CN117659170A - Monkey poxvirus detection method and kit - Google Patents

Abstract

The invention provides a monkey pox virus detection kit, which adopts a double-antibody sandwich method to detect monkey pox virus; the monkey pox virus detection kit can effectively improve the specificity and/or the detection rate of detection.

Description

Monkey poxvirus detection method and kit

Technical Field

The invention relates to the field of protein detection. In particular, the invention relates to methods and kits for detecting monkey poxviruses.

Background

Monkey pox (monkey pox) is a zoonotic disease caused by monkey pox virus (MPXV) and was first found in the original forests in western africa and in the middle region. Monkey pox virus is similar to vaccinia virus and smallpox virus and clinically manifests as fever after 10-12 days of exposure and eruption of skin after 2-3 days of fever. Rash changes, ranging from maculopapules, up to small blisters, pustules, and crusting around 10 days. Viruses can be transmitted to humans by direct intimate contact with animals, the transmission route mainly including blood, body fluids, skin or mucosal wounds. The secondary human transmission is mainly transmitted through air droplets, which can infect the mucous membranes of eyes, nose and throat.

At present, polymerase chain reaction PCR is a main detection method for detecting the monkey pox virus at home and abroad, but the PCR is dependent on equipment and is easy to produce pollution. Development of a simple and easy-to-popularize monkey poxvirus immunological detection method is needed.

Disclosure of Invention

The monkey poxvirus A29L protein (MKPVA 29L) is a surface envelope protein of the monkey poxvirus (NCBI sequence No. NP-536566.1), 110 amino acids in length, homologous to the vaccinia virus 162 protein (CPXV-162) and the vaccinia virus A27L protein (VACA 27L).

Through a great deal of theoretical research and experimental groping, the inventor fully considers the structural characteristics of A29L protein of the monkey poxvirus, and performs analysis and research on various A29L antigen sections to be detected and detection antibodies to obtain an A29L antigen region combination capable of being used for A29L detection.

In some embodiments, the invention may include one or more of the following:

1. a monkey poxvirus antibody comprising a first antibody of 19-25aa which binds to the a29L protein of the monkey poxvirus.

2. A monkey poxvirus antibody combination comprising the first antibody of item 1 or 2 and comprising a second antibody of 81-110aa binding to the A29L protein of monkey poxvirus,

3. the monkey poxvirus antibody combination of item 2, wherein the first antibody is a labeled antibody and the second antibody is a coated antibody; or the first antibody is a coating antibody when the second antibody is a labeled antibody.

4. The combination of antibodies of any one of items 2-3, wherein the labeled antibodies are labeled with a detectable label and/or a binding partner.

5. The antibody combination of any one of item 4, wherein the detectable label is selected from the group consisting of a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radiolabel, and an enzymatic label; for example, rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase labels; the binding partner is selected from biotin/avidin, biotin/streptavidin; for example, a labeled antibody is labeled with a detectable label via a binding partner.

6. The antibody combination according to any one of claims 2-3, wherein the coated antibody is attached to a solid phase, such as magnetic particles, latex particles, microtiter plates, nitrocellulose membranes or microfluidic chips, and/or binding partners; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the coated antibody is attached to the solid phase via a binding partner.

7. A kit comprising a first antibody; optionally, a second antibody; wherein the first antibody and the second antibody are defined in any one of items 1 to 6.

8. Use of the antibody of the monkey pox virus of item 1 or the combination of antibodies of any one of claims 2 to 6 for the preparation of a kit for the detection of a monkey pox virus.

9. The use according to claim 8, wherein the kit is for detecting the content of monkey poxvirus.

10. A method of making the antibody of item 1 or the antibody combination of item 2, comprising:

1) Immunization of animals using as antigen or hapten peptide fragments containing fragment 1 and/or fragment 2 as follows:

segment 1: 19-25aa of the monkey poxvirus A29L protein amino acid fragment;

fragment 2: monkey poxvirus a29L protein amino acid fragment 81-110aa;

and

2) Obtaining antibodies from said animal that bind to said fragment 1 or fragment 2, respectively.

Detailed Description

In some embodiments, the kits of the invention comprise a primary antibody that binds 19-25aa of the monkey poxvirus a29L protein.

In some embodiments, the kit of the invention further comprises an antibody that binds 81-110aa of the A29L protein.

In some embodiments, the invention can utilize antibodies that bind to different amino acid fragments to recognize multiple positions on an antigen, reducing the risk of missed detection, and increasing the detection rate.

In some embodiments, the kit includes a detection reagent card (test strip).

In some embodiments, the kit comprises an Elisa plate.

In some embodiments, the kit comprises magnetic beads.

In some embodiments, the invention may be performed by or include reagents for performing fluorescent immunochromatography. In some embodiments, the invention may be performed by or include reagents for performing colloidal gold immunochromatography. The invention may be performed by or include reagents for performing time-resolved fluorescence immunochromatography. The invention may be carried out by or include reagents for carrying out quantum dot chromatography. In some embodiments, the invention may be performed by or include reagents for performing magnetic bead immunochromatography.

The term "antibody" is used herein in the broadest sense. In some embodiments, the antibodies of the invention may be monoclonal antibodies or polyclonal antibodies. In some embodiments, the antibodies of the invention may be full length antibodies or antibody fragments. In some embodiments, the antibodies of the invention may be prepared using methods known in the art. In some embodiments, antibodies of the invention can be prepared by immunizing an animal with an antigen comprising an amino acid fragment described herein. To increase immunogenicity, carrier proteins (including but not limited to BSA, ovalbumin, KLH, etc.) can be coupled to immunoreactive substances (e.g., epitope peptides). Carrier proteins may include proteins or polypeptides, which may function as carriers for immunogens. These types of polypeptides include albumin, serum proteins, globulins, lens proteins, lipoproteins and/or fragments thereof. In some embodiments, immunoreactive substances (e.g., but not limited to, fragments of the monkey pox virus a29L protein) may be used to generate antibodies having affinity for the monkey pox virus a29L protein.

In some embodiments, the antibody binds to an amino acid fragment corresponding to the monkey poxvirus a29L protein, and binding may refer to an antibody being able to bind to the amino acid fragment, but the amino acid fragment is not necessarily the minimum binding fragment.

In some embodiments, the effects of the antibodies of the invention, such as binding activity and/or cross-reactivity, can be detected using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, and the like. In some embodiments, the assay may include, for example, ELISA, FACS binding assay, biacore, competitive binding assay, and the like. In some embodiments, the reactivity of the antibodies of the invention with antigen (antigenic peptide) binding is characterized, for example, in ELISA, and a reaction value of ≡0.5 read by peroxidase-labeled ELISA is determined to be better reactivity and can be used in immunoassays.

In some embodiments, the kit includes a first antibody; in some embodiments, the kit includes a first antibody, a second antibody; in some embodiments, the kit includes a first antibody, a second antibody, and a third antibody; in some embodiments, other antibodies may also be used as coating antibodies or labeling antibodies.

In some embodiments, the coated antibody is bound to a solid phase. In some embodiments, the manner in which the coated antibodies are bound to the solid phase may be direct or indirect. In some embodiments, the coated antibodies may be used to coat a solid support. In some embodiments, the solid support is not particularly limited, and may be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips. In some embodiments, the coated antibody binds to a binding partner, e.g., biotin/avidin, biotin/streptavidin; in some embodiments, the coated antibody is attached to the solid phase by a binding partner.

In some embodiments, the labeled antibody is labeled with a detectable label. In some embodiments, the labeled antibody and the detectable label may be directly or indirectly bound. In some embodiments, the detectable label is, for example, a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radiolabel, or an enzymatic label; in some embodiments, the detectable label may be, for example, rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, glycooxidase, glucose oxidase, galactose oxidase, or glucose-6-phosphate dehydrogenase label. In some embodiments, the labeled antibody is linked to a binding partner, e.g., biotin/avidin, biotin/streptavidin. In some embodiments, the labeled antibody is labeled with a detectable label via a binding partner.

In some embodiments, the kits of the invention comprise reagents suitable for performing an immunoassay. In some embodiments, the kits of the invention may be used to perform immunoassays, such as ELISA, indirect immunofluorescence assay IFA, radioimmunoassay RIA, and other non-enzyme linked antibody binding assays or methods.

In some embodiments, the invention provides a combination of antibodies for detecting the monkey poxvirus a29L protein, comprising a first antibody and a second antibody as defined previously; in some embodiments, a third antibody as defined above is also included.

In some embodiments, the invention provides methods of making antibodies that detect the monkey poxvirus a29L protein comprising immunizing an animal with an immunoreactive polypeptide comprising an amino acid fragment described herein as an antigen or hapten, respectively, to thereby make antibodies, such as monoclonal antibodies or polyclonal antibodies, that detect the monkey poxvirus a29L protein. Monoclonal or polyclonal antibodies can be made by methods known in the art.

In some embodiments, the invention provides the use of an immunoreactive polypeptide comprising an amino acid fragment described herein in the manufacture of a reagent or kit for detecting a monkey poxvirus a29L protein.

The present invention will be described in further detail with reference to the following examples. The following examples are provided to illustrate embodiments of the invention and are not intended to limit the invention. The invention may optionally include embodiments not shown by way of example.

1. Preparation of monkey poxvirus A29L protein monoclonal antibody

1. Immunization of animals: BALB/c mice of 8-12 weeks old and myeloma cell lines are taken, and are fully and uniformly mixed with A29L antigen containing 100 mug/serving of protein and equal amount of Freund's complete adjuvant, injected into the abdominal cavity of the mice, fully and uniformly mixed with 100 mug/serving of A29L antigen and equal amount of Freund's incomplete adjuvant every 2 weeks, and injected into the abdominal cavity of the mice for multiple times to strengthen immunity. The serum of the mice is detected (indirect ELISA method), the titer is more than 1:2000, the mice can be used for fusion, the mice are boosted in abdominal cavity for 3 days before fusion, and the dosage is 50 mug/mouse.

2. Preparation of feeder cells

BALB/c murine peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c mice were sacrificed by pulling the neck, immersed in 75% alcohol whole body, placed in a super clean bench, the abdominal skin was cut off with scissors under aseptic operation, the peritoneum was exposed, 5mL of RPMI 1640 basal medium was injected with a syringe, washing was repeated, the washing liquid was recovered, 1000rpm, centrifuged for 5 minutes, and the pellet was left, and the culture solution was screened with RPMI 1640 (HAT-containing RPMI 164)0 in complete medium), and cell concentration was adjusted to 1X 10 ⁵ mu.L/well of 96-well plate, 150. Mu.L/well, 37℃and 5% CO were added per mL ₂ Culturing overnight.

3. Preparation of immune spleen cells

Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a plate, rinsed once with RPMI 1640 basal culture solution, placed on a nylon mesh of a small beaker, and ground and filtered to prepare a cell suspension. Centrifugation, discarding supernatant, re-suspending the RPMI 1640 basal medium, repeating the above steps three times, and counting.

4. Cell fusion

(1) Taking 40mL of HAT culture solution, 15mL of DMEM serum-free culture solution and 1mL of 50% PEG (M12000), and respectively placing in a water bath at 37 ℃ for pre-heating;

(2) The mouse myeloma cells Sp2/0 (stored by the Phpeng organism Co., ltd.) were taken separately (2-5X 10) ⁷ ) The above-mentioned immune spleen cells (10) ⁸ ) The suspension was added to a 50mL centrifuge tube and mixed well, and DMEM serum-free medium was added to 40mL. Centrifuging for 10min, pouring out supernatant, and mixing;

(3) The centrifuge tube was placed in 37℃pre-warmed water, 0.7mL of pre-warmed 50% PEG solution was taken and allowed to stand for 90 seconds. Immediately dropwise adding 15mL of preheated serum-free culture solution at 37 ℃;

(4) The DMEM serum-free medium was added to 40mL, centrifuged for 10 minutes, and the supernatant was discarded. 40mL of HAT culture solution containing 15% -20% of fetal bovine serum is added. Mixing with a pipette, dripping into small holes of 4 96-well cell culture plates containing feeder cells, 2 drops per well, placing at 37deg.C and 7% CO ₂ Is cultured in an incubator of (a).

5. Selective culture of hybridoma cells

The immune mouse spleen cells and the mouse myeloma cells are treated by PEG to form a mixture of various cell components, wherein the mixture comprises unfused myeloma cells and immune spleen cells, synuclei of the myeloma cells and synuclei of the immune spleen cells, and synuclei of the myeloma cells and the immune spleen cells. Only the latter is able to form hybridoma cells. For this purpose, unfused cells and homofusion co-nuclei must be removed from the mixture of cells and true hybrid cells must be selected. Therefore, the HAT medium was used for the culture at 1,3,5, and 7 days after cell fusion.

6. Antibody detection and hybridoma cell cloning

The supernatant from each well was aspirated and wells containing antibodies to A29L protein were detected in the culture by indirect ELISA. Hybridoma cells were cloned by limiting dilution. Single cells can proliferate into homologous cell clones after culture; the cell strain which has better reactivity and stably secretes the anti-A29L monoclonal antibody is obtained through reactive screening.

Method of reactivity screening: at room temperature, the microtiter plates (Nunc, maxisorb) were coated with 100. Mu.L/well of coating buffer containing 2.5. Mu.g/mL of the monkey poxvirus A29L protein (as antigen) for 1 hour while stirring. Coating post-treatment incubation was performed in PBS buffer and 1% monoclonal antibody for 30min. Subsequently, washing is performed with a washing buffer. 100. Mu.L/well antibody sample was incubated for 1 hour at room temperature with stirring. And then washed 2 more times with the washing solution. Then incubated with 100. Mu.L/well of detection antibody peroxidase-conjugated goat anti-mouse IgG diluted 1:40000 in PBS buffer for another 1 hour at room temperature with stirring. After washing again with wash buffer, peroxidase activity was measured by conventional methods (e.g.by reading the reaction at 405nm with ELISA plate reader) and OD was retained ₄₀₅ Antibodies of ≡0.5 (better reactivity) gave 24 cell lines including 56#, 23#, 14#, 2#, 9# and 64 #.

2. Identification of antibody Cross-reactivity

Cross-reactivity of cell lines secreting antibodies to the monkey poxvirus A29L protein, the poxvirus 162 protein and the vaccinia virus A27L protein was identified by ELISA. The ELISA was performed as follows:

coating: the microtiter plates were coated with 0.5. Mu.g/ml and 0.05. Mu.g/ml MKPVA29L, VCA27L, CPXV162 at 100 ul/well, respectively, and incubated for 2h at 37 ℃.

Blocking 120ul of BSA blocking solution was used and incubated at 37℃for 2h.

Anti/sample: the cell lines secrete antibodies, all diluted to 0.5ug/ml, 0.05ug/ml,50 ul/well with PBS and incubated at 37℃for 30min.

Negative control: incubation with PBS

And (2) secondary antibody: 100 ul/well, goat anti-mouse IgG-HRP 5K diluted in HIV-ED, incubated at 37℃for 30min.

Color development: after 10min, 50ul of stop solution was added to each of the solutions of 50ul A and B, and the solution was read.

The results are shown in the following table:

TABLE 1

Six antibodies 56#, 23#, 14#, 2#, 9#, 64# and 52# all bind MKPVA29L, but only 23# and 64# bind the 17-49aa segment of MKPVA 29L; 56#, 14#, 2#, 9# and 52# bind to VACA27L and CPXV162, 23# and 64# do not bind to VACA27L and CPXV162, indicating that 56#, 14#, 2#, 9# and 52# bind to a common epitope of mkpva29L, VACA L and CPXV162, and 23# and 64# bind to a unique epitope of mkpva29L.

3. Colloidal gold platform detection and verification

Antibody pairing validation 1.

1. Labeling with colloidal gold: 10mL of 4/ten thousand (40 nm) colloidal gold was added to 60. Mu.L of 0.2. 0.2M K ₂ CO ₃ (pH is approximately equal to between 6.8 and 7.2), stirring is carried out for 5min, 100 mug of 14# antibody (volume of added antibody=100 mug/concentration of antibody) is added, stirring is carried out for 10min, 100 mug of 10% BSA is added for blocking and stopping, and labeling and stirring are carried out for 10min; centrifuging at 10000rpm for 5min, removing supernatant, precipitating with gold complex solution, re-dissolving, and finally fixing volume to 1mL (i.e. 1/10 colloidal gold solution volume) with gold complex solution to obtain colloidal gold labeled antibody solution No. 14.

2. Preparing a working solution of the golden seeds: and (3) finally diluting the colloidal gold-labeled No. 14 antibody solution prepared in the step (1) to 10OD by using gold seed diluent to prepare gold seed working solution, and paving the gold seed working solution on glass fibers.

3. Preparing dried golden seeds: the paved golden seeds are put into a freeze dryer for freeze drying (2 h) or are put into a drying room for drying overnight at 37 ℃.

4. Coating T lines: coating T1 line and T2 line with 56# antibody and 23# antibody diluted to 2.0mg/mL with coating diluent, respectively; placing in a 37 ℃ incubator for 60min.

5. Sample pad treatment: the colloidal gold-labeled antibody solution # 14 was sprayed onto the sample pad. The sample pad was 0.6cm wide by 10cm long and was spread at 280. Mu.L.

6. Preparing a gold mark strip: and cutting the gold standard strips according to the required width by using a strip cutting machine, and adding samples for detection after assembly.

MKPVA29L, VACA L and CPXV162 samples were tested, respectively. Note that, the readings of the colloid Jin Seka include 10 gradients of C1, C2, C3, C4, C5, C6, C7, C8, C9, and B, the 10 gradients sequentially lighten from left to right, C1 represents the color of the T line is the darkest, C9 represents the color of the T line is the lightest, B represents no color development, and b+ is almost no color development; "C3+" in Table 2 refers to a color that is slightly darker than C3 but does not reach C2, the other bands "+" and so on.

Antibody pairing validation 2.

The procedure is as above, except that in step 1, the 52# antibody is labeled with colloidal gold, in step 4, the T2 wire is coated with the 64# antibody, and the T1 wire is coated in parallel with the 2# antibody, the 9# antibody and the 56# antibody, respectively. The results of the antibody pairing tests for validation 1 and 2 are shown in Table 2 (the 52# antibody can be replaced by the 32# or 67# antibody with the same epitope, the three antibodies share the same epitope, and the experiment proves that the same way the 64# antibody can be replaced by the 23# antibody, and the two antibodies share the same epitope, and the experiment proves that for the double antibody sandwich method detection, the two antibodies can be used as labeled antibodies or coated antibodies interchangeably, and the final detection result is also equivalent.

TABLE 2

The results indicate that the combination of antibodies binding to the 17-49aa segment of the A29L protein of the monkey pox virus and antibodies binding to the A29L protein of the monkey pox virus but not to the 17-49aa segment of the A29L protein of the monkey pox virus is effective in detecting the A29L protein of the monkey pox virus and is distinguishable from the highly homologous A27L protein of vaccinia virus and the 162 protein of vaccinia virus.

4. Refinement identification of antibody binding fragments

The experimental method is the same as that of the second embodiment, the A29L antigen peptides of different sections are respectively coated on the micropores, PBS is used as diluent, the concentration of the monoclonal antibody is diluted to 0.5-2 mug/mL, goat anti-mouse IgG-HRP is used as secondary antibody, and the specific combined A29L fragment of the monoclonal antibody is determined according to the reaction condition of each monoclonal antibody on different antigens. The validation peptides synthesized are shown in Table 3 below, where A29LA1-A29LA4 were used to refine the identification of specific epitopes for the 64# antibody and the 23# antibody. A29LB-A29LF was used to refine the identification of specific epitopes of antibodies that bind to the A29L protein of the monkey poxvirus but not to the 17-49aa segment of the A29L protein of the monkey poxvirus, and the results of the validation are shown in tables 4 and 5. The specific epitopes of the 64# antibody and the 23# antibody are 19-25aa, while the specific epitopes of the 52# antibody and the like are 81-110aa.

TABLE 3 Table 3

TABLE 4 Table 4

TABLE 5

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (10)

1. A monkey poxvirus antibody comprising a first antibody of 19-25aa which binds to the a29L protein of the monkey poxvirus.

2. A combination of monkey poxvirus antibodies, comprising the first antibody of claim 1 and comprising a second antibody of 81-110aa binding to the monkey poxvirus a29L protein.

3. The combination of monkey poxvirus antibodies of claim 2, wherein the first antibody is a labeled antibody and the second antibody is a coated antibody; or the first antibody is a coating antibody when the second antibody is a labeled antibody.

4. The combination of antibodies of any one of claims 2 to 3, wherein the labeled antibodies are labeled with a detectable label and/or a binding partner.

5. The antibody combination of claim 4, wherein the detectable label is selected from the group consisting of a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radiolabel, and an enzymatic label; for example, rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase labels; the binding partner is selected from the group consisting of biotin/avidin, biotin/streptavidin.

6. The antibody combination according to any one of claims 2-3, wherein the coated antibodies are attached to a solid phase, such as magnetic particles, latex particles, microtiter plates, nitrocellulose membranes or microfluidic chips, and/or binding partners; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the coated antibody is attached to the solid phase via a binding partner.

7. A kit comprising a first antibody; optionally, a second antibody; wherein the first and second antibodies are defined in any one of claims 1-6.

8. Use of the antibody of the monkey poxvirus of claim 1 or the combination of antibodies of any one of claims 2 to 6 for the preparation of a kit for the detection of monkey poxviruses.

9. The use according to claim 8, wherein the kit is for detecting the level of monkey poxvirus.

10. A method of making the antibody of claim 1 or the antibody combination of claim 2, comprising:

1) Immunization of animals using as antigen or hapten peptide fragments containing fragment 1 and/or fragment 2 as follows:

segment 1: 19-25aa of the monkey poxvirus A29L protein amino acid fragment;

fragment 2: monkey poxvirus a29L protein amino acid fragment 81-110aa;

and

2) Obtaining antibodies from said animal that bind to said fragment 1 or fragment 2, respectively.