CN109810190B

CN109810190B - Monoclonal antibody and kit for detecting peste des petits ruminants virus

Info

Publication number: CN109810190B
Application number: CN201910275169.0A
Authority: CN
Inventors: 马宏伟; 金燕萍; 徐宏科; 李彤
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2018-08-16
Filing date: 2019-04-08
Publication date: 2020-12-22
Anticipated expiration: 2039-04-08
Also published as: CN112321705A; CN112321706A; CN112321706B; CN109810190A; CN112321705B

Abstract

The invention relates to a monoclonal antibody and a kit for detecting peste des petits ruminants virus. The monoclonal antibody of the invention is capable of specifically binding Peste des petits ruminants virus. The peste des petits ruminants diagnostic kit of the present invention comprises a substrate, and the monoclonal antibody of the present invention immobilized on the substrate, and can be used for detecting the presence of peste des petits ruminants virus in a biological sample of biological origin of a subject.

Description

Monoclonal antibody and kit for detecting peste des petits ruminants virus

Technical Field

The invention belongs to the technical field of biological detection and medical detection, and particularly relates to a monoclonal antibody and a kit for detecting peste des petits ruminants virus.

Background

Peste des petits ruminants (PPR) is commonly called as plague, also called pseudocattle plague (pseudocardipole disease), pneumoenteritis (pneumoenteritis) and stomatitis-pneumonitis complex (stomatitis-pneumonitis complex), is an acute viral infectious disease caused by peste des petits ruminants virus, mainly infects peste ruminants, and is characterized by fever, stomatitis, diarrhea and pneumonia.

The disease occurs on the coast of ivory for the first time in 1942, and then, the disease is reported in Saibogal, Ganna, Dougo, Beining and the like in Africa, and the disease also occurs in sheep and goats in Nigeria and causes great loss. This disease has also been reported in several countries in asia, and according to the world animal health Organization (OIE) report in 1993 "world animal health", goats in bangladesh have developed this disease, while sheep in parts of indian dela and maharashtra have developed a disease similar to cattle plague, and finally have been diagnosed as peste des petits ruminants, after which infection has also been reported in tamierra. In 1993, Peste des petits ruminants were reported for the first time in Israel, the source of infection was unknown, and to prevent the spread of the disease, Israel inoculated cattle plague vaccines to sheep and goats in northern regions. In 1992, this disease-specific antibody was found in jordan sheep and goats, and in 1993, 11 clinical cases occurred in farms, and more than 100 sheep and goats died. In 1993, 133 cases were first discovered by saudi arabia. In 2007 in 7 months, the epidemic disease is introduced into Tibet regions of China for the first time.

Peste des petits ruminants virus belongs to the genus Paramyxoviridae and the genus morbillivirus. Has similar physical, chemical and immunological properties with Rinderpest virus. Viruses are polymorphic and are generally roughly spherical in shape. The virus particles are larger than Rinderpest virus, and the nucleocapsid is a spiral hollow rod-shaped and characteristic subunit and is provided with an envelope. The virus can proliferate on testis cells and Vero cells of fetal sheep kidney, fetal sheep and newborn sheep, and generate cytopathic effect (CPE) to form syncytial.

The disease mainly infects small ruminants such as goats, sheep, white tailed deer and the like, and is prevalent in western, middle and parts of asia of africa. In epidemic areas, the disease occurs sporadically, and epidemic can occur when susceptible animals increase. The disease is mainly infected by direct contact, and the secretion and excrement of the sick livestock are sources of infection, so that the sick sheep in the subclinical diagnosis type are particularly dangerous. The incubation period of Peste des petits ruminants is 4-5 days, and the longest period is 21 days. The natural onset is seen only in goats and sheep. The goat has serious disease, and the sheep occasionally has serious disease. The lips of some recovered goats developed aphthous lesions. The clinical symptoms of the infected animals are similar to those of cattle plague. The acute body temperature can be raised to 41 ℃ and maintained for 3-5 days. The infected animals have dysphoria, dull hair, dry mouth and nose, and anorexia. Mucus flows through purulent rhinorrhea and smelly gas is exhaled. In the first 4 days of fever, the oral mucosa becomes engorged with blood, the buccal mucosa undergoes extensive damage, causing salivation, and then necrotic lesions appear, and the oral mucosa begins to appear as small rough red superficial necrotic lesions, and later turns pink, and the infected site includes lower lip, lower gum, and the like. Severe cases can be seen where necrotic lesions spread to the pad, palate, cheeks and papillae, tongue, etc. In the later period, watery diarrhea with blood, severe dehydration, emaciation and body temperature reduction follow. Cough and abnormal breathing occur. The incidence rate is up to 100%, the mortality rate is 100% in severe outbreaks, and the mortality rate is not more than 50% in mild outbreaks. The serious morbidity and mortality of the young animals are high, and the disease is a disease defined in China.

The virus has special affinity to gastrointestinal tract lymphocyte and epithelial cell, so that it can cause characteristic pathological changes. Eosinophilic cytoplasmic inclusion bodies and multinucleated giant cells are typically present in infected cells. In lymphoid tissues, peste des petits ruminants virus can cause lymphocyte necrosis. Spleen, tonsil, and lymph node cells were destroyed. Multinucleated giant cells containing eosinophilic cytoplasmic inclusion bodies appeared with few intranuclear inclusion bodies. In the digestive system, the virus causes necrosis of epithelial cells deep in the markia layer, infected cells generate nuclear compaction and nuclear rupture, and multinucleated giant cells containing eosinophilic plasma inclusion bodies are formed in the epidermal germinal layer.

At present, no effective method for treating peste des petits ruminants exists, and control can be performed only by adopting methods of vaccination, killing after epidemic situations occur and regular serum monitoring.

At present, peste des petits ruminants are mainly diagnosed by detecting peste des petits ruminants virus antibodies in biological samples. The peste des petits ruminants virus antibody detection methods recommended by the world animal health organization mainly include Virus Neutralization Test (VNT) and enzyme-linked immunoassay (ELISA). The detection result of the virus neutralization test method is accurate and is a gold standard for detecting the peste des petits ruminants virus, but the method has long detection time and is not suitable for detecting a large number of samples. In contrast, enzyme-linked immunoassay has high specificity and sensitivity, has a shorter detection time than virus neutralization tests, and is suitable for detecting a large number of samples, and therefore, is widely applied to the detection of peste des petits ruminants virus antibodies.

However, the presence of direct evidence in a biological sample that antibodies to peste des petits ruminants virus are not a target organism for peste des petits ruminants is an inherent deficiency of diagnostic methods for diagnosing peste des petits ruminants by detecting antibodies. Therefore, it would be desirable to have an ELISA method for directly detecting peste des petits ruminants virus in a biological sample.

However, the ELISA methods all require the use of monoclonal antibodies, and as mentioned above, Peste des petits ruminants virus is an enveloped virus, and monoclonal antibodies that can be used for detecting Peste des petits ruminants virus by ELISA methods are not readily available.

Disclosure of Invention

In view of the problems of the prior art, it is an object of the present invention to provide a monoclonal antibody capable of specifically binding to peste des petits ruminants virus, and a diagnostic kit for peste des petits ruminants using the monoclonal antibody, which has high specificity and sensitivity.

The present inventors have made intensive studies to solve the above-mentioned problems, and have prepared a rabbit anti-Peste des petits ruminants virus monoclonal antibody (code number: NMS-1-2H3L2) by immunizing a rabbit with a purified Peste des petits ruminants virus, which can specifically bind to Peste des petits ruminants virus and can be used to prepare a Peste des petits ruminants diagnostic kit based on the ELISA principle.

Namely, the present invention comprises:

a monoclonal antibody useful for detecting peste des petits ruminants virus comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:

(a) the amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1 is shown in the specification;

(b) the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2 is shown in the specification;

(c) the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 is shown in the specification;

(d) the amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4 is shown in the specification;

(e) the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5 is shown in the specification; and is

(f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6 is shown in the specification;

wherein the content of the first and second substances,

SEQ ID NO: 1: GIDLNSAT (one letter code of amino acid, the same below)

SEQ ID NO：2：IVSGSTN

SEQ ID NO：3：ARYAGTIAYQWYFNI

SEQ ID NO：4：ESISSI

SEQ ID NO：5：RTS

SEQ ID NO：6：QQGVSNSNVDNI；

Or three heavy chain complementarity determining regions (CDR-H4, CDR-H5 and CDR-H6) and three light chain complementarity determining regions (CDR-L4, CDR-L5 and CDR-L6), wherein:

(g) the amino acid sequence of CDR-H4 is shown in SEQ ID NO: 9 is shown in the figure;

(h) the amino acid sequence of CDR-H5 is shown in SEQ ID NO: 10 is shown in the figure;

(i) the amino acid sequence of CDR-H6 is shown in SEQ ID NO: 11 is shown in the figure;

(j) the amino acid sequence of CDR-L4 is shown in SEQ ID NO: 12 is shown in the specification;

(k) the amino acid sequence of CDR-L5 is shown in SEQ ID NO: 13 is shown in the figure; and is

(l) The amino acid sequence of CDR-L6 is shown in SEQ ID NO: 14 is shown in the figure;

wherein the content of the first and second substances,

SEQ ID NO：9：GIDLGGYA

SEQ ID NO：10：IDTTDS

SEQ ID NO：11：ARYAGDGGGGYFFDY

SEQ ID NO：12：QSVYNNNC

SEQ ID NO：13：GAS

SEQ ID NO：14：VGAYIGSNYA；

or three heavy chain complementarity determining regions (CDR-H7, CDR-H8 and CDR-H9) and three light chain complementarity determining regions (CDR-L7, CDR-L8 and CDR-L9), wherein:

(m) the amino acid sequence of CDR-H7 is set forth in SEQ ID NO: 17 is shown;

(n) the amino acid sequence of CDR-H8 is set forth in SEQ ID NO: 18 is shown in the figure;

(o) the amino acid sequence of CDR-H9 is set forth in SEQ ID NO: 19 is shown in the figure;

(p) the amino acid sequence of CDR-L7 is set forth in SEQ ID NO: 10 is shown in the figure;

(q) the amino acid sequence of CDR-L8 is set forth in SEQ ID NO: 21 is shown in the figure; and is

(r) the amino acid sequence of CDR-L9 is set forth in SEQ ID NO: 22;

wherein the content of the first and second substances,

SEQ ID NO：17：GIDLGGYA

SEQ ID NO：18：IDTTDS

SEQ ID NO：19：ARYAGDGGGGYFFDY

SEQ ID NO：10：QSIYNN

SEQ ID NO：21：EAS

SEQ ID NO：22：QQGYSITNVDNT。

the monoclonal antibody described above, which comprises a heavy chain and a light chain, wherein,

the amino acid sequence of the heavy chain comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) is set forth in SEQ ID NO: 7 is shown in the specification;

the amino acid sequence of the light chain comprising three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3) is set forth in SEQ ID NO: 8 is shown in the specification;

wherein the content of the first and second substances,

SEQ ID NO：7：

CQSLEESGGRLVTPGGSLTLTCTVSGIDLNSATVGWVRQAPGKGLEWIGDIVSGSTNTDYANWASGRFTISKTSSTTVDLKMTSLTTEDTATYFCARYAGTIAYQWYFNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

SEQ ID NO：8：

DVVMTQTPASVEVAVGGTVTIKCQASESISSILAWYQQKPGQRPNLLMYRTSTLASGVSSRFKGSGSGTDFTLTISGVQCDDAATYYCQQGVSNSNVDNIFGGGTEVVVTGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

the amino acid sequence of the heavy chain comprising three heavy chain complementarity determining regions (CDR-H4, CDR-H5, and CDR-H6) is set forth in SEQ ID NO: 15 is shown in the figure;

the amino acid sequence of the light chain comprising three light chain complementarity determining regions (CDR-L4, CDR-L5 and CDR-L6) is set forth in SEQ ID NO: 16 is shown in the figure;

wherein the content of the first and second substances,

SEQ ID NO：15：

CQSLEESGGRLVTPGGSLTLTCTVSGIDLGGYAVGWVRQAPGKGLEYIGIIDTTDSTYYASWAKGRFTSSKTSSTTVDLKMTSLTTEDTATYFCARYAGDGGGGYFFDYWGSGTLVTITSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

SEQ ID NO：16：

AAVLTQTPSPISAAVGGTVTINCQSSQSVYNNNCLSWYQQKPGQPPKFLIYGASTLASGVPSRFKGSGSGTEFTLTISDVQCADAATYYCVGAYIGSNYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

the amino acid sequence of the heavy chain comprising three heavy chain complementarity determining regions (CDR-H7, CDR-H8, and CDR-H9) is set forth in SEQ ID NO: 23 is shown;

the amino acid sequence of the light chain comprising three light chain complementarity determining regions (CDR-L7, CDR-L8 and CDR-L9) is set forth in SEQ ID NO: shown at 24;

wherein the content of the first and second substances,

SEQ ID NO：23：

SEQ ID NO：24：

AAVLTQTPSPISAAVGGTVTINCQSSQSVYNNNCLSWYQQKPGQPPKFLIYGASTLASGVPSRFKGSGSGTEFTLTISDVQCADAATYYCVGAYIGSNYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC。

a detection device comprises a substrate and the monoclonal antibody fixed on the substrate.

The detection device is used for detecting whether the peste des petits ruminants virus exists in a biological sample of a biological source of a subject.

The detection device as described above, wherein the subject organism is a goat or a sheep.

The above detection device, wherein the biological sample is whole blood, plasma or serum.

A diagnostic kit for Peste des petits ruminants is used for diagnosing whether a subject organism suffers from Peste des petits ruminants by detecting the presence or absence of Peste des petits ruminants virus in a biological sample of biological origin of the subject.

The diagnostic kit for Peste des petits ruminants as described above, wherein said subject organism is a goat or a sheep.

The diagnostic kit for Peste des petits ruminants, wherein the biological sample is whole blood, plasma or serum.

Detailed description of the invention

Example 1 purification of Peste des petits ruminants Virus used as antigen

1.1 purification and concentration system: a desktop membrane filtration system, an AKTA Pure protein purification chromatography system, a high-speed refrigerated centrifuge, an ultrarefrigerated centrifuge, a 500K hollow fiber column and a 100kd ultrafiltration centrifugal tube.

1.2 main reagents: 0.02mol/L PBS, 0.5N NaOH solution sephrose 6FF chromatography medium, sucrose.

1.3 antigen preparation and clarification

Under aseptic conditions, 2000mL peste des petits ruminants virus antigen was centrifuged at 5000rpm for 30min at 4 ℃. The precipitate was discarded and the supernatant was taken for ultrafiltration concentration.

1.4 concentration and purification

1.4.1 installing 500K hollow fiber column and pipeline, circularly cleaning 500K hollow fiber column with 2% NaOH alkali solution, soaking overnight, washing residual alkali solution in hollow fiber column with purified water to neutrality, and measuring water flux. The hollow fiber column was equilibrated with sterile PBS solution.

1.4.2 the liquid inlet and the liquid return of the hollow fiber column are aseptically connected and then concentrated, and the pump revolution is kept between 10 percent and 15 percent during treatment. Sampling and detecting when each batch of samples are respectively concentrated by multiple times to 6 times, continuously washing and filtering 6 times of concentrated solution by sterile PBS for 4 times, calculating the washing and filtering times by using the washing and filtering liquid (namely finishing the washing and filtering when the volume of the washing and filtering liquid is 2 times of that of the concentrated solution by 5 times), and respectively taking at least 5 samples after each washing and filtering.

1.4.3 gel filtration chromatography purification

24mL of the PPR ultrafiltration concentrate was sampled at a volume flow rate of 5mL/min, eluted with 0.02mol/L PBS (pH 7.4, Cond 18.0ms/cm), and collected as a UV 280 absorption peak.

1.4.4100 KD ultrafiltration membrane concentration

The absorption peak # 1 was concentrated 5-fold using a Merck-Millipop 100KD ultrafiltration centrifuge tube.

1.4.5 sucrose Density gradient centrifugation

1mL of the sample was added to a discontinuous sucrose density gradient (15% -80%) w/v solution, centrifuged at 35000rpm/min at 4 ℃ for 3h, and 1 tube per 0.5mL, 23 tubes in total, numbered 1-23 in sequence. 70uL of each tube was examined by SDS-PAGE.

1.5 detection

For tubes 1-23, 70uL of each tube was run on SDS-PAGE. The detection result shows that the protein concentration is highest in the tubes 11-14. Therefore, the samples in the tubes 11 to 14 were pooled as antigens for immunization.

Example 2 antigen immunization, B cell screening and preparation of monoclonal antibodies

Antigen immune rabbit, B cell screening and monoclonal antibody preparation were committed to Hangzhou Bailing Biotechnology GmbH. The rabbit was immunized with the antigen for immunization prepared in the above example 1, individual B cells secreting monoclonal antibody against Peste des petits ruminants virus were selected from peripheral blood of the immunized rabbit, and the nucleotide sequence encoding the monoclonal antibody was obtained by sequencing (so that the amino acid sequence of the monoclonal antibody can be known, see SEQ ID NO: 1-8). The monoclonal antibody NMS-1-2H3L2 is obtained by cloning the coding nucleotide sequence into a suitable expression vector and introducing the expression vector into a suitable host cell for expression and purification.

Example 3 preparation and application of Peste des petits ruminants diagnostic kit (detection device)

The solution of the monoclonal antibody NMS-1-2H3L2 is spotted on a solid phase carrier for conventional ELISA by a conventional method, and a PB dot is spotted as a negative quality control dot to prepare a detection device (diagnostic kit).

Detection procedure (antibody sandwich method)

(1) Then, 20 Xconcentrated wash (TBST: 0.4M Tris-HCl, 2.74M NaCl, 2% Tween20, pH 7.2. + -. 0.2) was diluted at 1:20 with purified water to obtain a wash. To completely wet the surface of the test device, about 200 μ L of the cleaning solution was applied to the surface of the test device with a pipette and the test device was soaked for a certain time.

(2) The serum samples to be tested were diluted 1:50 with a sample diluent (0.05MPBS, 1% BSA, 0.2% PVP, 0.5% Tween20, pH7.2. + -. 0.2).

(3) Then, 200. mu.L of the diluted serum sample was aspirated and added to the detection device with the surface completely wet, from the detection device from which the washing solution was discarded.

(4) The detection device was incubated for 30 minutes at 150 rpm in a constant temperature shaker at room temperature.

(5) The serum sample is discarded and the surface of the test device is cleaned with a cleaning solution.

(6) After washing, 200. mu.L of a solution of HRP-labeled monoclonal antibody NMS-1-2H3L2 was added to the test device, and the test device was incubated in a constant temperature shaker at 150 rpm for 30 minutes at room temperature.

(7) And discarding the enzyme-labeled antibody solution, and cleaning the surface of the detection device by using a cleaning solution.

(8) After the completion of the washing, 20. mu.L of a luminescent substrate solution (Thermo, Prod #37074) was uniformly spread on the surface of the detection device.

(9) And carrying out chemiluminescence imaging on the detection device by using a gel imager, and judging the result.

(10) And judging the result: for each serum, whether the monoclonal antibody NMS-1-2H3L2 on the detection device has response (i.e. signal-to-noise ratio (SNR) is more than or equal to 2) is counted and judged. That is to say that the first and second electrodes,

when the monoclonal antibody NMS-1-2H3L2 is detected to respond, the peste des petits ruminants virus is judged to be positive; otherwise, the result is judged to be negative.

Wherein, the signal-to-noise ratio is (polypeptide dot signal value-negative control dot signal value)/negative control dot signal value. The polypeptide dot signal value refers to the chemiluminescence intensity value of the polypeptide dot read by software, and the negative control dot signal value refers to the chemiluminescence intensity value of the negative control dot read by software.

The diagnostic kit and viral nucleic acid amplification were used to detect 800 goat and sheep serum samples from various regions in China, and the detection results are shown below.

TABLE 1800 goat and sheep serum sample test results

Sensitivity 493/560 x 100% 88%

Specificity 224/240 93%

From the above, the sensitivity and specificity of the diagnostic kit are both more than 85% (and the diagnostic kit is verified by adopting a nucleic acid amplification method, but not by adopting other control kit methods), and the requirements of epidemic disease diagnosis are completely met.

Example 4 purification of Peste des petits ruminants Virus used as antigen

1.2 main reagents: 0.02mol/L PBS, 0.5N NaOH solution sephrose 6FF chromatography medium and sucrose.

1.3 antigen preparation and clarification

1.4 concentration and purification

1.4.3 gel filtration chromatography purification

1.4.4100 KD ultrafiltration membrane concentration

1.4.5 sucrose Density gradient centrifugation

1.5 detection

Example 5 antigen immunization, B cell screening and preparation of monoclonal antibodies

Antigen immune rabbit, B cell screening and monoclonal antibody preparation were committed to Hangzhou Bailing Biotechnology GmbH. The rabbit was immunized with the antigen for immunization prepared in the above example 4, individual B cells secreting monoclonal antibody against Peste des petits ruminants virus were selected from peripheral blood of the immunized rabbit, and the nucleotide sequence encoding the monoclonal antibody was obtained by sequencing (so that the amino acid sequence of the monoclonal antibody can be known, see SEQ ID NOS: 9-16). The monoclonal antibody NMS-1-17H6L3 is obtained by cloning the coding nucleotide sequence into a suitable expression vector and introducing the expression vector into a suitable host cell for expression and purification.

Example 6 preparation and application of Peste des petits ruminants diagnostic kit (detection device)

The solution of the monoclonal antibody NMS-1-17H6L3 is spotted on a solid phase carrier for conventional ELISA by a conventional method, and a PB spot is spotted as a negative quality control spot to prepare a detection device (diagnostic kit).

Detection procedure (antibody sandwich method)

(6) After washing, 200. mu.L of a solution of the HRP-labeled monoclonal antibody NMS-1-17H6L3 was added to the test device, and the test device was incubated in a constant temperature shaker at 150 rpm for 30 minutes at room temperature.

(10) And judging the result: for each serum, whether the monoclonal antibody NMS-1-17H6L3 on the detection device has response (i.e., the signal-to-noise ratio (SNR) is greater than or equal to 2) is counted and judged. That is to say that the first and second electrodes,

when the monoclonal antibody NMS-1-17H6L3 is detected to respond, the peste des petits ruminants virus is judged to be positive; otherwise, the result is judged to be negative.

TABLE 2800 test results for goat and sheep serum samples

Sensitivity 505/560 ═ 100%

Specificity 226/240 94%

Example 7 purification of Peste des petits ruminants Virus used as antigen

1.3 antigen preparation and clarification

1.4 concentration and purification

1.4.3 gel filtration chromatography purification

1.4.4100 KD ultrafiltration membrane concentration

1.4.5 sucrose Density gradient centrifugation

1.5 detection

Example 8 antigen immunization, B cell screening and preparation of monoclonal antibodies

Antigen immune rabbit, B cell screening and monoclonal antibody preparation were committed to Hangzhou Bailing Biotechnology GmbH. The rabbits were immunized with the antigen for immunization prepared in the above example 7, individual B cells secreting monoclonal antibodies against Peste des petits ruminants virus were selected from peripheral blood of the rabbits after completion of the immunization, and the nucleotide sequence encoding the monoclonal antibody was obtained by sequencing (so that the amino acid sequence of the monoclonal antibody can be known, see SEQ ID NOS: 17-24). The monoclonal antibody NMS-1-21H1L3 is obtained by cloning the coding nucleotide sequence into a suitable expression vector and introducing the expression vector into a suitable host cell for expression and purification.

Example 9 preparation and application of diagnostic kit (detection device) for Peste des petits ruminants

The solution of the monoclonal antibody NMS-1-21H1L3 is spotted on a solid phase carrier for conventional ELISA by a conventional method, and a PB spot is spotted as a negative quality control spot to prepare a detection device (diagnostic kit).

Detection procedure (antibody sandwich method)

(6) After washing, 200. mu.L of a solution of HRP-labeled monoclonal antibody NMS-1-21H1L3 was added to the test device, and the test device was incubated in a constant temperature shaker at 150 rpm for 30 minutes at room temperature.

(10) And judging the result: for each serum, whether the monoclonal antibody NMS-1-21H1L3 on the detection device has response (i.e., signal-to-noise ratio (SNR) is greater than or equal to 2) is counted and judged. That is to say that the first and second electrodes,

when the monoclonal antibody NMS-1-21H1L3 is detected to have response, the peste des petits ruminants virus is judged to be positive; otherwise, the result is judged to be negative.

TABLE 3800 test results for goat and sheep serum samples

Sensitivity 487/560 × 100%

Specificity 221/240 92%

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalent changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Suzhou nanotechnology and nano-bionic institute of Chinese academy of sciences

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Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val

145 150 155 160

Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser

165 170 175

Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val

180 185 190

Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro

210 215 220

Thr Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe

260 265 270

Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu

275 280 285

Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro

290 295 300

Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val

305 310 315 320

His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg

340 345 350

Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala

370 375 380

Glu Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg

405 410 415

Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys

435 440

<210> 8

<211> 214

<212> PRT

<213> Oryctolagus cuniculus

<400> 8

Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly

1 5 10 15

Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Ser Ser Ile

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Asn Leu Leu Met

35 40 45

Tyr Arg Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Val Gln Cys

65 70 75 80

Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Asn Ser Asn

85 90 95

Val Asp Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val Thr Gly Asp

100 105 110

Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val

115 120 125

Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro

130 135 140

Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly

145 150 155 160

Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn

165 170 175

Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys

180 185 190

Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser

195 200 205

Phe Asn Arg Gly Asp Cys

210

<210> 9

<211> 8

<212> PRT

<213> Oryctolagus cuniculus

<400> 9

Gly Ile Asp Leu Gly Gly Tyr Ala

1 5

<210> 10

<211> 6

<212> PRT

<213> Oryctolagus cuniculus

<400> 10

Ile Asp Thr Thr Asp Ser

1 5

<210> 11

<211> 15

<212> PRT

<213> Oryctolagus cuniculus

<400> 11

Ala Arg Tyr Ala Gly Asp Gly Gly Gly Gly Tyr Phe Phe Asp Tyr

1 5 10 15

<210> 12

<211> 8

<212> PRT

<213> Oryctolagus cuniculus

<400> 12

Gln Ser Val Tyr Asn Asn Asn Cys

1 5

<210> 13

<211> 3

<212> PRT

<213> Oryctolagus cuniculus

<400> 13

Gly Ala Ser

1

<210> 14

<211> 10

<212> PRT

<213> Oryctolagus cuniculus

<400> 14

Val Gly Ala Tyr Ile Gly Ser Asn Tyr Ala

1 5 10

<210> 15

<211> 443

<212> PRT

<213> Oryctolagus cuniculus

<400> 15

Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Gly Gly Tyr

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Ile Ile Asp Thr Thr Asp Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ser Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys

65 70 75 80

Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg

85 90 95

Tyr Ala Gly Asp Gly Gly Gly Gly Tyr Phe Phe Asp Tyr Trp Gly Ser

100 105 110

Gly Thr Leu Val Thr Ile Thr Ser Gly Gln Pro Lys Ala Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val

165 170 175

Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr

180 185 190

Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr

210 215 220

Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr

260 265 270

Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg

275 280 285

Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile

290 295 300

Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His

305 310 315 320

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg

325 330 335

Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu

340 345 350

Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu

370 375 380

Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr

385 390 395 400

Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly

405 410 415

Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys

435 440

<210> 16

<211> 214

<212> PRT

<213> Oryctolagus cuniculus

<400> 16

Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Ile Ser Ala Ala Val Gly

1 5 10 15

Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr Asn Asn

20 25 30

Asn Cys Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Phe

35 40 45

Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Val

65 70 75 80

Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Val Gly Ala Tyr Ile Gly

85 90 95

Ser Asn Tyr Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Asp

100 105 110

Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val

115 120 125

Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro

130 135 140

Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly

145 150 155 160

Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn

165 170 175

Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys

180 185 190

Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser

195 200 205

Phe Asn Arg Gly Asp Cys

210

<210> 17

<211> 8

<212> PRT

<213> Oryctolagus cuniculus

<400> 17

Gly Ile Asp Leu Gly Gly Tyr Ala

1 5

<210> 18

<211> 6

<212> PRT

<213> Oryctolagus cuniculus

<400> 18

Ile Asp Thr Thr Asp Asn

1 5

<210> 19

<211> 15

<212> PRT

<213> Oryctolagus cuniculus

<400> 19

Ala Arg Tyr Ala Gly Asp Gly Gly Gly Gly Tyr Phe Phe Asp Tyr

1 5 10 15

<210> 20

<211> 6

<212> PRT

<213> Oryctolagus cuniculus

<400> 20

Gln Ser Ile Tyr Asn Asn

1 5

<210> 21

<211> 3

<212> PRT

<213> Oryctolagus cuniculus

<400> 21

Glu Ala Ser

1

<210> 22

<211> 12

<212> PRT

<213> Oryctolagus cuniculus

<400> 22

Gln Gln Gly Tyr Ser Ile Thr Asn Val Asp Asn Thr

1 5 10

<210> 23

<211> 443

<212> PRT

<213> Oryctolagus cuniculus

<400> 23

Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Gly Gly Tyr

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Ile Ile Asp Thr Thr Asp Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ser Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys

65 70 75 80

Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg

85 90 95

Tyr Ala Gly Asp Gly Gly Gly Gly Tyr Phe Phe Asp Tyr Trp Gly Ser

100 105 110

Gly Thr Leu Val Thr Ile Thr Ser Gly Gln Pro Lys Ala Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val

165 170 175

Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr

180 185 190

Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr

210 215 220

Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr

260 265 270

Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg

275 280 285

Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile

290 295 300

Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His

305 310 315 320

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg

325 330 335

Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu

340 345 350

Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu

370 375 380

Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr

385 390 395 400

Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly

405 410 415

Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys

435 440

<210> 24

<211> 214

<212> PRT

<213> Oryctolagus cuniculus

<400> 24

Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Ile Ser Ala Ala Val Gly

1 5 10 15

Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr Asn Asn

20 25 30

Asn Cys Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Phe

35 40 45

Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Val

65 70 75 80

Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Val Gly Ala Tyr Ile Gly

85 90 95

Ser Asn Tyr Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Asp

100 105 110

Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val

115 120 125

Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro

130 135 140

Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly

145 150 155 160

Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn

165 170 175

Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys

180 185 190

Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser

195 200 205

Phe Asn Arg Gly Asp Cys

210

Claims

1. A monoclonal antibody useful for detecting peste des petits ruminants virus comprising three heavy chain complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 and three light chain complementarity determining regions CDR-L1, CDR-L2 and CDR-L3, wherein:

(f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO: and 6.

2. The monoclonal antibody of claim 1, comprising a heavy chain and a light chain, wherein,

the amino acid sequence of the heavy chain comprising the three heavy chain complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 is set forth in SEQ ID NO: 7 is shown in the specification;

the amino acid sequence of a light chain comprising three light chain complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 is set forth in SEQ ID NO: shown in fig. 8.

3. A detection device comprising a substrate, and the monoclonal antibody of claim 1 or 2 immobilized on the substrate.