CN119390828A

CN119390828A - Human immunodeficiency virus HIV antibody and its application and detection product

Info

Publication number: CN119390828A
Application number: CN202411981015.0A
Authority: CN
Inventors: 张舟; 何其; 朱伟伟; 刘春龙
Original assignee: Tianjin Longsheng Biotechnology Co ltd
Current assignee: Tianjin Longsheng Biotechnology Co ltd
Priority date: 2024-12-31
Filing date: 2024-12-31
Publication date: 2025-02-07
Also published as: CN120574318A

Abstract

The invention provides a pair of HIV antibodies, application thereof and detection products thereof. The invention provides a1 pair of HIV antibodies, comprising antibody c and/or antibody n, and amino acid sequences of the heavy chain complementarity determining regions and the light chain complementarity determining regions of the antibodies. The HIV antibody has good specificity, high bioactivity, high stability and high affinity with HIV, and can be used for preparing products for detecting HIV. The HIV detection kit provided by the invention has the advantages of simplicity and convenience in operation, rapid response, high sensitivity, strong specificity, suitability for on-site rapid detection, economy, practicability and the like, can be used for rapidly screening when viruses flow, and can rapidly, accurately and safely detect a large number of asymptomatic virus carrier groups including suspected cases, asymptomatic infection cases in a latent period and higher risks in time.

Description

HIV antibody of human immunodeficiency virus, application thereof and detection product thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a Human Immunodeficiency Virus (HIV) antibody and application thereof.

Background

Acquired immunodeficiency syndrome, abbreviated as aids, caused by human immunodeficiency virus (human immunodeficiency virus, HIV) has become one of the important public health problems that is prevalent worldwide and severely threatens public health in various countries. By the end of 2023, 128.3 thousands of existing live HIV infected persons/HIV patients reported in China, 10.78 thousands of newly discovered HIV infected persons/HIV patients in 2022 are newly discovered, and nearly 300 HIV infected persons/HIV patients are newly discovered every day on average. The existing detection technology and diagnosis level can not discover acute HIV infected persons as early as possible, and can not take effective intervention measures in time, so that the method is probably one of the main reasons for the high HIV epidemic rate at present.

In general, the clinical symptoms and biological characteristics of an individual after infection with HIV are divided into 3 major phases. Stage 1 is the acute phase, and is characterized by rapid proliferation and transmission of the virus in vivo, mostly occurring at 2-4 weeks after infection. At this stage, virus replication bursts, and the p24 antigen (Ag) in the blood drops off and peaks. Stage 2 is a chronic or asymptomatic phase in which the virus continues to propagate, but at a lower level. The host immune system also begins to produce antibodies (abs) while Viral Load (VL) drops to steady state. As VL decreases, p24 antigen levels also decrease. This is due to the fact that the p24 antigen binds to the antibody to form an antibody-p 24 antigen complex, thereby reducing the level of free p24 antigen in the blood. The period of time from infection to the appearance of antibodies (seroconversion) is referred to as the "window period". If the patient is not treated, as the virus continues to replicate, the CD4 cells that are the target cells for the replication of the virus are gradually destroyed, resulting in a decrease in the number of CD4 cells. Stage 3 is AIDS stage, in which the host immune system is weakened and the level of free p24 antigen in the blood gradually increases as the virus continues to replicate and CD4 cells are depleted.

Currently, the detection method of HIV mainly comprises a serological detection method, a CD4 cell detection method, an HIV nucleic acid detection method and the like. The most common method in serology detection is antibody detection, but the antibody and CD4 cells are obviously changed in the 2 nd stage, the latency period is longer, the significance for early diagnosis is limited, and nucleic acid detection is the most sensitive means for detecting HIV at present, but the nucleic acid detection is difficult to apply to daily detection work due to higher cost of required reagents and instruments. After HIV-1 infects the organism, the earliest detectable virus marker in blood is p24 antigen, and the detection can shorten the window period of HIV infection detection to 12-15 d. Therefore, the detection of the p24 antigen has important significance in early diagnosis of HIV infection, prediction of disease progress, judgment of prognosis, screening and evaluation of curative effects of anti-HIV medicines, and the combined detection of the antigen and the antibody can predict infection stage. In addition, the chromatographic method supporting self-test is adopted to detect the p24 antigen in blood, so that the method has the advantages of simple operation, low equipment dependence, short detection time and the like, and is beneficial to early diagnosis. In order to further shorten the window period, antibodies with strong affinity can be screened for detection, and the sensitivity of the product is improved.

Disclosure of Invention

The invention aims at:

The first object of the present invention is to provide a method for preparing an antibody against HIV, which can prepare an antibody specifically recognizing HIV and realize efficient pairing.

It is a second object of the present invention to provide antibodies to HIV, which specifically recognize HIV.

A third object of the present invention is to provide an application of the above-mentioned HIV antibody for detecting HIV of human immunodeficiency virus.

A fourth object of the present invention is to provide a test strip kit for HIV detection by human immunodeficiency virus, which solves at least one of the above problems.

To achieve the above object, the present invention provides a human immunodeficiency virus HIV antibody comprising antibody c and/or antibody n;

The amino acid sequence of the heavy chain complementarity determining region of the antibody c comprises CDR1-VH with the amino acid sequence shown as SEQ ID NO.4, CDR2-VH with the amino acid sequence shown as SEQ ID NO.5 and CDR3-VH with the amino acid sequence shown as SEQ ID NO. 6;

the amino acid sequence of the light chain complementarity determining region of the antibody c comprises CDR1-VL with the amino acid sequence shown as SEQ ID NO.7, CDR2-VL with the amino acid sequence shown as SEQ ID NO.8 and CDR3-VL with the amino acid sequence shown as SEQ ID NO. 9;

The amino acid sequence of the heavy chain complementarity determining region of the antibody n comprises CDR1-VH with the amino acid sequence shown as SEQ ID NO.10, CDR2-VH with the amino acid sequence shown as SEQ ID NO.11 and CDR3-VH with the amino acid sequence shown as SEQ ID NO. 12;

the amino acid sequence of the light chain complementarity determining region of the antibody n comprises CDR1-VL with the amino acid sequence shown as SEQ ID NO.13, CDR2-VL with the amino acid sequence shown as SEQ ID NO.14 and CDR3-VL with the amino acid sequence shown as SEQ ID NO. 15.

Preferably, the amino acid sequence of the heavy chain variable region of the antibody c is shown as SEQ ID NO.16, and the amino acid sequence of the light chain variable region of the antibody c is shown as SEQ ID NO. 17;

preferably, the amino acid sequence of the heavy chain variable region of the antibody n is shown as SEQ ID NO.18, and the amino acid sequence of the light chain variable region of the antibody n is shown as SEQ ID NO. 19.

Preferably, any of the above is that the HIV antibody is murine.

Preferably, the light chain of the antibody c is an amino acid sequence shown in SEQ ID NO. 20.

Preferably, the heavy chain of the antibody c is an amino acid sequence shown in SEQ ID NO. 21.

Preferably, any one of the above-mentioned methods is that the light chain of the antibody n has an amino acid sequence shown in SEQ ID NO. 22.

Preferably, the heavy chain of the antibody n is an amino acid sequence shown in SEQ ID NO. 23.

Preferably, the amino acid sequence of the light chain constant region of the antibody c or antibody n is shown in SEQ ID NO. 24.

Preferably, the amino acid sequence of the heavy chain constant region of the antibody c or antibody n is shown in SEQ ID NO. 25.

The invention also provides an immunogenic protein of human immunodeficiency virus HIV, said immunogenic protein comprising immunogenic protein C and/or immunogenic protein N.

Preferably, the immunogenic protein C comprises the amino acid sequence shown in SEQ ID NO. 2.

Preferably, any of the above mentioned immunogenic proteins N comprises the amino acid sequence shown in SEQ ID NO. 3.

The invention also provides application of the immunogen protein in preparing the HIV antibody of any one of the human immunodeficiency virus.

Preferably, the antibody C is obtained from an immunogenic protein C by animal immunization.

Preferably, the antibody N is obtained from an immunogenic protein N by animal immunization.

The invention also provides a preparation method of the HIV antibody, which comprises 2 kinds of immunogen proteins;

The human immunodeficiency virus HIV immunogenic protein is selected from the group consisting of marker proteins, comprising immunogenic protein C and immunogenic protein N;

The amino acid sequence of the immunogen protein C is shown as SEQ ID NO.2, and the amino acid sequence of the immunogen protein N is shown as SEQ ID NO. 3.

The invention also provides a marker protein of HIV, which is selected from p24 protein of virus, comprising an amino acid sequence shown as SEQ ID NO.2 and an amino acid sequence shown as SEQ ID NO.3, wherein the amino acid sequence of the marker protein is shown as SEQ ID NO. 1.

The invention provides a human being immunodeficiency virus HIV antibodies specifically recognize the marker protein.

The invention also provides application of the HIV antibody in preparation of HIV detection products.

The invention also provides a human immunodeficiency virus HIV detection test strip, which comprises a bottom plate, and a sample pad, a marking pad, a detection pad and a sample absorbing pad which are sequentially overlapped on the bottom plate, wherein the marking pad contains any one of the human immunodeficiency virus HIV antibodies, and the human immunodeficiency virus HIV antibodies are marked with markers.

Preferably, the label comprises at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres, or quantum dot microspheres.

The particle size of the colloidal gold is preferably 40 to 100nm, more preferably 40, 50, 60, 80, 100nm and the range therebetween.

Any of the above-mentioned colored microspheres preferably have a particle size of 100 to 300nm, more preferably 100, 200, 300nm and their ranges.

Any of the above-mentioned particles preferably has a particle size of 100 to 300nm, more preferably 100, 200, 300nm and the range therebetween.

Any one of the above is preferable that the particle size of the quantum dot microsphere is 100 to 300nm, and more preferable is 100, 200, 300nm and the range therebetween.

Preferably, the mass ratio of the HIV antibody to the colloidal gold is (0.04-0.32): 1, more preferably 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1 and ranges thereof.

Preferably, the mass ratio of the HIV antibody to the color microspheres is (0.1-0.4): 1, more preferably 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the ranges therebetween.

Preferably, the mass ratio of the HIV antibody to the time-resolved fluorescent microsphere is (0.1-0.4): 1, more preferably 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the ranges thereof.

Preferably, the mass ratio of the HIV antibody to the quantum dot microsphere is (0.1-0.4): 1, more preferably 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the ranges thereof.

Preferably, the detection pad is provided with a detection line and a quality control line, the detection line is coated with the HIV antibody, and the quality control line is coated with the goat anti-mouse IgG polyclonal antibody.

Preferably, any one of the above steps is that the detection line is coated with 0.5-5 mg/mL of the HIV antibody. Further preferred, the detection line is coated with 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mg/mL and ranges therebetween of the HIV antibodies.

Preferably, any one of the above materials is that the quality control line is coated with 0.5-5 mg/mL goat anti-mouse IgG polyclonal antibody. Further preferably, the quality control line is coated with 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mg/mL and ranges therebetween of goat anti-mouse IgG polyclonal antibodies.

Preferably, the dilution of HIV antibody and goat anti-mouse IgG polyclonal antibody is 10-50 mM PB buffer solution containing trehalose, and each 100mL of dilution contains 0.1-1.0 g of trehalose. Further preferably, the diluent is 10, 20, 30, 40, 50mM and ranges therebetween PB buffer comprising trehalose, further preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0g and ranges therebetween trehalose is included per 100mL of diluent.

The detection line and the quality control line are preferably arranged on the detection pad, the detection line is coated with the HIV antibody, the quality control line is coated with the sheep anti-chicken IgY, the marking pad contains the HIV antibody, the HIV antibody on the marking pad is marked with a marker, and the marker is marked with chicken IgY.

Preferably, any of the above-mentioned HIV antibodies on the label pad and HIV antibodies on the detection pad are different from each other.

Preferably, any of the above-mentioned HIV antibodies on the label pad and HIV antibodies on the detection pad are different from each other. Preferably, the HIV antibody on the labeling pad is an antibody c, the HIV antibody on the detection pad is an antibody n, preferably, the HIV antibody on the labeling pad is an antibody n, and the HIV antibody on the detection pad is an antibody c.

The invention also provides a Human Immunodeficiency Virus (HIV) detection test strip and a shell, wherein the HIV detection test strip comprises any one of the above components, and the HIV detection test strip is arranged inside the shell.

Preferably, the housing includes an upper cover and a lower cover detachably connected.

Preferably, any one of the above is provided with a viewing window and a loading well.

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

The epitope contained in the immunogen protein in the antibody preparation method is strong in conservation property, is difficult to cause omission, is a dominant epitope, is easy to generate a high-affinity antibody, and is easy to realize efficient pairing when being respectively positioned at the C end and the N end of the p24 antigen.

The HIV antibody provided by the invention has good specificity, high bioactivity, strong stability and high affinity with HIV, and can be used for preparing products for detecting HIV.

The HIV detection kit provided by the invention has the advantages of simplicity and convenience in operation, rapid response, high sensitivity, strong specificity, suitability for on-site rapid detection, economy, practicability and the like, and has important significance in early diagnosis of HIV infection, prediction of disease progress, judgment of prognosis, screening and evaluation of curative effects of anti-HIV drugs and the like.

Drawings

FIG. 1 shows a top cover of a HIV detection kit according to a preferred embodiment 2 of the present invention.

FIG. 2 is a bottom cover of the HIV detection kit according to the preferred embodiment 2 of the present invention.

FIG. 3 is a block diagram showing a test strip for detecting HIV of human immunodeficiency virus according to a preferred embodiment 2 of the present invention.

FIG. 4 is an electrophoresis chart of HIV monoclonal antibodies against HIV in the preferred embodiment 1 of the present invention.

FIG. 5 is a graph showing the results of the test in the preferred embodiment 3 of the present invention.

The icons comprise a 1-observation window, a 2-sample adding hole, a 3-detection clamping strip area, a 4-bottom plate, a 5-detection pad, a 6-sample absorbing pad, a 7-marking pad, an 8-sample pad, a 9-quality control line and a 10-detection line.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy chain or the light chain of the antibody. The variable domain of a heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The light or heavy chain variable region is composed of framework regions interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework regions of antibodies, i.e., the framework regions that make up the combination of the essential light and heavy chains, function to locate and align the CDRs, which are primarily responsible for binding to the antigen.

"Framework" or "FR" regions mean regions of the antibody variable domain other than those defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR 1, FR2, FR3, and FR 4) separated by CDRs.

Typically, the variable regions VL/VH of the heavy and light chains are obtained by connecting the CDRs numbered from FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a combined arrangement with the FRs.

In the present invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three hypervariable regions of the heavy chain variable region, respectively, and CDR1-VL, CDR2-VL and CDR3-VL refer to the three hypervariable regions of the light chain variable region, respectively.

An immunogenic protein of human immunodeficiency virus HIV selected from the group consisting of a marker protein comprising immunogenic protein C and immunogenic protein N;

In a first aspect, the invention provides a marker protein of HIV, the amino acid sequence of which is shown as SEQ ID NO.1, and in addition, the invention provides 2 immunogenic proteins of HIV, comprising an immunogenic protein C and an immunogenic protein N, wherein the amino acid sequence of the immunogenic protein C is shown as SEQ ID NO.2, and the amino acid sequence of the immunogenic protein N is shown as SEQ ID NO.3.

The amino acid sequences shown in SEQ ID NO. 1-3 are shown in Table 1.

TABLE 1

In a second aspect, the invention provides a human immunodeficiency virus HIV antibody selected from antibody c and/or antibody n;

The antibody c comprises a heavy chain complementarity determining region CDR1-VH, CDR2-VH and CDR3-VH with amino acid sequences shown in SEQ ID NO. 4-6 in sequence, and a light chain complementarity determining region CDR1-VL, CDR2-VL and CDR3-VL with amino acid sequences shown in SEQ ID NO. 7-9 in sequence;

The antibody n comprises a heavy chain complementarity determining region CDR1-VH, a CDR2-VH and a CDR3-VH, the amino acid sequences of which are shown as SEQ ID NO. 10-12 in sequence, and a light chain complementarity determining region CDR1-VL, a CDR2-VL and a CDR3-VL, the amino acid sequences of which are shown as SEQ ID NO. 13-15 in sequence.

The amino acid sequences shown in SEQ ID NO. 4-15 are shown in Table 2.

TABLE 2

In some preferred embodiments, the antibody c comprises a heavy chain variable region and a light chain variable region with amino acid sequences shown in SEQ ID NO. 16-17;

The antibody n comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID NO. 18-19 in sequence;

preferably, the HIV antibody is murine.

The amino acid sequences shown in SEQ ID NO. 16-17 and SEQ ID NO. 18-19 are shown in Table 3.

TABLE 3 Table 3

In some preferred embodiments, the light chain of antibody c is an amino acid sequence as shown in SEQ ID No. 20. The heavy chain of the antibody c is an amino acid sequence shown as SEQ ID NO. 21.

In some preferred embodiments, the light chain of antibody n is an amino acid sequence as shown in SEQ ID No. 22. The heavy chain of the antibody n is an amino acid sequence shown as SEQ ID NO. 23.

In some preferred embodiments, the amino acid sequence of the light chain constant region of antibody c or antibody n is as shown in SEQ ID NO. 24.

In some preferred embodiments, the amino acid sequence of the heavy chain constant region of antibody c or antibody n is as shown in SEQ ID NO. 25.

The amino acid sequences shown in SEQ ID No. 20-SEQ ID No.25 are shown in Table 4.

TABLE 4 Table 4

In a third aspect, the invention provides the use of the HIV antibody for human immunodeficiency virus in the preparation of a HIV detection product.

In a fourth aspect, the invention provides a Human Immunodeficiency Virus (HIV) detection test strip, which comprises a bottom plate, and a sample pad, a marking pad, a detection pad and a sample absorbing pad which are sequentially overlapped and arranged on the bottom plate;

The marking pad contains the HIV antibody, the HIV antibody is marked with a marker, and the marker can identify the position or the concentration of the marker by detection.

The HIV detection test strip has the advantages of simple operation, rapid response, high sensitivity, strong specificity, suitability for on-site rapid detection, economy, practicability and the like, and has important significance in early diagnosis of HIV infection, prediction of disease progress, judgment of prognosis, screening and evaluation of curative effects of anti-HIV drugs and the like.

In some preferred embodiments, the label includes, but is not limited to, at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres, or quantum dot microspheres;

Preferably, the particle size of the colloidal gold may be, for example, but not limited to, 40 nm, 60 nm, 80 nm, or 100 nm;

preferably, the particle size of the colored microspheres, time-resolved fluorescent microspheres, and quantum dot microspheres can be, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm;

Preferably, the mass ratio of the HIV antibody to the coupling colloidal gold is (0.04-0.32): 1, and the mass ratio of the HIV antibody to the color microsphere, the time-resolved fluorescence microsphere or the quantum dot microsphere is (0.1-0.4): 1.

The sensitivity of the detection test paper strip is higher by adjusting the size and the dosage of the marker particles.

In some preferred embodiments, the detection pad is provided with a detection line (T line) and a quality control line (C line);

the detection line is coated with 0.5-5 mg/mL of HIV antibody;

the quality control line is coated with 0.5-5 mg/mL goat anti-mouse IgG polyclonal antibody;

Preferably, the dilution of HIV antibody and goat anti-mouse IgG polyclonal antibody is 10-50 mM PB buffer solution containing trehalose, and each 100mL dilutions contains 0.1-1.0 g trehalose.

In some preferred embodiments, the HIV antibody on the label pad and the HIV antibody on the test pad are different from each other.

For example, the protein on the label pad is HIV antibody c, wherein antibody c is human immunodeficiency virus HIV monoclonal antibody 1, and the protein on the test pad is HIV antibody n, wherein antibody n is human immunodeficiency virus HIV monoclonal antibody 2.

The test strip provided by the invention is prepared by using colloidal gold, color microspheres, time-resolved fluorescent microspheres and quantum dots to mark HIV monoclonal antibodies according to the principle of antigen-antibody reaction, and then solidifying the markers on a glass cellulose film. The detection pad (such as NC membrane) is coated with another monoclonal antibody of HIV, and the detection can be carried out by naked eyes or matched instruments in the detection time based on the principle of antigen-antibody reaction. If the human immunodeficiency virus HIV exists in the sample, a double-antibody sandwich structure is formed, a macroscopic strip is formed or a light intensity signal exists in the instrument, and if the human immunodeficiency virus HIV does not exist in the sample, the strip does not exist on an NC film or the light intensity signal does not exist in the instrument. And carrying out negative and positive judgment according to the existence of the signal or carrying out the prejudgment of the viral load according to the intensity of the light intensity signal.

The test strip provided by the invention is used for detecting, and the detection result can be obtained in the whole process for 10-30min, so that the test strip is rapid and efficient, is beneficial to medical staff to obtain the detection result in time, and can be comprehensively judged and treated in time according to the result, thereby avoiding panic and reducing epidemic spread.

In a fifth aspect, the invention provides a HIV detection kit, comprising the HIV detection test strip and a housing, wherein the HIV detection test strip is disposed inside the housing.

The HIV detection kit provided by the invention contains the HIV detection test strip, so that the HIV detection test strip has all the beneficial effects.

In some preferred embodiments, the housing comprises a removably attachable upper cover and lower cover;

the upper cover is provided with an observation window and a sample adding hole.

In the invention, the shapes of the observation window and the sample adding hole are not particularly limited, the observation window can be square, and is positioned above the test strip detection line and the quality control line and used for observing the detection result, and the sample adding hole can be a circular hole with the diameter of 0.5-1cm and is positioned above the sample pad.

Kit test sample loading:

in the following examples, unless otherwise indicated, dilutions of HIV antibodies and goat anti-mouse IgG polyclonal antibodies were 20mM g of trehalose in PB buffer, and 0.5g of trehalose was included in each 100: 100 mL dilution.

The preparation method of the monoclonal antibody in the present invention is the content of the prior art, and only a simple description is given here, and the specific operation method can be performed according to the operation method described in the prior art:

(1) Mixing immunogen and Freund's adjuvant in equal volume to a proper volume, emulsifying completely, and performing animal immunization on mice by intraperitoneal injection, wherein 50 μg of immunogen is injected into each mouse, the injection volume is 100 μl/mouse, and the injection is performed 1 time per week;

(2) After 4 times of immunization, testing the titer of antibodies in serum by adopting an ELISA indirect method, and screening mice with OD values greater than 1.0 detected by using 16000 times of serum diluent;

(3) Fusing spleen cells of the screened mice with myeloma cells, and performing plating culture on the fused cells by a limiting dilution method;

(4) Screening monoclonal cell holes, culturing and amplifying, and taking the monoclonal cell hole with the highest detection OD value of the cell culture supernatant as a target hybridoma cell;

(5) Amplifying the target hybridoma cells, injecting the amplified target hybridoma cells into the abdominal cavity of a mouse, collecting ascites, and purifying monoclonal antibodies in the ascites by using ProteinA to obtain the target monoclonal antibodies.

Preferably, the antibody c is prepared from the immunogenic protein c and the antibody n is prepared from the immunogenic protein n by the method described above.

Example 1

The HIV antibodies include HIV monoclonal antibody 1 (antibody c) and HIV monoclonal antibody 2 (antibody n), and the variable region sequences are shown in Table 3.

A12% SDS-PAGE gel was prepared according to the conventional method, and the above antibodies were loaded at 5. Mu.g and run against a protein molecular weight standard as a reference, and as a result, 2 anti-HIV monoclonal antibodies showed two characteristic bands of about 25 KD and 55 KD, which were the light and heavy chains of IgG, respectively (FIG. 4). The content of the antibody in the strip after scanning is above 90%.

In FIG. 4, 1:anti-1; 2:anti-2, wherein Anti-1 is Anti-HIV monoclonal antibody 1 and Anti-2 is Anti-HIV monoclonal antibody 2.

The antibodies used in the following examples are the same as in example 1.

Example 2

A human immunodeficiency virus HIV detection kit is shown in fig. 1-3, and comprises a human immunodeficiency virus HIV detection test strip and a shell, wherein the human immunodeficiency virus HIV detection test strip is arranged inside the shell. The HIV detection test strip comprises a base plate 4, a sample pad 8, a marking pad 7, a detection pad 5 and a sample absorbing pad 6 which are sequentially overlapped on the base plate, wherein a detection line 10 and a quality control line 9 are arranged on the detection pad, the shell comprises an upper cover and a lower cover which are detachably connected, the upper cover is provided with an observation window 1 and a sample adding hole 2, and the lower cover is provided with a detection clamping strip area 3.

Example 3

Example 3 provides a kit for detecting HIV antigen of human immunodeficiency virus prepared by using colloidal gold labeled antibody.

1. Main materials

1.1 Antibody murine monoclonal antibody c and antibody n of the invention are respectively labeled and coated, and goat anti-mouse IgG antibody Shenzhen Phellinus biological Co., ltd, which is used for nitrocellulose membrane quality control line coating.

1.2 Nitrocellulose membrane NC membrane is a product of Sidoris company.

1.3 Other consumables, such as PVC plates, are manufactured by Bi Kenlai Bo company, and the common reagents are all analytically pure reagents.

1.4 Acquisition of recombinant antigen protein recombinant antigen with amino acid sequence shown as seq ID No.1 was commercially synthesized by the biological engineering (Shanghai) Co., ltd according to the HIV data published by the database NCBI.

2. Method of

2.1 Preparation of colloidal gold-labeled pad:

The preparation method of the colloidal gold labeled pad comprises the following steps:

(1) Taking 1mL of colloidal gold solution with the particle size of 40 nm, and adjusting the pH value to 8.5 by using 0.2M K ₂CO₃;

(2) Adding 25 mug monoclonal antibody 1, wherein the mass ratio of the antibody to the colloidal gold particles is 0.1:1, regulating a rotary table to a certain rotating speed, rotationally marking 1.5 h at room temperature, and adding 20 mug of sealing liquid;

(3) 12000 rpm centrifuging 15: 15min and discarding the supernatant;

(4) Adding 100 mu L of colloidal gold complex solution;

(5) Diluting the concentrated solution according to the proportion of 1:7, spraying metal, and then placing the diluted concentrated solution in a drying oven at 37 ℃ for drying 2h for later use;

2.2 NC film coating:

the monoclonal antibody 2 is diluted to 1.5mg/mL by 0.02M PB containing 0.5% trehalose, the goat anti-mouse IgG antibody is diluted to 2 mg/mL, then a film spraying instrument is used for respectively marking a detection line T and a quality control line C on a nitrocellulose film, and the NC film is dried for use in an oven at 37 ℃ for 24: 24h after coating is completed.

2.3 Assembly of the kit:

Placing the coated nitrocellulose membrane in the middle of a plastic supporting plate in a drying chamber, pasting, overlapping a marker colloidal gold pad (1/3 of the colloidal gold pad) on one side of a nitrocellulose membrane T line, pasting, overlapping a sample pad (1/5 of the colloidal gold pad) on the other side of the colloidal gold pad, overlapping a sample absorbing pad (1/10 of the sample absorbing pad) on one side of a nitrocellulose membrane C line, cutting the pasted plastic plate into test strips with a certain width by a cutting machine, and then putting the test strips into a detection card to form the HIV antigen detection kit.

2.4 Detection:

Step 1, taking out the kit and a sample to be detected, and balancing to room temperature;

step 2, opening a sealed aluminum foil bag, and taking out the kit and placing the kit on a table top;

Step 3, 2 drops of sample (about 80-100 mu L) are dripped into the sample adding hole in the figure 1;

Step 4, counting by a timer, and judging the result after 10 minutes, wherein if the sample is not laterally chromatographed or percolated for 1 minute after the sample is added, the sample is probably too viscous, and the sample needs to be pretreated by normal saline.

3. Results

Under the action of lateral chromatography, when the human immunodeficiency virus HIV exists in the sample, the detection line is colored, and meanwhile, the quality control line is colored (a in fig. 5), when the human immunodeficiency virus HIV does not exist in the sample, the detection line is not colored, the quality control line is colored (b in fig. 5), after the sample is loaded, the quality control line is not colored, and whether the detection line is not colored, the result is invalid (c in fig. 5 and d in fig. 5) is judged.

4. The test of a blood sample from an HIV patient was performed using the kit provided in example 3, consistent with the results shown in 3. The effectiveness of the marker proteins, the antibodies c and the antibodies n provided by the invention in HIV detection is demonstrated.

Example 4

Example 4 provides a kit for the detection of HIV antigen by time-resolved fluorescent microsphere labeled antibodies.

The invention provides a kit for detecting HIV antigen of a human immunodeficiency virus by using 1 pair of HIV monoclonal antibodies (antibody c and antibody n), which comprises a detection card and a test strip, wherein the detection card is divided into an upper cover and a lower cover, the test strip is embedded with a time-resolved fluorescent microsphere marked monoclonal antibody 1 (antibody c) in a fluorescent pad, a detection line is coated with a monoclonal antibody 2 (antibody n), and the HIV antigen of the human immunodeficiency virus in a sample is quantitatively detected by using a double antibody sandwich method.

1. The preparation operation process of the kit comprises the following steps:

the monoclonal antibody 1 prepared by the method is marked on the surface of the time-resolved fluorescence microsphere. Specific examples are as follows:

Time-resolved fluorescence microsphere antibody labelling 1mL 1% carboxyl time-resolved fluorescence microsphere, adding 9 mL MES buffer, adding 25. Mu.L EDC solution (10 mg/mL) and 25. Mu.L NHS solution (10 mg/mL), shaking at room temperature for reaction 30min, centrifuging and collecting precipitate. HEPES complex solution was added, 1mL of 1mg/mL monoclonal antibody 1 was added after uniform ultrasonic dispersion, the reaction was performed by shaking at room temperature, 120 min was performed, and the precipitate was collected by centrifugation. Then adding 1mL of blocking solution, and vibrating at room temperature to react 120 min. Centrifuging to collect microsphere precipitate, and redissolving with redissolution.

And (3) preparing the fluorescent pad, namely diluting the marked time-resolved fluorescent microsphere by using microsphere complex solution, spraying the fluorescent pad by using a metal spraying film drawing instrument, and spraying 3 mu L/cm with a spraying interval of 6mm. And after the spraying is finished, the materials are put into a low-humidity oven at 37 ℃ and dried for 2 hours (30%).

NC film coated CT line, T line using monoclonal antibody 2 with concentration of 1.5 mg/mL, C line using goat anti-mouse IgG antibody with concentration of 1mg/mL, streaking with 1 μL/cm, and drying at 37deg.C with low humidity (< 30%) for 24 hr.

Sample pad treatment, wherein the sample pad treatment liquid consists of buffer salt, slow release agent, cosolvent, sealing agent and the like. The specific formula is 20 mM Tris buffer solution, and each 100 mLTris buffer solution contains 1g BSA, 0.5g Tween 20 and 2g sucrose. Treatment was performed with 1mL of sample treatment fluid per 20 cm ². After the treatment is uniform, the mixture is put into a low-humidity oven at 37 ℃ for 2 hours (< 30%).

And (3) assembling the test strip, namely sequentially adhering an NC film, a sample absorbing pad, a fluorescent pad and a sample pad on a PVC plate, wherein the sample absorbing pad and the fluorescent pad respectively press the NC film for 1-2 mm, and the sample pad presses the fluorescent pad for 1-2 mm. Cutting the assembled test strip into a width of 4+/-0.4 mm, and packaging the test strip into a clamping shell. The clamping shell and the drying agent are put into an aluminum foil bag together and then sealed. And (5) labeling and boxing to obtain the finished product detection card.

Preparation of test samples the recombinant antigen of commercial synthetic protein of the biological engineering (Shanghai) stock company was subjected to gradient dilution and then tested. The results are shown in Table 5.

TABLE 5

2. Kit detection process

And placing the detection card on a clean and flat table top, sucking 80-100 mu L of processed samples, dripping the samples into a sample adding end of the detection card, and simultaneously setting a PBS control group.

After the test strip standard curve is introduced into the sample, 10 min, a fluorescence immunoassay instrument is used for scanning a detection area to obtain a fluorescence signal, the concentration value corresponding to HIV antigen of the human immunodeficiency virus is displayed after detection, and the measurement results are shown in the following tables 6 and 7.

TABLE 6 test results of diluent samples

TABLE 7 recombinant antigen test results

Example 5

Example 5 provides a kit for the detection of HIV antigen by color microsphere-labeled antibodies.

The 1 pair HIV monoclonal antibody provided by the invention is used for preparing a HIV antigen detection test strip of the HIV, the test strip is embedded with a monoclonal antibody 1 (antibody c) marked by a color microsphere on a marking pad, a detection line is coated with a monoclonal antibody 2 (antibody n), and the HIV antigen in a sample is quantitatively detected by utilizing a double-antibody sandwich method.

1. Preparation of test strips:

1.1 color microsphere labeled antibodies

(1) The pH of the colored microspheres with the particle size of 100nm is regulated to 8.0 by 0.1mol/L K ₂CO₃;

(2) Labeling the microsphere by using a monoclonal antibody 1, namely taking 1mL of the solution with the pH adjusted, adding 1 mug of the monoclonal antibody, wherein the mass ratio of the monoclonal antibody to the color microsphere is 0.3:1, reacting for 1h at room temperature, centrifuging, and discarding the supernatant;

Chicken IgY antibody marked microsphere, 1mL of the solution with the PH adjusted is added with 5 mug of chicken IgY, the mass ratio of the antibody to the color microsphere is 0.05:1, and after reacting for 1h at room temperature, the supernatant is centrifugally discarded;

(3) Blocking for 2h with 20% BSA 1mL, centrifuging, and discarding supernatant;

(4) After re-dissolving the microspheres with 100 mu L of a re-solution with the pH of 8.0, mixing the two microspheres according to the ratio of 5:1, and diluting the mixture with the re-solution according to the ratio of 15%;

1.2 preparation of marking pad

The dilution of the labeled colored microsphere antibody complex was sprayed on the labeling pad using a gold spraying and film drawing instrument at a spray coating pitch of 6 mm and 7.5. Mu.L/cm. And after the spraying is finished, the materials are put into a low-humidity oven at 37 ℃ and dried for 2 hours (30%).

1.3 Sample pad preparation

The sample pad treatment fluid consists of buffer salt, slow release agent, cosolvent, sealing agent and the like. The specific formulation is 20mM Tris, 1% BSA, 0.5% Tween 20, 2% sucrose. Treatment was performed with 1mL of sample treatment fluid per 20 cm ². After the treatment is uniform, the mixture is put into a low-humidity oven at 37 ℃ for 2 hours (< 30%).

1.4 C/T wire coating

The T line was streaked with monoclonal antibody 2 (C line was streaked with goat anti-chicken IgY antibody at a concentration of 2mg/mL, 1. Mu.L/cm), and after completion, was dried at 37℃with low humidity (< 30%) for 24 hours.

1.5 Test strip Assembly

The sample absorbing pad, the NC film and the marking pad are sequentially stuck on the PVC plate, the sample absorbing pad and the marking pad are respectively pressed by 1-2 mm, and the sample pad is pressed by 1-2 mm. Cutting the assembled test strip into a width of 4+/-0.4 mm, and packaging the test strip into a clamping shell. The clamping shell and the drying agent are put into an aluminum foil bag together for molding. And labeling and boxing to obtain the finished product kit.

1.6 Preparation of test samples commercial synthetic protein recombinant antigen from Biotechnology (Shanghai) Co., ltd were used for the test after gradient dilution. The results are shown in Table 8.

TABLE 8

2. Detection of

And placing the detection card on a clean and flat table top, sucking 80-100 mu L of prepared sample of the recombinant antigen, dripping the sample into the sample loading end of the detection card, and setting a PBS control group. After 10min, the inspection card window is observed, 2 lines in the window are positive, and only one C line is negative. The test results are shown in Table 9, wherein the test antigen compliance rate of the test strip assembled by the monoclonal antibody is 100%.

TABLE 9

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.

Claims

1. Human immunodeficiency virus HIV antibody, characterized by comprising antibody c and/or antibody n;

The amino acid sequence of the heavy chain complementary determining region of the antibody c includes: CDR1-VH with an amino acid sequence as shown in SEQ ID NO.4, CDR2-VH with an amino acid sequence as shown in SEQ ID NO.5, and CDR3-VH with an amino acid sequence as shown in SEQ ID NO.6;

The amino acid sequence of the light chain complementary determining region of the antibody c includes: CDR1-VL with an amino acid sequence as shown in SEQ ID NO.7, CDR2-VL with an amino acid sequence as shown in SEQ ID NO.8, and CDR3-VL with an amino acid sequence as shown in SEQ ID NO.9;

The amino acid sequence of the heavy chain complementary determining region of the antibody n includes: CDR1-VH with an amino acid sequence as shown in SEQ ID NO.10, CDR2-VH with an amino acid sequence as shown in SEQ ID NO.11, and CDR3-VH with an amino acid sequence as shown in SEQ ID NO.12;

The amino acid sequence of the light chain complementary determining region of the antibody n includes: CDR1-VL as shown in SEQ ID NO.13, CDR2-VL as shown in SEQ ID NO.14, and CDR3-VL as shown in SEQ ID NO.15.

2. The human immunodeficiency virus (HIV) antibody according to claim 1, characterized in that:

The amino acid sequence of the heavy chain variable region of the antibody c is shown in SEQ ID NO.16; the amino acid sequence of the light chain variable region of the antibody c is shown in SEQ ID NO.17;

And/or, the amino acid sequence of the heavy chain variable region of the antibody n is shown as SEQ ID NO.18; the amino acid sequence of the light chain variable region of the antibody n is shown as SEQ ID NO.19.

3. An immunogenic protein of human immunodeficiency virus (HIV), used for preparing the human immunodeficiency virus (HIV) antibody according to claim 1 or 2, characterized in that the immunogenic protein comprises immunogenic protein C and/or immunogenic protein N; the immunogenic protein C comprises the amino acid sequence shown in SEQ ID NO.2; and/or the immunogenic protein N comprises the amino acid sequence shown in SEQ ID NO.3.

4. A marker protein of human immunodeficiency virus HIV, selected from the p24 protein of the virus, comprising the amino acid sequence shown in SEQ ID NO.2 and the amino acid sequence shown in SEQ ID NO.3, characterized in that the amino acid sequence of the marker protein is as shown in SEQ ID NO.1, and the human immunodeficiency virus HIV antibody according to claim 1 or 2 specifically recognizes the marker protein.

5. Use of the human immunodeficiency virus (HIV) antibody according to claim 1 or 2 in the preparation of a human immunodeficiency virus (HIV) detection product.

6. A human immunodeficiency virus (HIV) test strip, comprising a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked in sequence on the base plate, characterized in that the marking pad contains the human immunodeficiency virus (HIV) antibody according to claim 1 or 2, and the human immunodeficiency virus (HIV) antibody is marked with a marker.

7. The human immunodeficiency virus (HIV) test strip according to claim 6, wherein the marker comprises at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres.

8. The human immunodeficiency virus (HIV) test strip according to claim 6, characterized in that a test line and a quality control line are provided on the test pad; the test line is coated with 0.5-5 mg/mL of the human immunodeficiency virus (HIV) antibody; and the quality control line is coated with 0.5-5 mg/mL of goat anti-mouse IgG polyclonal antibody.

9. The human immunodeficiency virus (HIV) test strip according to claim 8, wherein the human immunodeficiency virus (HIV) antibodies on the labeling pad are different from the human immunodeficiency virus (HIV) antibodies on the detection pad.

10. A human immunodeficiency virus (HIV) detection kit, characterized in that it comprises the human immunodeficiency virus (HIV) detection test strip according to any one of claims 6 to 9 and a shell, wherein the human immunodeficiency virus (HIV) detection test strip is arranged inside the shell.