CN116041489A

CN116041489A - Monkey pox virus binding protein and application thereof, and monkey pox virus detection kit

Info

Publication number: CN116041489A
Application number: CN202211188887.2A
Authority: CN
Inventors: 刘春龙; 李梅; 张艳霞; 盛长忠; 粟艳; 周泽奇
Original assignee: Dana Hunan Biotechnology Co ltd; Dana Tianjin Medical Laboratory Co ltd; Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dana Hunan Biotechnology Co ltd; Dana Tianjin Medical Laboratory Co ltd; Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-05-02

Abstract

The invention provides a monkey pox virus binding protein and application thereof, and a monkey pox virus detection kit, and relates to the technical field of biology. The monkey poxvirus binding proteins provided by the invention are selected from protein a, protein b, protein c, protein d, protein e or protein f and define the amino acid sequences of the heavy and light chain complementarity determining regions of the binding proteins. The monkey pox virus binding protein has good specificity, high biological activity, strong stability and high affinity with the monkey pox virus, and can be used for preparing products for detecting the monkey pox virus. The monkey pox virus 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 viruses when the viruses are pandemic, and can be used for rapidly, accurately and safely detecting a large number of asymptomatic virus carrier groups including suspected cases, asymptomatic infection cases in a latent period and higher risks in time.

Description

Monkey pox virus binding protein and application thereof, and monkey pox virus detection kit

Technical Field

The invention relates to the technical field of biology, in particular to a monkey pox virus binding protein and application thereof, and a monkey pox virus detection kit.

Background

The early correct diagnosis of infectious diseases is the basis of timely isolation and effective treatment, and can effectively prevent the spread of the infectious diseases. The detection of infectious diseases includes isolated culture of pathogenic microorganisms, detection of pathogenic microorganism antigens, and nucleic acid detection of pathogenic microorganisms. The separation culture of pathogenic microorganisms and viruses is an important detection method of infectious diseases, and the prediction of the mutation condition of pathogenic bacteria or viruses of the infectious diseases obtained by the separation culture is significant; the development of molecular biology technology has led to the popularization of nucleic acid detection, mainly according to the characteristics of specific nucleic acid sequences and DNA sequences of each organism, only if the gene sequences of specific viruses or pathogenic microorganisms in human bodies are detected, the infection of the pathogenic microorganisms or viruses is proved, and the degree of the infection is determined according to the intensity of detected signals, so that the method has the advantages of high sensitivity and good specificity, but needs professional operators to carry out. The antigen detection of pathogenic microorganisms is an important detection means of serology, has no special requirements on laboratories, has the advantages of early screening and early diagnosis, and is suitable for large-scale screening of basic communities.

Monkey pox, also known as smallpox, is a zoonotic disease caused by the monkey pox virus (MPXV), which can be transmitted by intimate contact or by contact with virus-contaminated materials, and can be transmitted from person to person and from animal to animal. The virus spreads gradually across the geographical limit of the natural epidemic source region in the world at present, becomes the most serious disease related to orthopoxvirus infection after smallpox extinct, has huge diagnosis at present and is increased in several stages. At present, no effective medicine aiming at clinical verification of the monkey pox virus exists, and the transmission of the monkey pox virus is effectively restrained, so that the early diagnosis of the monkey pox is particularly important.

Laboratory tests for infection with monkey pox include general tests and etiology tests, which are mainly performed by virus culture and nucleic acid tests, the virus culture being performed in three or more biosafety laboratories, the nucleic acid tests being identified and detected mainly by means of the nuclear amplification of rashes, blisters, crusts, oropharynx snuff secretions.

The method has the problems of complicated detection steps and long detection period, is not beneficial to rapid screening when viruses are in pandemic, and cannot 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.

In view of this, the present invention has been made.

Disclosure of Invention

It is a first object of the present invention to provide a monkey poxvirus binding protein which is capable of specifically recognizing a monkey poxvirus.

A second object of the present invention is to provide the use of the above-mentioned monkey pox virus binding protein in the preparation of a monkey pox virus detection product.

A third object of the present invention is to provide a monkey pox virus test cassette to solve at least one of the above problems.

In a first aspect, the invention provides a monkey poxvirus binding protein selected from protein a, protein b, protein c, protein d, protein e or protein f;

the protein a comprises a heavy chain complementarity determining region CDR1-VH, CDR2-VH and CDR3-VH with amino acid sequences shown in SEQ ID NO. 1-3 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. 4-6 in sequence;

the protein b comprises a heavy chain complementarity determining region CDR1-VH, CDR2-VH and CDR3-VH with amino acid sequences shown in SEQ ID NO. 7-9 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. 10-12 in sequence;

the protein c comprises a heavy chain complementarity determining region CDR1-VH, CDR2-VH and CDR3-VH with amino acid sequences shown in SEQ ID NO. 13-15 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. 16-18 in sequence;

the protein d comprises a heavy chain complementarity determining region CDR1-VH, CDR2-VH and CDR3-VH with amino acid sequences shown in SEQ ID NO. 19-21 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. 22-24 in sequence;

the protein e comprises a heavy chain complementarity determining region CDR1-VH, CDR2-VH and CDR3-VH with amino acid sequences shown in SEQ ID NO. 25-27 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. 28-30 in sequence;

the protein f comprises a heavy chain complementarity determining region CDR1-VH, CDR2-VH and CDR3-VH with amino acid sequences shown in SEQ ID NO. 31-33 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. 34-36 in sequence.

As a further technical scheme, the protein a 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. 37-38 in sequence;

the protein b comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are shown in SEQ ID NO. 39-40 in sequence;

the protein c comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are shown in SEQ ID NO. 41-42 in sequence;

the protein d comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are shown in SEQ ID NO. 43-44 in sequence;

the protein e comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are shown in SEQ ID NO. 45-46 in sequence;

the protein f comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are shown in SEQ ID NO. 47-48 in sequence;

preferably, the monkey poxvirus binding protein is of rabbit origin.

In a second aspect, the invention provides the use of said monkey pox virus binding proteins in the preparation of a monkey pox virus detection product.

In a third aspect, the invention provides a monkey pox virus detection test strip, which comprises a bottom plate, and a sample loading pad, a marking pad, a detection pad and a sample absorbing pad which are sequentially overlapped and arranged on the bottom plate;

the labeling pad contains the monkey pox virus binding protein, and the monkey pox virus binding protein is labeled with a label.

As a further technical scheme, the marker comprises at least one of colloidal gold, color microspheres, time-resolved fluorescent microspheres or quantum dot microspheres;

preferably, the particle size of the colloidal gold is 40-100 nm;

preferably, the particle sizes of the color microspheres, the time-resolved fluorescence microspheres and the quantum dot microspheres are respectively and independently 100-300 nm;

preferably, the mass ratio of the monkey poxvirus binding eggs to the colloidal gold is: (0.04-0.32): 1, a step of; the mass ratio of the monkey pox virus binding egg to the color microsphere, the time-resolved fluorescent microsphere or the quantum dot microsphere is as follows: (0.1-0.4): 1.

as a further technical scheme, the detection pad is provided with a detection line and a quality control line;

the detection line is coated with 1-2 mg/ml of the monkey pox virus binding protein;

the quality control line is coated with 1-2 mg/ml sheep-derived IgG polyclonal antibody;

preferably, the dilutions of the monkey pox binding proteins and sheep derived IgG polyclonal antibodies are 15 to 25mM PB buffer comprising trehalose, comprising 0.1 to 0.9g trehalose per 100ml dilution.

As a further technical scheme, the monkey pox virus binding protein on the label pad and the monkey pox virus binding protein on the detection pad are different from each other.

In a fourth aspect, the invention provides a monkey pox virus detection kit, which comprises a monkey pox virus detection test strip and a shell, wherein the monkey pox virus detection test strip is arranged in the shell.

As a further technical scheme, the shell comprises an upper cover and a lower cover which are detachably connected;

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

As a further technical scheme, the detection sample of the monkey pox virus detection kit comprises at least one of blood, saliva, pustule fluid at an infection site or scab;

preferably, the monkey pox virus detection kit further comprises a sample extract;

the extracting solution of the blood sample is 45-55 mM PB buffer solution, and each 100mL of 45-55 mM PB buffer solution comprises 0.4-0.6 g Triton x-100, 0.1-0.3 g Proclin300, 0.4-0.6 g casein sodium and 0.8-1 g NaCl;

the extracting solution of the saliva sample and the pustule liquid sample at the infection part is 90-110 Mm Tris buffer, each 100mL of the 90-110 Mm Tris buffer comprises 0.9g NaCl, 0.4-0.6 g Triton x-100 and 0.4-0.6 g tween 20, and the pH of the extracting solution is 8.5.

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

the monkey pox virus binding protein provided by the invention has the advantages of good specificity, high biological activity, strong stability and high affinity with the monkey pox virus, and can be used for preparing products for detecting the monkey pox virus.

The monkey pox virus 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 viruses when the viruses are pandemic, rapidly, accurately and safely detecting a large number of asymptomatic virus carrier groups including suspected cases, asymptomatic infection cases in a latent period and higher risk in time, and indirectly solving the problem of insufficient detection capability of monkey pox nucleic acid and reducing the potential infection risk.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the top cover of the monkey pox virus detection kit of the invention;

FIG. 2 shows the lower cover of the monkey pox virus detection kit of the invention;

FIG. 3 is a block diagram of a test strip for detecting monkey pox virus of the present invention;

FIG. 4 is an electrophoresis pattern of anti-monkey poxvirus monoclonal antibodies;

fig. 5 is a diagram showing the detection result.

Icon: 1-a viewing window; 2-a sample adding hole; 3-detecting the clamping strip area; 4-a bottom plate; 5-a detection pad; 6-a sample absorbing pad; 7-marking the pad; 8-loading pad; 9-a quality control line; 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 joining the CDRs numbered below with the FR in a combination arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

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.

In the present invention, A29L, A35R, M R is the surface envelope protein of the monkey pox virus.

The amino acid sequences shown in SEQ ID NOS.1 to 36 are shown in Table 1.

TABLE 1

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In some preferred embodiments, the protein a comprises a heavy chain variable region and a light chain variable region having the amino acid sequences shown in SEQ ID NOS.37 to 38;

preferably, the monkey poxvirus binding protein is of rabbit origin.

The amino acid sequences shown in SEQ ID NOS.37 to 48 are shown in Table 1.

TABLE 2

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the label pad contains the monkey pox virus binding protein, the monkey pox virus binding protein is labeled with a label, and the label can be used for detecting the position or the concentration of the identification label.

The monkey pox virus detection test strip provided by the invention has the advantages of simplicity and convenience in operation, quick response, high sensitivity, strong specificity, suitability for on-site quick detection, economy, practicability and the like, can be used for quick screening when viruses are pandemic, can be used for quickly, accurately and safely detecting a large number of asymptomatic virus carrier groups including suspected cases, asymptomatic infection cases in a latent period and higher risk in time, and can be used for indirectly solving the problem of insufficient detection capability of monkey pox nucleic acid and reducing the potential infection risk.

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, 40nm, 60nm, 80nm, or 100nm;

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

preferably, the mass ratio of the monkey poxvirus binding egg-coupled colloidal gold is: (0.04-0.32): 1, a step of; the mass ratio of the monkey pox virus binding egg to the color microsphere, the time-resolved fluorescent microsphere or the quantum dot microsphere is as follows: (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);

In some preferred embodiments, the monkey pox binding proteins on the label pad are different from the monkey pox binding proteins on the test pad.

For example, the protein on the label pad is a or c or e, where a is the monkey pox virus a29L monoclonal antibody 1, c is the monkey pox virus a35R monoclonal antibody 1, e is the monkey pox virus M1R monoclonal antibody 1, and the protein on the test pad is in turn the monkey pox binding protein b or d or f, where b is the monkey pox virus a29L monoclonal antibody 2, d is the monkey pox virus a35R monoclonal antibody 2, f is the monkey pox virus M1R monoclonal antibody 2.

According to the principle of antigen-antibody reaction, the test strip provided by the invention uses colloidal gold, color microspheres, time-resolved fluorescent microspheres and quantum dots to mark the monkey pox virus monoclonal antibody, and then the marker is solidified on a glass cellulose film. The detection pad (such as NC membrane) is coated with another monoclonal antibody of the monkey pox virus, 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 monkey pox virus 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 monkey pox virus does not exist in the sample, the strip does not exist on the 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 15-20min in the whole process, 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.

The monkey pox virus detection kit provided by the invention contains the monkey pox virus detection test strip, so that the monkey pox virus detection kit has all the beneficial effects of the monkey pox virus detection test strip.

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

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 adding pad.

In some preferred embodiments, the detection sample of the monkey poxvirus detection kit includes, but is not limited to, blood, saliva, pustule fluid at the site of infection, or scab; the sample may be a sample collected in a test tube or may be a sample attached to a swab.

Preferably, the monkey pox virus detection kit further comprises a sample extracting solution, wherein the sample extracting solution can treat samples such as vesicles, pustule liquid swabs and the like of saliva, blood and skin tissue infection parts, and then the monkey pox virus detection kit is used for detection.

The blood sample extract is prepared by using 45-55 mM PB buffer solution, and each 100mM LPB buffer solution comprises: 0.4-0.6 g Tritonx-100, 0.1-0.3 g Proclin300, 0.4-0.6 g casein sodium, 0.8-1 g NaCl;

the extracting solution of the saliva sample and the pustule liquid sample at the infection part is prepared by adopting 90-110 Mm Tris buffer solution, wherein each 100ml Tris buffer solution comprises 0.9g NaCl, 0.4-0.6 g Triton x-100 and 0.4-0.6 g Tween 20, and the pH value of the extracting solution is 8.5;

taking a serum sample as an example, a serum sample diluent and a sample adding mode are as follows:

preparation of the diluent:

dilutions of a sample of a blood sample of a monkey pox antigen detection kit were prepared using 100ml as an example:

(1) 1.79g of Na was weighed out by a balance ₂ HPO ₄ 、0.78g NaH ₂ PO ₄ 0.5g of sodium caseinate,0.9g NaCl, dissolved in water;

(2) Weighing 0.5g Tritonx-100 and 0.1g Proclin300 by using an EP tube respectively, and repeatedly cleaning the EP tube by using the solution in the step 1 to completely dissolve in water;

(3) Adjusting the pH value to 8.0 by using 4M NaOH or HCl, adding water to 100ml, and fixing the volume by using a volumetric flask of 100 ml;

kit test sample loading:

(1) When testing blood samples (including blood plasma, serum or whole blood), 1 drop (about 40-50 ul) of the prepared blood sample diluent is quickly added to react after 1 drop (about 40-50 ul) of the low-cost 1 drop of the sample is added in the sample adding hole in FIG. 1;

(2) After 15min the results were analyzed according to different methodologies.

Taking saliva, vesicle and pustule swab samples as examples, saliva, vesicle and pustule swab extracting solutions and sample adding modes are as follows:

preparation of the extract:

taking 100ml as an example, preparing a monkey pox virus antigen detection kit swab extracting solution:

(1) 12.1g Ttis and 8.7g NaCl are weighed and dissolved in 80ml water;

(2) Weighing 0.5g Tritonx-100 and 0.5g Tween 20 by using an EP tube, and repeatedly cleaning the EP tube by using the solution in the step 1 to completely dissolve in water;

(3) Adjusting the pH value to 8.5 by using 4M NaOH or HCl, adding water to 100ml, and fixing the volume by using a volumetric flask of 100 ml;

saliva, vesicle, pustule swab sample treatment and loading:

(1) Sampling the saliva, vesicle and pustule by using a sampling swab;

(2) Immersing the sampled swab in an extraction tube under stirring for 6-8 or immersing the sampled swab in the extraction liquid for 1min;

(3) After the cover of the extraction tube is covered, 2-3 drops of the mixed liquid (about 100 ul) of the extraction sample are dripped into the sample adding hole;

(4) After 15min, the detection results were analyzed according to different methodologies.

The detection effect of the extracting solution after the corresponding sample is treated is good.

The invention is further illustrated by the following specific examples and comparative examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and should not be construed as limiting the invention in any way.

In the following examples, unless otherwise indicated, dilutions of monkey pox binding proteins and goat anti-rabbit IgG polyclonal antibodies were 20mM PB buffer containing trehalose, with 0.5g of trehalose per 100ml of dilution;

the extract of the blood sample was prepared using 50mM PB buffer, containing per 100mM LPB buffer: 0.5g Tritonx-100, 0.2g Proclin300, 0.5g sodium caseinate, 0.9g NaCl;

the extracts of saliva samples and pustule fluid samples at the affected sites were prepared using 100mM Tris buffer containing 0.9g NaCl, 0.5g Triton x-100, 0.5g Tween 20 per 100mM Tris buffer, pH 8.5.

Example 1

The monkey poxvirus binding proteins include: the variable region sequences of the monkey poxvirus A29L monoclonal antibody 1, the monkey poxvirus A29L monoclonal antibody 2, the monkey poxvirus A35R monoclonal antibody 1, the monkey poxvirus A35R monoclonal antibody 2, the monkey poxvirus M1R monoclonal antibody 1 and the monkey poxvirus M1R monoclonal antibody 2 are shown in Table 2.

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, the 6 anti-monkey poxvirus monoclonal antibodies showed two characteristic bands of about 25KD and 55KD, 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-MPXV-A29L-1;2: anti-MPXV-A29L-2;3: anti-MPXV-A35R-1; 4: anti-MPXV-A35R-2;5: anti-MPXV-M1R-1;6: anti-MPXV-M1R-2. Wherein, the Anti-MPXV-A29L-1 is Anti-monkey pox virus A29L monoclonal antibody 1; anti-MPXV-A29L-2 is Anti-monkey poxvirus A29L monoclonal antibody 2; anti-MPXV-A35R-1 is Anti-monkey poxvirus A35R monoclonal antibody 1; anti-MPXV-A35R-2 is Anti-monkey poxvirus A35R monoclonal antibody 2; anti-MPXV-M1R-1 is Anti-monkey poxvirus-M1R monoclonal antibody 1; anti-MPXV-M1R-2 is Anti-monkey poxvirus M1R monoclonal antibody 2.

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

Example 2

A monkey pox virus detection kit is shown in figures 1-3, and comprises a monkey pox virus detection test strip and a shell, wherein the monkey pox virus detection test strip is arranged inside the shell. The monkey pox virus detection test strip comprises a bottom plate 4, and a sample loading pad 8, a marking pad 7, a detection pad 5 and a sample absorbing pad 6 which are sequentially overlapped and arranged on the bottom plate; the detection pad is provided with a detection line 10 and a quality control line 9; the shell comprises an upper cover and a lower cover which are detachably connected; the upper cover is provided with observation window 1 and application of sample hole 2, and the lower cover is provided with detects card dress strip district 3.

Example 3 preparation of antibody labeled with colloidal gold monkey pox antigen detection kit

1 main material

1.1 antibodies: the rabbit anti-A29L, A35R, M R monoclonal antibodies are self-developed antibodies of the company and are rabbit-derived monoclonal antibodies, and are respectively marked and coated; goat anti-rabbit IgG antibody: shenzhen Fengpeng biological stock Co., ltd, which is used for the coating of nitrocellulose membrane quality control lines respectively.

1.2 nitrocellulose membrane: NC film is a product of the company cerdolis.

1.3 other consumables: other consumables such as PVC plates are manufactured by Bi Kenlai Bo company; the reagents are all analytically pure.

1.4 acquisition of recombinant antigen: A29L, A35R, M R protein recombinant antigen was commercially synthesized by the company Shanghai, inc. of biological engineering, according to the data disclosed in database NCBI for monkey poxviruses.

2 method

2.1 preparation of colloidal gold-labeled pad:

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

(1) 1ml of a 40nm colloidal gold solution was taken and treated with 0.2. 0.2M K ₂ CO ₃ Adjusting the pH value to 8.5;

(2) 25ugA L monoclonal antibody 1 (or A35R monoclonal antibody 1 or M1R monoclonal antibody 1) is added, and the mass ratio of the antibody to the colloidal gold particles is as follows: 0.1:1, regulating a rotary table to a certain rotating speed, rotationally marking for 1.5 hours at room temperature, and then adding 20ul of sealing liquid;

(3) Centrifuging at 12000rpm for 15min, and discarding supernatant;

(4) Adding 100ul of colloidal gold complex solution;

(5) The concentrate was prepared according to 1:7, after diluting in proportion, spraying metal, and then placing in a drying oven at 37 ℃ for drying for 2 hours for standby;

2.2NC film coating:

A29L monoclonal antibody 2 (A35R monoclonal antibody 2 is selected here if the colloidal gold-labeled antibody is A35R monoclonal antibody 1, and M1R monoclonal antibody 2 is selected here if the colloidal gold-labeled antibody is M1R monoclonal antibody 1) was diluted to 1.5mg/ml and goat anti-rabbit IgG was diluted to 2mg/ml with 0.02M PB containing 0.5% trehalose, respectively; and then a film spraying instrument is used for respectively marking a detection line T and a quality control line C on the nitrocellulose film, and the NC film is dried in an oven at 37 ℃ for 24 hours for standby 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 for pasting, overlapping a marker colloidal gold pad (1/3 of the overlapping colloidal gold pad) on one side of a nitrocellulose membrane T line for pasting, and overlapping a sample loading pad (1/5 of the overlapping 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; then cutting the plastic plate into test strips with certain width by a cutting machine, and then putting the test strips into a detection card to form the monkey pox virus 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, adding 50ul of serum into a sample adding hole by using a liquid transfer device, then rapidly adding 50ul of sample diluent or repeatedly stirring a sampling swab into an extraction tube for 6-7 times, then taking out the swab, and then covering the extraction tube with a cover and dripping 3 drops of samples; step 4, timing by a timer, and judging a result in 15 minutes; note that if the sample is not laterally chromatographed or diafiltered for 1min after loading, it may be due to the sample being too viscous, requiring pretreatment of the sample with physiological saline.

3 results

Under the action of lateral chromatography, when the monkey pox virus exists in the sample, the detection line is colored, and the quality control line is colored (a in fig. 5); when the sample does not contain the monkey pox virus, the detection line does not develop, and the quality control line develops (b in fig. 5); after loading, the quality control line was not developed, and whether the detection line was developed or not developed, the results were judged to be invalid (c in fig. 5, d in fig. 5).

Example 4 time resolved fluorescent microsphere labeled antibody A monkey pox virus antigen detection kit was prepared.

The kit 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 A29L monoclonal antibody 1 (or A35R monoclonal antibody 1 or M1R monoclonal antibody 1) in a fluorescent pad, the detection line is coated with A29L monoclonal antibody 2 (or A35R monoclonal antibody 2 or M1R monoclonal antibody 2), and the monkey pox virus antigen in a sample is detected by utilizing a double-antibody sandwich method.

1 preparation and operation processes of the kit:

the A29L monoclonal antibody 1 (or the A35R monoclonal antibody 1 or the M1R monoclonal antibody 1) prepared by the method is marked on the surface of the time-resolved fluorescent microsphere. Specific examples are as follows:

time-resolved fluorescent microsphere antibody labeling: 1mL of 1% carboxyl time-resolved fluorescent microsphere, 9mL of MES buffer was added, 25. Mu.L of EDC solution (10 mg/mL) and 25. Mu.L of NHS solution (10 mg/mL) were added, and the reaction was performed at room temperature with shaking for 30min, and the precipitate was collected by centrifugation. HEPES complex solution was added, 1mL of 1mg/mL of A29L monoclonal antibody 1 (or A35R monoclonal antibody 1 or M1R monoclonal antibody 1) was added after uniform ultrasonic dispersion, the reaction was performed by shaking at room temperature for 120min, and the precipitate was collected by centrifugation. Then 1mL of sealing solution is added, and the reaction is carried out for 120min at room temperature in a shaking way. Centrifuging to collect microsphere precipitate, and redissolving with redissolution.

Preparing a fluorescent pad: the marked time-resolved fluorescent microspheres are diluted by microsphere compound solution, a metal spraying and film drawing instrument is used for spraying fluorescent mats, and the spraying distance is 6mm. And after the spraying is finished, the mixture is put into a low-humidity oven at 37 ℃ for 2 hours (< 30%).

NC film coating CT line: A29L monoclonal antibody 2 (A35R monoclonal antibody 2 was selected here if the above-mentioned colloidal gold-labeled antibody was A35R monoclonal antibody 1, and M1R monoclonal antibody 2 was selected here if the above-mentioned colloidal gold-labeled antibody was M1R monoclonal antibody 1) was used at a concentration of 1.5mg/mL for the T line, a goat anti-rabbit IgG antibody at a concentration of 1mg/mL was used for the C line, a 1. Mu.L/cm line was drawn, and after completion, the mixture was dried at 37℃with low humidity (< 30%) for 24 hours.

Sample pad treatment: the sample pad treatment fluid consists of buffer salt, slow release agent, cosolvent, sealing agent and the like. The specific formulation is 20mM Tris buffer, containing 1g BSA, 0.5g Tween 20, 2g sucrose per 100ml Tris buffer. According to every 20cm ² Treatment was performed using 1mL of sample treatment fluid. After even treatment, the mixture is put into a low humidity chamber with the temperature of 37 DEG C<30%) for 2h.

Assembling a test strip: NC film, sample absorbing pad, fluorescent pad and sample loading pad are sequentially stuck on the PVC plate, the sample absorbing pad and the fluorescent pad respectively press the NC film by 1-2 mm, and the sample loading pad presses the fluorescent pad 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 and then sealed. And (5) labeling and boxing to obtain the finished product detection card.

Test sample preparation: the recombinant antigen of A29L, A35R, M R protein is commercially synthesized by the division of biological engineering (Shanghai) and subjected to gradient dilution for testing.

2 detection procedure of kit

Placing the detection card on a clean and flat table top, sucking 90-100 mu L of processed sample, dripping the sample into the sample loading end of the detection card, and simultaneously setting a PBS control group.

After the test strip standard curve is introduced for 15min, a fluorescence immunoassay instrument is used for scanning a detection area to obtain a fluorescence signal, the concentration value corresponding to the monkey pox virus antigen is displayed after detection, and the measurement results are shown in the following tables 3 and 4.

TABLE 3 test results of diluent samples

TABLE 4 results of Positive recombinant antigen test

Example 5 preparation of antibody labeled with colored microspheres monkey pox virus antigen detection kit

3 pairs of monkey pox virus monoclonal antibodies independently developed by the company are used for preparing a monkey pox virus antigen detection test strip, the test strip is embedded with a color microsphere marked A29L monoclonal antibody 1 (or A35R monoclonal antibody 1 or M1R monoclonal antibody 1) on a marking pad, a detection line is coated with A29L monoclonal antibody 2 (or A35R monoclonal antibody 2 or M1R monoclonal antibody 2), and the monkey pox virus antigen in a sample is quantitatively detected by using a double antibody sandwich method.

1, preparing a test strip:

1.1 color microsphere labeled antibodies

(1) The 100 nm-diameter color microspheres are treated with 0.1mol/L K ₂ CO ₃ Modulating the pH to 8.0;

(2) Adding 30ug of A29L monoclonal antibody 1 (or A35R monoclonal antibody 1 or M1R monoclonal antibody 1) into 1mL of the solution with the pH adjusted, wherein the mass ratio of the antibody to the color microspheres is as follows: 0.3:1, after reacting for 1h at room temperature, centrifuging and discarding the supernatant; 1mL of the PH-adjusted solution is added with chicken IgY 5ug, and the mass ratio of the antibody to the color microsphere is 0.05:1, after reacting for 1h at room temperature, centrifuging and discarding the supernatant;

(3) 1mL of 20% BSA was added and blocked for 2 hours, and the supernatant was centrifuged off;

(4) After re-dissolving the above microspheres with 100ul of a complex solution of ph8.0, two microspheres were prepared according to a ratio of 5:1, mixing, and diluting the mixture with a compound solution according to the proportion of 15%;

1.2 preparation of marking pad

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

1.3 sample pad preparation

The sample loading pad treatment solution consists of buffer salt, a slow release agent, a cosolvent, a 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 the sample treatment solution per 20cm 2. After the treatment is uniform, the mixture is put into a low humidity (< 30%) at 37 ℃ and dried for 2 hours.

1.4C/T wire coating

A29L monoclonal antibody 2 with a concentration of 1.5mg/mL was used as the T line (1.2 mg/mL of A35R monoclonal antibody 2 was selected here if the above-mentioned colloidal gold-labeled antibody was A35R monoclonal antibody 1, 2.1mg/mL of M1R monoclonal antibody 2 was selected here if the above-mentioned colloidal gold-labeled antibody was M1R monoclonal antibody 1), a sheep anti-chicken IgY antibody with a concentration of 2mg/mL was used as the C line, 1. Mu.L/cm was streaked, and after the completion, the mixture was put into a low humidity of 37 ℃ (< 30%) and dried for 24 hours.

1.5 test strip Assembly

The PVC plate is sequentially stuck with a sample sucking pad, NC films and a marking pad, the sample sucking pad and the marking pad are respectively pressed by the NC films by 1-2 mm, and the sample sucking pad and the marking pad are pressed by the marking pad 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: the recombinant antigen of A29L, A35R, M R protein is commercially synthesized by the division of biological engineering (Shanghai) and used for testing after gradient dilution.

2 detection

The detection card is placed on a clean and flat table top, 90-100 mu L of prepared sample of recombinant antigen is sucked and dripped into the sample adding end of the detection card, and a PBS control group is arranged. After 10min, the inspection card window is observed, 2 lines in the window are positive, and only one C line is negative. The detection results are as follows: the results show that the test antigen coincidence rate of the test strip assembled by the three pairs of monoclonal antibodies is 100 percent;

TABLE 5

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A monkey poxvirus binding protein selected from the group consisting of protein a, protein b, protein c, protein d, protein e, and protein f;

2. The monkey pox virus binding protein of claim 1 in which the binding protein is a variant of a monkey pox virus,

the protein a comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are shown in SEQ ID NO. 37-38 in sequence;

preferably, the monkey poxvirus binding protein is of rabbit origin.

3. Use of a monkey poxvirus binding protein according to claim 1 or 2 for the preparation of a monkey poxvirus detection product.

4. The monkey pox virus detection test strip is characterized by comprising a bottom plate, and a sample loading pad, a marking pad, a detection pad and a sample absorbing pad which are sequentially overlapped and arranged on the bottom plate;

the label pad contains the monkey poxvirus binding protein of claim 1 or 2, which is labeled with a label.

5. The monkey pox virus detection test strip of claim 4 wherein the label comprises at least one of colloidal gold, colored microspheres, time resolved fluorescent microspheres, or quantum dot microspheres;

preferably, the particle size of the colloidal gold is 40-100 nm;

6. the monkey pox virus detection test strip of claim 4 wherein the detection pad is provided with a detection line and a quality control line;

the detection line is coated with 1-2 mg/ml of the monkey poxvirus binding protein of claim 1 or 2;

7. The test strip of claim 6, wherein the monkey pox binding protein on the label pad and the monkey pox binding protein on the test pad are different from each other.

8. A monkey pox virus detection kit comprising the monkey pox virus detection test strip of any one of claims 4 to 7 and a housing, wherein the monkey pox virus detection test strip is disposed within the housing.

9. The monkey pox virus detection kit of claim 8 wherein the housing comprises a removably attached upper cover and lower cover;

10. The monkey poxvirus detection kit of claim 8, wherein the detection sample of the monkey poxvirus detection kit comprises at least one of blood, saliva, pustule fluid at the site of infection, or crusting;