeypox epidemic are also the main high-risk groups for the spread of AIDS. In the 1980s, 80% of HIV-infected people in the United States were men who had sex with men. According to the survey on the knowledge and influencing factors of monkeypox prevention among Chinese men who have sex with men, 3563 MSM were effectively surveyed this time, 53.4% (1903/3563) had engaged in male-male sex within 1 month before the survey, 25.7% (916/3563) reported HIV infection, and 2.7% had a history of travel to monkeypox epidemic areas.

At present, the monkeypox virus nucleic acid detection kit (fluorescence PCR method) is the main detection method for monkeypox virus at home and abroad, but this detection method is highly dependent on equipment, has a long detection time, requires certain professionalism from the detection personnel, and is more prone to contamination.

When testing for monkeypox virus, the low awareness of HIV infection may result in potential infected people not actively testing for HIV, and thus not knowing whether they are at risk of contracting AIDS, which invisibly increases the risk of HIV transmission. In addition, the detection and diagnosis of monkeypox and HIV usually use single tests, which are laborious and time-consuming, and often require the use of specific instruments and equipment.

Colloidal gold immunochromatography has the advantages of simple and convenient testing method, fast detection speed, easy storage of the detection kit, which can be stored for 18 months at room temperature, no need for a refrigerator, no need for any instruments and special technical operation training, reliable results, good repeatability, etc. It is easy to be mastered by grassroots organizations and promoted on a large scale, and can play a positive role in various fields.

Currently, there are few colloidal gold detection reagents for detecting monkeypox antigen on the market. Although there are many colloidal gold test strips for detecting HIV, most of them detect HIV antibodies or HIV antigens alone. In addition, single tests may result in false negatives due to low loads of antigens or antibodies as the test objects during a certain period of time.

Summary of the invention

In view of the technical problems existing in the prior art, the present invention provides a combined detection test strip for monkeypox antigen, HIV1/2 antibody and P24 antigen, which for the first time introduces the detection of monkeypox antigen, HIV 1/2 antibody and P24 antigen into the same colloidal gold test strip, thus realizing triple detection.

The present invention achieves the above technical objectives through the following technical solutions:

The invention provides a joint test strip for monkeypox antigen, HIV1/2 antibody and P24 antigen, the test strip comprising a PVC bottom plate with adhesive, a sample pad, a gold label pad 2, a gold label pad 1, a chromatography membrane and a water absorption pad arranged in sequence from bottom to top along the width direction of the bottom plate, and adjacent components are connected to each other;

The gold label pad 1 is solidified with colloidal gold-labeled biotinylated HIV1/2 antigen and colloidal gold-labeled biotinylated P24 antibody, and the gold label pad 2 is solidified with colloidal gold-labeled biotinylated monkeypox antibody and streptavidin-labeled colloidal gold;

The chromatographic membrane comprises detection lines and quality control lines which are arranged in parallel and spaced from the end close to the sample pad to the end close to the absorbent pad. The detection lines include at least three, which are respectively a T1 detection line coated with mouse anti-monkeypox monoclonal antibodies that can specifically bind to monkeypox antigens, a T2 detection line coated with mouse anti-HIV P24 monoclonal antibodies that can specifically bind to HIV P24 antigens, and a T3 detection line coated with HIV 1/2 antigens that can specifically bind to HIV 1/2 antibodies.

Preferably, the chromatography membrane is a nitrocellulose membrane, and the transit time of the nitrocellulose membrane is 130 to 150 seconds.

Preferably, the sample pad is prepared by treating with a sample pad treatment solution, wherein the sample pad treatment solution is prepared as follows: prepare 10-100 mM Tris-HCl as a base solution, then add 0.1-1% m/v casein and 0.1-0.3% m/v skim milk powder to dissolve for 8-24 hours, then add 0.1-0.8% v/v Tween 20, 0.1-0.8% m/v surfactant S9, 0.5-5% m/v PVP-10, 0.05-0.15% v/v PC300, 0.1-0.4% m/v EDTA-2Na, 0.05-3 mg/mL active heterophilic antibody blocker, 0.1-0.4 mg/mL anti-RBC monoclonal antibody, and adjust the pH to 7.0-9.0.

Preferably, the sample pad is selected from one of glass cellulose membrane, non-woven fabric, polyester cellulose membrane, and whole blood filtration membrane. Further, the sample pad is glass cellulose membrane.

Preferably, the method for labeling antigen/antibody with colloidal gold comprises the following steps:

First, the antigen or antibody is biotinylated to obtain a biotinylated antigen or antibody, and then it is added to a colloidal gold solution adjusted to a preset pH range, polyethylene glycol is added after stirring, and the solution is stirred again and the precipitate is collected, and the precipitate is redissolved with a colloidal gold solution and mixed to obtain a biotinylated antigen or antibody;

When colloidal gold is used to label biotinylated monkeypox antibody, the preset pH of the colloidal gold is 8.0-8.4; when colloidal gold is used to label biotinylated P24 antibody, the preset pH of the colloidal gold is 8.8-9.2; when colloidal gold is used to label biotinylated HIV1/2 antigen, the preset pH of the colloidal gold is 8.3-8.7; further, in the method of colloidal gold labeling antigen/antibody, the particle size of the colloidal gold is 20 nm. If the particle size of the colloidal gold is increased, there may be steric hindrance, which will affect the specific binding of the antibody and antigen.

Preferably, the method for labeling colloidal gold with streptavidin comprises the following steps: adjusting the pH of the colloidal gold solution to 8.8-9.2, adding streptavidin, adding polyethylene glycol after stirring, stirring again and collecting the precipitate, re-dissolving the precipitate with a colloidal gold re-solution and mixing, and obtaining; further, the particle size of the colloidal gold in the streptavidin-labeled colloidal gold is 40 nm. Since the streptavidin labeling can be used in three projects at the same time, the use of colloidal gold with a large particle size can improve the detection sensitivity of the three projects.

Preferably, the preparation method of the gold label pad 1 comprises the following steps: treating the gold label pad with a gold label pad treatment solution to obtain a conjugate pad, spraying a colloidal gold-labeled biotinylated HIV1/2 antigen and a colloidal gold-labeled biotinylated P24 antibody on the conjugate pad at concentrations of OD 1.1 to 1.3 and 0.8 to 1.2 and a gold spraying volume of 2 to 4 μL/cm, and baking to obtain the conjugate pad.

Preferably, the preparation method of the gold label pad 2 comprises the following steps: treating the gold label pad with a gold label pad treatment solution to obtain a conjugate pad, spraying the colloidal gold-labeled biotinylated monkeypox antibody and streptavidin-labeled colloidal gold on the conjugate pad at concentrations of OD 0.8 to 1.2 and OD 0.6 to 0.8, and a gold spraying volume of 2 to 4 μL/cm, and baking to obtain the conjugate pad.

Preferably, the gold label pad is selected from glass cellulose membrane and polyester cellulose membrane.

Preferably, the gold label pad treatment solution comprises: 0.1-0.3% v/v Triton X-100, 0.5-1.5% m/v BSA, 15-25% m/v sucrose, 3-7% m/v trehalose, and 0.1 M Tris-HCl at pH 7.5.

Compared with the prior art, the present invention has the following beneficial effects:

(1) For the first time, the detection of monkeypox antigen, HIV 1/2 antibody and P24 antigen was introduced into the same colloidal gold test strip, and through optimization, a rapid triple test of monkeypox antigen, HIV 1/2 antibody and P24 antigen was realized, which can detect HIV while detecting monkeypox. For high-risk groups infected with monkeypox and HIV at the same time, due to the long window period of HIV, it may be difficult to be detected when performing single HIV antigen or antibody tests. While detecting monkeypox, HIV antigen and antibody joint tests were performed, which not only shortened the window period of HIV detection, but also roughly judged the infection stage of the infected person based on the HIV antigen and antibody test results, providing a basis for further diagnosis and treatment;

(2) Furthermore, the combined test strip combines colloidal gold immunochromatography technology with the biotin-avidin system, uses 20nm colloidal gold to label antigens/antibodies to ensure strong specificity during the immunochromatography reaction, and uses 40nm colloidal gold and streptavidin alone to achieve amplification of the detection signal and enhanced color development in the triple test. The combination of biotin and avidin is stable and specific, and is not affected by reagent concentration, pH environment, or organic solvents such as protein denaturants. Each avidin can combine 4 molecules of biotin to construct a multi-level amplification system to amplify the detection signal, improve color development, and solve the problem of poor detection sensitivity that may exist in the combined test. It can be used for sensitive detection of oral exudate, and has the advantages of being non-invasive, rapid, no need for special detection sites, no need for special detection instruments, and the remaining samples do not need to be treated as biohazard samples. It is more easily accepted by the general public, expanding the detection coverage of the two, timely discovering potential infected persons, and reducing the risk of transmission;

(3) Spray the streptavidin with colloidal gold label on the gold label pad and stick it under the sample pad with an overlap of 2 mm. In this way, the colloidal gold-labeled streptavidin can be used in all test items of the test strip, avoiding repeated development of different test items and improving the utilization rate of raw materials;

(4) Oral exudate was used for testing, and the sensitivity of monkeypox antigen detection could reach 7pg/mL, and the sensitivity of HIV1P24 antigen detection could reach 0.15625IU/mL. Compared with other detection methods, this combined test strip has the advantages of rapid detection, easy operation, no reliance on instruments and equipment, no need for special professional skills training, and simple and reliable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic structural diagram of a colloidal gold test strip prepared in Example 1 of the present invention;

FIG. 2 is a diagram showing the positive detection results in Example 2 of the present invention.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

The following embodiments are only used to illustrate the present invention, but are not used to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

In the examples of the present invention, the raw materials used are all conventional commercially available products.

Example 1

The embodiment of the present invention provides a method for preparing a colloidal gold immunochromatographic test strip for detecting monkeypox antigen, HIV1/2 antibody and P24 antigen, which is specifically as follows:

The treated sample pad, gold label pad (biotinylated antigen/antibody labeled with colloidal gold, streptavidin labeled with colloidal gold), NC membrane (coated with mouse anti-monkeypox antigen, mouse anti-HIV1/2 antibody, HIV1/2 antigen and goat anti-mouse IgG), and absorbent pad were sequentially pasted on the 8cm PVC bottom plate. The details are as follows:

(1) Preparation of sample pad:

JY-J101 Ahlstrom 8964 glass fiber (purchased from Shanghai Jieyi Biotechnology Co., Ltd.) was soaked in the sample pad treatment solution at room temperature. After 12 hours, the glass fiber membrane was taken out and weighed to control the amount of solution carried by each glass cellulose membrane (at 47±0.3g). The glass fiber was baked in an oven at 37°C for 24 hours to obtain the sample pad.

The preparation method of the sample pad treatment solution is as follows: prepare 50mM Tris-HCl as the base solution, add 0.5% (m/v) casein and 0.2% (m/v) skim milk powder to dissolve for 12 hours, then add 0.2% (v/v) Tween20, 0.2% (m/v) surfactant S9, 1.5% (m/v) PVP-10 (polyvinyl pyrrolidone), 0.1% (v/v) PC-300, 0.3% (m/v) EDTA-2Na, 1.0mg/mL active heterophilic antibody blocker (purchased from Roche Diagnostics (Shanghai) Co., Ltd.), 0.3mg/mL anti-RBC monoclonal antibody (purchased from Beijing Mozhidong Biotechnology Co., Ltd.), and adjust the pH to 8.0 _.

(2) Preparation of gold label pad:

The labeled monkeypox antibody (purchased from Feipeng Biotechnology Co., Ltd.), P24 antibody (purchased from BiotechAtlantic InC.), and HIV1/2 antigen (purchased from CTK Biotechnology Co., Ltd., USA) were added to 10mM PBS buffer respectively, dialyzed at 4°C overnight, and then pre-activated biotin was used to label the monkeypox antibody, P24 antibody, and HIV1/2 antigen to obtain biotinylated monkeypox antibody, biotinylated P24 antibody, and biotinylated HIV1/2 antigen. The specific operation is as follows: 1 mg of labeled raw material (antibody or antigen) was taken, and biotin (purchased from Shenzhen Newbon Biotechnology Co., Ltd.) was added at a molar ratio of 1:100 to the labeled raw material/biotin, with a total volume of 1mL, incubated at room temperature for 4h, diluted to 5 times the volume of the reaction solution with 10mM, pH7.4 PB buffer, dialyzed at 4°C for 72h, and taken out for use.

a. Biotinylated monkeypox antibody labeling: Use a measuring cylinder to measure the required amount of 20 nm colloidal gold solution into a clean beaker, place it on a magnetic stirrer and stir while adjusting the pH to 8.2 with 0.2 mol/L potassium carbonate solution, take biotinylated monkeypox antibody, and label it with colloidal gold at 6 μg/mL, add the biotinylated monkeypox antibody to the colloidal gold, stir on a magnetic stirrer for 30 minutes, add 0.5‰ (m/v) polyethylene glycol, stir for another 30 minutes and then centrifuge, collect the precipitate, and redissolve it with a colloidal gold reconstitution solution at a volume percentage of 3%, stir on a magnetic stirrer until mixed evenly, and obtain a colloidal gold-biotinylated monkeypox antibody conjugate reconstitution solution with a volume percentage of 3%, and measure the absorbance at a wavelength of 520 nm, OD: 1.8-2.1, and store it at 4°C for use;

b. Biotinylated P24 antibody labeling: Use a measuring cylinder to measure the required amount of 20 nm colloidal gold solution into a clean beaker, place it on a magnetic stirrer and stir while adjusting the pH to 9.0 with 0.2 mol/L potassium carbonate solution, take biotinylated P24 antibody, and label it with colloidal gold at 6 μg/mL, add biotinylated P24 antibody to colloidal gold, stir on a magnetic stirrer for 30 minutes, add 0.5‰ (m/v) polyethylene glycol, stir for another 30 minutes and then centrifuge, collect the precipitate, and redissolve it with colloidal gold reconstitution solution at 3% by volume, stir on a magnetic stirrer until mixed evenly, and obtain a colloidal gold-biotinylated P24 antibody conjugate reconstitution solution with a volume percentage of 3%, measure the absorbance at a wavelength of 520 nm, and store it at 4° C. for use;

c. Biotinylated HIV1/2 antigen labeling: Use a measuring cylinder to measure the required amount of 20nm colloidal gold solution into a clean beaker, place it on a magnetic stirrer, and adjust the pH to 8.5 with 0.2mol/L potassium carbonate solution while stirring. Take biotinylated HIV1/2 antigen and add it to colloidal gold, and label the colloidal gold at 5μg/mL. Add biotinylated HIV1/2 antigen to colloidal gold, stir on a magnetic stirrer for 30 minutes, add 0.5‰ (m/v) polyethylene glycol, stir for another 30 minutes and then centrifuge, collect the precipitate, and re-dissolve it with colloidal gold re-solution at a volume percentage of 3%, stir on a magnetic stirrer until mixed evenly, and obtain a colloidal gold-biotin HIV1/2 antigen conjugate re-solution with a volume percentage of 3%, and measure the absorbance at a wavelength of 520nm. OD and store it at 4°C for use;

d. Preparation of streptavidin-labeled colloidal gold: Use a measuring cylinder to measure the required labeled amount of 40 nm colloidal gold solution into a clean beaker, place it on a magnetic stirrer and stir while adjusting the pH to 9.0 with 0.2 mol/L potassium carbonate solution, add streptavidin (final concentration 12.4 μg/mL), stir on a magnetic stirrer for 30 minutes, add 0.5‰ (m/v) polyethylene glycol, stir for another 30 minutes and then centrifuge, collect the precipitate, re-dissolve it with a colloidal gold re-solution at a volume percentage of 3%, stir on a magnetic stirrer until mixed evenly, and obtain a colloidal gold-streptavidin re-solution with a volume percentage of 3%, and measure the absorbance at a wavelength of 540 nm, OD: 1.8-2.1, and store it at 4°C for use after measuring OD.

The preparation method of the gold label pad is as follows:

After soaking 8955 glass cellulose membrane (purchased from Shanghai Jieyi Biotechnology Co., Ltd.) in the treatment solution for 4 hours, the amount of solution carried by each glass cellulose membrane was taken out and weighed to control the amount (at 42±0.2 g), and then baked in an oven at 37°C for 24 hours to obtain a binding pad, wherein the treatment solution includes 0.2% (v/v) Triton X-100, 1% (m/v) BSA (bovine serum albumin), 20% (m/v) sucrose, 5% (m/v) trehalose, and 0.1 M Tris-HCl at pH 7.5;

Colloidal gold-labeled biotinylated HIV1/2 antigen and colloidal gold-labeled biotinylated P24 antibody were diluted with colloidal gold complex solution, respectively, and then mixed and sprayed on the same treated binding pad at a concentration of OD 1.2 and 1.0, and a gold spraying volume of 3 μL/cm, and then baked in an oven at 37°C for 24 hours to prepare a gold-labeled pad 1;

Colloidal gold-labeled biotinylated monkeypox antibody and streptavidin-labeled colloidal gold were diluted with colloidal gold diluent (i.e., colloidal gold complex solution) respectively, and then mixed and sprayed on the same treated conjugate pad at a concentration of OD 1.0 and 0.8 and a gold spraying volume of 3 μL/cm, and then baked in a 37° C. oven for 24 hours to prepare a gold surface pad 2.

The colloidal gold solution preparation method is as follows: the colloidal gold is prepared by heating 100 mL of 0.01% chloroauric acid solution to boiling, rapidly adding 1% sodium citrate solution (to prepare 20 nm and 40 nm colloidal gold solutions, 2.5 mL and 1 mL are added respectively) while stirring at high speed, and reacting under constant temperature reflux under stirring. After stopping heating, stirring needs to be continued for 15 minutes, and the prepared colloidal gold solution is stored at 4°C for use.

The preparation method of colloidal gold complex solution is as follows: prepare 20mM Tris-HCl as the base solution, add 2% (m/v) BSA, 0.2% (v/v) Tween20, 10% (m/v) sucrose, 2% (m/v) trehalose, 1% (m/v) PVP-10 (polyvinyl pyrrolidone), 0.1% (v/v) PC-300, and adjust the pH to 8.0 _.

(3) NC membrane coating:

The NC membrane used was CN140 (purchased from Shanghai Jieyi Biotechnology Co., Ltd.), which was pasted on an 8 cm colloidal gold-specific PVC base plate and then coated. The NC membrane had a paired mouse anti-monkeypox monoclonal antibody (purchased from Feipeng Biotechnology Co., Ltd.) for coating. After diluting with coating diluent, it was coated on the nitrocellulose membrane at a final concentration of 1.3 mg/mL and a film volume of 1 μL/cm to form a T1 detection line;

The paired mouse anti-HIV P24 monoclonal antibody (purchased from Biotech Atlantic Inc.) used for coating was diluted with coating diluent and coated on the nitrocellulose membrane at a final concentration of 1.5 mg/mL and a film volume of 1 μL/cm to form a T2 detection line;

HIV1/2 antigen (purchased from CTK Biotech, USA) was diluted with coating diluent and coated on nitrocellulose membrane at a final concentration of 1.5 mg/mL and a film volume of 1 μL/cm to form a T3 detection line;

The goat anti-mouse antibody (purchased from Feipeng Biotechnology Co., Ltd.) was diluted with coating diluent and coated on the nitrocellulose membrane at a final concentration of 0.6 mg/mL and a film volume of 1 μL/cm to form a quality control line (C line);

The coated NC membrane was dried at 37° C. and humidity not higher than 30% for 8 hours, and then dried at 45° C. and humidity not higher than 25% for 36 hours. The coated membrane was sealed and stored at room temperature.

The preparation method of the coating diluent is as follows: prepare 20 mM boric acid buffer as the base solution, add 1% (m/v) NaCl, 0.8% (v/v) Tween20, 5% (m/v) trehalose, and 0.1% (v/v) PC-300, and adjust the pH to 8.2 _.

(4) Assembly of test strips:

On an 8 cm colloidal gold-specific PVC bottom plate that has been pasted with NC membrane and coated with antibodies, the treated single-layer sample pad, gold label pad 2, gold label pad 1, NC membrane coated with antibodies, and absorbent paper are pasted in sequence, staggered 2 mm each other. After pasting, a test paper board is obtained, and then the test paper strip is obtained by cutting according to the required width. For details, please refer to Figure 1. The test paper strip can be used directly after packaging, or the test paper strip can be installed in the reagent kit to make a reagent card and used with a small dropper.

Example 2

This example provides a method for detecting a combined test strip of monkeypox antigen, HIV1/2 antibody and P24 antigen, specifically using the test strip prepared in Example 1 for detection, the method is as follows:

Use an oral exudate sampler to take samples. Use a pipette to draw 4 drops (about 120 mL) of the sample from the sampler vertically into the sample well. Observe the displayed results after 15 minutes. The results displayed after 30 minutes are invalid. The judgment method is as follows. According to the judgment method, record the test results. The positive test results that need attention are shown in Figure 2:

(1) Only T1 and C show color. This means the test is valid, the patient is infected with monkeypox virus but not HIV;

(2) Only T2 and C show color. This means the test is valid, not infected with monkeypox virus, but infected with HIV;

(3) Only T3 and C show color. This means the test is valid, not infected with monkeypox virus, but infected with HIV;

(4) Only T1, T2, and C show color. This means the test is valid, and the patient is infected with monkeypox virus or HIV;

(5) Only T1, T3, and C show color. This means the test is valid, and the patient is infected with monkeypox virus or HIV;

(6) T1, T2, T3, and C all show color. This means the test is effective, and the patient is infected with monkeypox virus or HIV;

(7) C does not show color, so the test is invalid and needs to be retested.

After the test, the used reagent cards, samplers and pipettes shall be disposed of as biomedical waste.

Example 3

The test strips in Example 1 were used to test 100 clinical samples according to the method in Example 2. The results are shown in the following table:

Table 1 Test results of 100 oral exudate samples

-: no color development; +: color development; P: positive; N: negative.

In the test of 100 oral exudate samples, only sample No. 71 T3 showed obvious color development, indicating that the donor of sample No. 71 may be infected with HIV (HIV antibodies are present in the saliva). The human immunodeficiency virus (HIV-1/2) saliva antibody detection kit (colloidal gold method) of Beijing Jiele Biotechnology Co., Ltd. and the human immunodeficiency virus antibody (HIV1/2) oral mucosal exudate detection reagent (immunochromatography method) of Guangzhou Wondfo Biotechnology Co., Ltd. were used for retesting at the same time, and the results were consistent with the results of the present invention, indicating that the present invention has high sensitivity.

Example 4

The combined test strips described in Example 1 were used to detect purchased antigens and national reference products, wherein monkeypox antigen was purchased from Feipeng Biological Co., Ltd., cowpox antigen was purchased from Feipeng Biological Co., Ltd., and vaccinia antigen was purchased from Feipeng Biological Co., Ltd.; HIV antibody was a national reference product; HIV1P24 antigen was a national reference product.

(1) Oral exudate with negative test results was used as a matrix to dilute the purchased monkeypox antigen, cowpox antigen, and vaccinia antigen. The detection method was referred to Example 2. The test results are shown in Tables 2-4 below:

Table 2 Results of the gradient dilution test on monkeypox antigen

Antigen concentration T1 T2 T3 C Effectiveness Monkeypox HIV 10ng/ml + - - + efficient P N 500pg/ml + - - + efficient P N 100pg/ml + - - + efficient P N 50pg/ml + - - + efficient P N 10pg/ml + - - + efficient P N 7pg/ml + - - + efficient P N 4pg/ml - - - + efficient N N 0pg/ml - - - + efficient N N

-: no color; +: color; P: positive; N: negative

Table 3 Results of the gradient dilution test of vaccinia antigen

Antigen concentration T1 T2 T3 C Effectiveness Monkeypox HIV 10ug/ml - - - + efficient N N 500ng/ml - - - + efficient N N 100ng/ml - - - + efficient N N 10ng/ml - - - + efficient N N 500pg/ml - - - + efficient N N 50pg/ml - - - + efficient N N 0pg/ml - - - + efficient N N

-: no color; +: color; P: positive; N: negative

Table 4 Results of the gradient dilution test of vaccinia antigen

Antigen concentration T1 T2 T3 C Effectiveness Monkeypox HIV 10ug/ml - - - + efficient N N 500ng/ml - - - + efficient N N 100ng/ml - - - + efficient N N 10ng/ml - - - + efficient N N 500pg/ml - - - + efficient N N 50pg/ml - - - + efficient N N 0pg/ml - - - + efficient N N

-: no color; +: color; P: positive; N: negative

It can be concluded from Tables 2-4 that the oral exudate with negative monkeypox antigen detection was used as a matrix to dilute monkeypox antigen, cowpox antigen, and vaccinia antigen. Among them, monkeypox antigen 7pg/ml and above was positive, and the sensitivity was good. Cowpox antigen and vaccinia antigen were negative at various concentrations, indicating that the test strip of the present invention had good specificity.

(2) Testing of reference materials

HIV antibody national reference substance (colloidal gold): developed by the National Institute for the Control of Pharmaceutical and Biological Products. It includes 20 positive sera, 20 negative sera, 3 sensitive sera, and 1 precision serum. Take out the HIV antibody national reference substance (colloidal gold) serum plate, balance it to room temperature, and then use this reagent card for detection.

HIV1 P24 antigen national reference material, including 10 positive sera, 20 negative sera, 10 sensitive sera, 2 precision sera, take out the national reference serum plate, balance to room temperature and then use this reagent card for testing. The test results are shown in Table 5 and Table 6.

Table 5 Test results of national reference materials for HIV antibodies

-: no color; +: color; P: positive; N: negative;

The results of the HIV antibody national reference material (colloidal gold) test were completely consistent with expectations and passed the national standard regulations.

Table 6 Test results of HIV1 P24 antigen national reference material

-: no color; +: color; P: positive; N: negative

The test of HIV1 P24 antigen national reference material showed that the test strip of the present invention passed the test of national reference negative and positive reference materials, and the P24 antigen detection sensitivity was 0.15625 IU/ml (sensitivity sample 8).

It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. the joint inspection test strip of monkeypox antigen, HIV1/2 antibody and P24 antigen, it is characterized in that, described test strip comprises the PVC bottom plate with viscose, the sample pad that is arranged successively from bottom to top along the bottom plate width direction , gold standard pad 2, gold standard pad 1, chromatographic membrane and absorbent pad, and the adjacent components are connected to each other; Colloidal gold-labeled biotinylated HIV1/2 antigen and colloidal gold-labeled biotinylated P24 antibody are immobilized on the gold label pad 1, and colloidal gold-labeled biotinylated monkeypox antibody is immobilized on the gold label pad 2 and streptavidin-labeled colloidal gold; The chromatographic membrane includes a detection line and a quality control line arranged in parallel and spaced from each other from the end close to the sample pad to the end close to the water-absorbent pad. There are at least three detection lines, which are respectively coated and Monkeypox antigen-specific binding mouse anti-monoclonal antibody T1 detection line, coated with HIV P24 antigen-specific binding mouse anti-HIV P24 monoclonal antibody T2 detection line, coated with HIV 1/2 antibody specificity Sex-binding HIV 1/2 antigen T3 test line. 2. the joint inspection test strip of monkeypox antigen, HIV1/2 antibody and P24 antigen according to claim 1, is characterized in that, described chromatographic membrane is nitrocellulose membrane, and the passage time of described nitrocellulose membrane 130-150s. 3. the joint inspection test strip of monkeypox antigen, HIV1/2 antibody and P24 antigen according to claim 1, is characterized in that, sample pad is prepared after being processed by sample pad treatment solution, wherein the preparation method of sample pad treatment solution As follows: Prepare 10-100mM Tris-HCl as the base solution, then add 0.1-1% m/v casein, 0.1-0.3% m/v skimmed milk powder to dissolve for 8-24 hours, then add 0.1-0.8% v/v Tween 20, 0.1～0.8% m/v Surfactant S9, 0.5～5% m/v PVP-10, 0.05～0.15% v/v PC300, 0.1～0.4% m/v EDTA-2Na, 0.05～3mg/ mL active heterophile antibody blocking agent, 0.1-0.4mg/mL anti-RBC monoclonal antibody, adjust the pH to 7.0-9.0. 4. the joint inspection test strip of monkeypox antigen according to claim 1, HIV1/2 antibody and P24 antigen is characterized in that, described sample pad is selected from glass cellulose film, non-woven fabric, polyester cellulose film , One of the whole blood filtration membranes. The sample pad is a glass cellulose membrane. 5. the joint inspection test strip of monkeypox antigen according to claim 1, HIV1/2 antibody and P24 antigen is characterized in that, the method for colloidal gold labeling antigen/antibody comprises the following steps: Firstly, the antigen or antibody is biotinylated to obtain biotinylated antigen or antibody, and then added to the colloidal gold solution adjusted to the preset pH range, after stirring, polyethylene glycol is added, stirred again and the precipitate is collected. Redissolve and mix the precipitate with colloidal gold reconstitution solution to obtain; Among them, when colloidal gold is labeled with biotinylated monkeypox antibody, the preset pH of colloidal gold is 8.0-8.4; when colloidal gold is labeled with biotinylated P24 antibody, the preset pH of colloidal gold is 8.8-9.2; For HIV1/2 antigen, the preset pH of colloidal gold is 8.3-8.7; and/or Colloidal gold labeling method for antigen/antibody, the particle size of colloidal gold is 20nm. 6. the joint inspection test strip of monkeypox antigen according to claim 1, HIV1/2 antibody and P24 antigen is characterized in that, the method for streptavidin labeling colloidal gold comprises the following steps: Adjust the pH of the colloidal gold solution to 8.8 to 9.2, add streptavidin, stir and add polyethylene glycol, stir again and collect the precipitate, redissolve the precipitate with colloidal gold reconstitution solution and mix well to obtain the product; and/ or The particle size of the colloidal gold in the streptavidin-labeled colloidal gold is 40nm. 7. the joint inspection test strip of monkeypox antigen, HIV antibody and P24 antigen according to claim 1, is characterized in that, the preparation method of gold standard pad 1 comprises the steps: The gold-labeled pad is treated with the gold-labeled pad treatment solution to obtain the conjugated pad, and the colloidal gold-labeled biotinylated HIV1/2 antigen and the colloidal gold-labeled biotinylated P24 antibody are prepared at concentrations of OD 1.1-1.3 and OD 0.8-1.2 , spray gold with the parameters of 2-4μL/cm mixed spray on the bonding pad, after baking. 8. the joint inspection test strip of monkeypox antigen, HIV antibody and P24 antigen according to claim 1, is characterized in that, the preparation method of gold standard pad 2 comprises the steps: Treat the gold-labeled pad with the gold-labeled pad treatment solution to obtain the conjugated pad. Spray colloidal gold-labeled biotinylated monkeypox antibody and streptavidin-labeled colloidal gold at concentrations of OD 0.8-1.2 and OD 0.6-0.8. A gold volume of 2-4 μL/cm is mixed and sprayed on the bonding pad, and it is obtained after baking. 9. according to claim 1 or 7 or the joint inspection test strip of described monkeypox antigen, HIV antibody and P24 antigen, it is characterized in that, gold standard pad is selected from one of glass cellulose film, polyester cellulose film kind. 10. The joint inspection test strip of monkeypox antigen, HIV antibody and P24 antigen according to claim 7 or 8 or 9, characterized in that the gold standard pad treatment solution comprises: 0.1～0.3% v/v Triton X-100 , 0.5-1.5% m/v BSA, 15-25% m/v sucrose, 3-7% m/v trehalose, 0.1M Tris-HCl at pH 7.5.

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