CN111638329B

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Info

Publication number: CN111638329B
Application number: CN202010519967.6A
Authority: CN
Inventors: 王文敬; 李金峰; 任慧; 黎诚耀
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2021-06-01
Anticipated expiration: 2040-06-09
Also published as: CN111638329A

Abstract

Translated fromChinese

本发明公开了一种用于检测布鲁氏菌病的ELISPOT检测试剂盒及其应用。一种用于检测布鲁氏菌病的ELISPOT检测试剂盒，所述ELISPOT检测试剂盒包括：支持介质、捕获抗体、检测抗体、阴性对照和阳性对照和特异性刺激剂，所述ELISPOT检测试剂盒还包括特异性抑制剂，以通过特异性刺激剂和特异性抑制剂双向来测定是否存在布鲁氏菌感染。本发明ELISPOT检测试剂盒通过正反实验来测定是否存在布鲁氏菌感染，检测结果更为客观、准确，更能真实反映体内免疫水平，以解决现行布鲁氏菌感染检测中存在的准确率低、周期长等问题。The invention discloses an ELISPOT detection kit for detecting brucellosis and its application. A kind of ELISPOT detection kit for detecting brucellosis, described ELISPOT detection kit comprises: support medium, capture antibody, detection antibody, negative control and positive control and specific stimulator, described ELISPOT detection kit Specific inhibitors are also included to determine the presence or absence of Brucella infection bidirectionally by specific stimulators and specific inhibitors. The ELISPOT detection kit of the invention determines whether there is Brucella infection through positive and negative experiments, and the detection results are more objective and accurate, and can more truly reflect the immune level in the body, so as to solve the existing accuracy rate in the current detection of Brucella infection Low, long cycle and other issues.

Description

ELISPOT detection kit for detecting brucellosis and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to an ELISPOT detection kit for detecting brucellosis and application thereof.

Background

Brucellosis (Brucellosis, abbreviated as Brucellosis) is a common infectious disease of human and animal life threatening the health of human and various animals. Epidemic livestock such as sheep and cattle with diseases are the main infection source of brucellosis, and brucella can be transmitted through damaged skin mucous membrane, digestive tract and respiratory tract. In acute stage, the symptoms of fever, hypodynamia, hyperhidrosis, muscle pain, arthralgia, hepatomegaly, splenomegaly and lymphadenectasis are the main symptoms. The chronic cases are often manifested as joint damage, etc. The disease is the infectious disease B specified in infectious disease prevention and treatment law in China. According to the statistical report of CDC 2019 in China, the number of distributed diseases of people is increased to 37947, and the incidence rates of the two types of infectious diseases, namely, live A and B are 10. The epidemic trend of epidemic disease distribution is obviously changed, namely the epidemic disease distribution area spreads from a pasturing area to a semi-farming and semi-pasturing area, a farming area and a city; the form is a multiple and scattered punctate epidemic to replace a large-scale outbreak epidemic; besides the invasion of professional people, the infection rate of non-professional people is relatively increased, which becomes one of the most serious public health problems.

At present, the conventional quarantine means of the epidemic diseases is mainly bacteriology and serology detection technology, the bacteriology detection is the most credible method for determining the in-vivo bacteria carrying, but the positive detection rate is very low. Moreover, the Brucella grows slowly, mostly needs at least 7-15 days, and is limited by experimental environment and professional operators. Currently, serological diagnostic methods for cloth sickness mainly include agglutination assays, enzyme-linked immunosorbent assays (ELISA), complement fixation assays (CFT), and the like. Agglutination tests include tiger red plate agglutination test (RBPT), whole Milk Ring Test (MRT), test tube agglutination test (SAT), card test, and buffer plate agglutination test (BBAT), etc., where RBPT and MRT become primary screening methods in international trade and in cow disease detection, human disease serological diagnosis is primary screening with agglutination test RBPT, clinical primary-diagnosis patient SAT 1: more than 100+ + is confirmed as the disease, and the bacteriology positive is the clinical standard of infection.

The serological detection method is an important detection method due to the advantages of simplicity, rapidness, easy implementation and the like. As the effective component of the antigen used in the traditional Brucella test is mainly Lipopolysaccharide (LPS), the antigen has cross reaction with gram-negative bacteria, particularly Yersinia O:9, and the accuracy of the result is interfered. In addition, in the early stage of infection or at a time when the antibody titer is low, particularly in 12 to 16 days after the infection with brucella, it is difficult to detect the brucella infection by detecting the antibody. Furthermore, brucella is an intracellular parasitic bacterium, and in the late stage of brucella infection, there are cases where a brucella marker cannot be detected in serum.

In summary, no rapid, accurate and clinically effective detection method exists in the aspect of pathogen detection of brucella.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide an ELISPOT detection kit for detecting brucellosis and application thereof. Compared with the existing brucellosis detection technology, the ELISPOT detection kit has the characteristics of high accuracy, simplicity, convenience, quickness and the like.

The technical problem to be solved by the invention is realized by the following technical scheme:

in a first aspect, an ELISPOT assay kit for detecting brucellosis, comprising: a support medium, a capture antibody, a detection antibody, a negative control, a positive control and a specific stimulant, and the ELISPOT detection kit also comprises a specific inhibitor so as to determine whether the brucella infection exists through the forward specific stimulant and the reverse specific inhibitor.

The ELISPOT detection kit for detecting brucellosis as described above, wherein the specific inhibitor comprises brucella lipidated outer membrane protein 16 (L-OMP 16) or polypeptide of the protein.

The ELISPOT detection kit for detecting brucellosis comprises a specific stimulant, wherein the specific stimulant comprises one or more of brucellosis Lipopolysaccharide (LPS), a specific protein and a specific protein polypeptide.

The ELISPOT detection kit for detecting brucellosis is characterized in that the capture antibody is an IFN-gamma capture antibody.

The ELISPOT detection kit for detecting brucellosis is characterized in that the detection antibody is an IFN-gamma detection antibody marked by biotin or alkaline phosphatase. More preferably, the label is a biotin label. The marking process is a conventional marking method.

The above-mentioned "ELISPOT" is a short name for Enzyme-linked Immunospot (Enzyme-linked Immunospot).

It should be noted that, a microporous filter membrane is arranged on the contact surface of the supporting medium and the reagent, the capture antibody is coated on the supporting medium or the capture antibody and the supporting medium are respectively placed, that is, the microporous filter membrane plate can be coated with the capture antibody in advance, or only a blank microporous filter membrane plate and the capture antibody can be provided, and an operator can capture the antibody on the microporous filter membrane plate by a conventional method before detection; and the capture antibody and the detection antibody can jointly generate antigen-antibody binding action with IFN-gamma secreted by cells. Wherein the antibody in the capture antibody and the detection antibody is human IFN-gamma monoclonal antibody. The human IFN- γ monoclonal antibody of the present invention is not particularly limited, and can be obtained by a person skilled in the art according to a conventional method for producing a conventional monoclonal antibody.

In one embodiment of the present invention, the support medium is a microporous filter membrane sheet. More preferably, the support medium is a PVDF-coated membrane micropore culture plate.

The ELISPOT detection kit also comprises one or more of the following reagents: translation of characters

(1) Enzyme-labeled avidin;

(2) a color developing solution;

(3) concentrated PBS buffer, confining liquid, cell culture solution and washing liquid.

The reagents are all general reagents in elispot detection, and are not limited by specific detection items, so that the reagents can be selectively added into the detection method according to needs, and can be configured by an operator or purchased independently. By way of specific example, but not limitation, the enzyme-labeled avidin is, for example, an avidin-alkaline phosphatase conjugate; the washing solution is phosphate buffer solution or phosphate Tween buffer solution, and concentrated or unconcentrated washing solution can be selected according to the requirement; the color development liquid is liquid nitrogen blue tetrazole/5-bromo-4-chloro-3-indole phosphoric acid (NBT/BCIP) and the like; the blocking liquid is skimmed milk powder, FBS, BSA or casein; the cell culture solution is 10%1640 culture solution. The detection kit can simultaneously comprise enzyme-labeled avidin, substrate solution and washing solution, and can also selectively comprise other general reagents required by ELISPOT detection, such as confining liquid, cell culture solution and the like.

It should be noted that in the ELISPOT detection kit of the present invention, each reagent is separately stored.

In the second invention, the detection method for detecting a sample by using the ELISPOT detection kit comprises the following steps:

s1, coating a microporous filter membrane plate with a capture antibody;

s2, preparing a sample cell suspension;

s3, adding the following reagents into the capture antibody coated microporous filter membrane plate: adding a specific stimulant into a stimulation hole, adding a specific inhibitor into a suppression hole, adding a cell culture solution into a negative control hole, adding a lectin into a positive control hole, setting at least 3 repeated holes for each condition, adding a sample cell suspension into each hole, and finally culturing in an incubator;

s4, removing the culture supernatant, washing the plate, adding a labeled IFN-gamma detection antibody into each hole, and incubating at constant temperature;

s5, washing the plate, adding an enzyme-labeled avidin diluent into each hole, and incubating at constant temperature;

s6, adding a color developing solution into each hole, and placing the mixture in a dark place at room temperature for color development; terminating the color development;

s7, plate reading and analysis, counting purple spots in each reaction well using an inverted microscope, each spot representing one IFN- γ secreting T cell; or the microporous membrane filtering plate is put into an ELISPOT instrument, and the experimental result is scanned and counted.

Counting spots in the micropores, taking the average value of the multiple pores of each condition, and judging the result method: the negative control hole is not less than 10 and not more than 50, the positive control hole is more than 50, the detection result is considered to be effective, otherwise, the detection result is considered to be invalid. On the premise that the detection result is effective, if the inhibition hole is less than 10 and the stimulation hole is more than 50, the brucella infection can be determined; if the inhibition well is less than 10 and the stimulation well is less than or equal to 50, no Brucella infection is determined.

In a third aspect, the invention also provides the use of the above-described detection kit in the detection of brucella infection for non-diagnostic purposes, including epidemiological analysis and research, ex vivo tissue detection, epitope identification studies, and qualitative and semi-quantitative testing of secretion of brucella antigen-specific IFN- γ.

The use of a detection kit as described above for the detection of brucella infection for non-diagnostic purposes, in the detection of early or chronic stages of brucella infection or of secretion of brucella antigen-specific IFN- γ.

In a fourth aspect, the invention also provides any one of the following applications of the brucella lipidated outer membrane protein 16 as a specific inhibitor:

(1) the application in preparing an ELISPOT detection kit for detecting brucellosis;

(2) the application of the compound in preparing reagents or medicines for diagnosing, preventing or treating diseases caused by brucella.

In a fifth aspect, the invention also provides any one of the following applications of the polypeptide of the brucella lipidated outer membrane protein 16 as a specific inhibitor:

The invention has the following beneficial effects:

in the process of carrying out ELISPOT (enzyme-linked immunosorbent assay) for detecting brucellosis, the inventor unexpectedly discovers that the brucella lipidated outer membrane protein 16 (L-OMP 16) has a peptidoglycan related domain and has a stronger immunosuppressive trigger effect compared with other proteins, and can be a new potential pathogenic factor of Brucella, and verifies that the brucella lipidated outer membrane protein 16 (L-OMP 16) and the polypeptide thereof can really play a role in inhibiting IFN-gamma secretion of PBMC, and the inhibition of IFN-gamma secretion of PBMC by L-OMP16 can help Brucella escape from host cell immune surveillance, reduce cellular immune response, enable the Brucella lipidated outer membrane protein to survive in cells and further cause long-term chronic infection, so whether some related researches on L-OMP16 can change the mechanism of Brucella immune escape to avoid carrying the Brucella in a life. Furthermore, the inventor determines whether the brucellosis infection exists or not through a specific stimulant and specific inhibitor experiment, the detection result is more objective and accurate, the in-vivo immunity level can be reflected more truly, and the probability of false positive is reduced, so that the detection kit greatly improves the specificity and sensitivity of brucellosis detection, and is a quick and efficient detection means. Because only live bacteria exist continuously and cause T cell sensitization, and the T cell can generate IFN-gamma after being stimulated by exogenous specific antigen or peptide, the detection result can be used as the judgment of whether the T cell is in an active infection stage or not and also can be used as the evaluation index of whether live bacteria are carried after treatment.

The detection time of the method needs about 24 hours, which is much shorter than that of a bacterial culture method (the bacterial culture method is 3-5 days at the fastest speed, generally 15 days, and the longest time can be 20 days or one month), the positive detection rate of the method is about 98 percent and is much higher than that of the bacterial culture method (according to statistics, the positive detection rate of the existing bacterial culture method for an acute stage is about 53 percent, and the positive detection rate of a chronic stage is about 19 percent), and for the same sample, even if the bacterial culture method judges to be negative, the detection method of the detection kit can still detect the positive.

Moreover, although the current Immunofluorescence (IFS) detection method is accurate, the operation is too complex; although the current Flow Cytometer (FCM) detection method is simple, the current method is still in a searching stage, and results judge that too many variable factors exist; the detection kit disclosed by the invention indirectly reacts the condition with live bacteria through elispot, so that not only can whether brucella infection exists or not be known, but also the immunity of an organism can be preliminarily evaluated. The existing ELISA detection method needs body fluid dilution, so that the sensitivity is reduced, the survival state of the bacterium in the cell is unknown, and the immune level of an organism is difficult to truly reflect. And because each living cell forms a spot, the detection method can be used as semi-quantitative detection for detecting the brucella infection of the body.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Example 1 preparation of ELISPOT assay kit

Specifically, the steps of assembling the ELISPOT detection kit are as follows: and respectively packaging and assembling a 96-hole filter membrane plate, an IFN-gamma capture antibody, a biotin labeled IFN-gamma detection antibody, a specific stimulant, a specific inhibitor, a negative control (10% 1640 culture solution) and a positive control (phytohemagglutinin PHA) into a kit.

Further, the ELISPOT detection kit is assembled into: one or more of enzyme-labeled avidin (avidin-alkaline phosphatase conjugate), NBT/BCIP color developing solution, concentrated PBS buffer solution, blocking solution, cell culture solution, phosphate buffer solution or phosphate Tween buffer solution.

Example 2 detection of human peripheral blood samples Using the ELISPOT detection kit of example 1

The detection method of the embodiment comprises the following steps:

2.1 IFN-. gamma.capture antibody was diluted to 15. mu.g/ml using sterile pH7.4 calcium magnesium ion free 1 XPBS (i.e., DPBS) (0.22 μm filter filtration).

2.2 Millipore 96-well PVDF ELISPOT plate (cat: MSIPS4510, Millipore, 96-well filter plate) was removed and treated for 2min with 15. mu.l 35% ethanol per well.

2.3 washing the plates 5 times with sterile water (0.22 μm filter), adding 200 μ l each time, discarding the liquid in the last pass, and draining gently on sterile paper.

2.4 mu.l of the capture antibody solution diluted in step 2.1 was added per well and incubated overnight at 4-8 ℃.

2.5 removing the antibody coating solution in the filter membrane plate, washing the filter membrane plate for 5 times by sterile PBS (PBS), wherein each time the filter membrane plate is washed by 200 mu l, and after the liquid is discarded in the last time, slightly drying the filter membrane plate on sterile paper.

2.6 sealing the filter membrane plate: the reaction plate was blocked by adding 200. mu.l of 10%1640 medium per well and incubated at room temperature for at least 1 h.

2.7 human Peripheral Blood Mononuclear Cells (PBMCs) isolated from human peripheral blood were prepared in 10%1640 medium at a concentration of 3.4 x 10⁶cell/ml cell suspension.

2.8 remove the blocking solution, add 90ul of the cell suspension of step 2.7 per well, and add specific stimulator (OMP 22 and U-OMP16, 10 ug/ml) to the stimulation wells, specific inhibitor (L-OMP 16, 10 ug/ml) to the inhibition wells, phytohemagglutinin (PHA, 10 ug/ml) to the positive control wells, 10%1640 culture to the negative control wells, 3 replicate wells per condition well, taking care of the sterile procedure. Put in 5% CO at 37 DEG C₂The incubator is used for 16-24 h.

2.9 remove the liquid from the wells, then wash 4-6 times with PBST (0.05% Tween-20) to completely remove the cells, 200. mu.l per well each time.

2.10 adding diluted biotin-labeled IFN-gamma detection antibody working solution into each well at a concentration of 100. mu.L/well; incubate at 37 ℃ for 1 h. Then, PBST was washed 4 to 6 times.

2.11 the diluted working solution of the enzyme-labeled avidin is added to each well at 100. mu.L/well, and incubated at 37 ℃ for 1h in the absence of light. Then, PBST was washed 4 to 6 times.

2.12 color development: the prepared developing solution was added to each well at 100. mu.L/well, and left to stand at room temperature in the dark for 15 min.

2.13 end of color development: pouring out the liquid in the hole, uncovering the base of the plate, washing the front and back sides and the base for 3-5 times by using tap water, and stopping color development. The plate is placed in a cool place at room temperature, and the base is closed after the plate is naturally dried.

2.14 plates were read, spots in microwells were counted and statistically analyzed.

Example 3 detection of human peripheral blood samples Using the ELISPOT detection kit of example 1

The detection method of the embodiment comprises the following steps:

2.8 remove the blocking solution, add 90ul of the cell suspension of step 2.7 per well, and add specific stimulator (OMP 31 and U-OMP19, 10 ug/ml) to the stimulation wells, specific inhibitor (L-OMP 16 polypeptide AVAGCASKKNLPN, 10 ug/ml) to the inhibition wells, phytohemagglutinin (PHA, 10 ug/ml) to the positive control wells, 10%1640 medium to the negative control wells, 3 replicate wells per conditioned well, taking care of the sterility. Put in 5% CO at 37 DEG C₂The incubator is used for 16-24 h.

Example 4 detection of human peripheral blood samples Using the ELISPOT detection kit of example 1

The detection method of the embodiment comprises the following steps:

2.8 remove the blocking solution, add 90ul of the cell suspension of step 2.7 per well, and add specific stimulator (OMP 25 and bp26, 10 ug/ml) to the stimulation wells, specific inhibitor (L-OMP 16 polypeptide MRRIQSIARSPI, 10 ug/ml) to the inhibition wells, phytohemagglutinin (PHA, 10 ug/ml) to the positive control wells, 10%1640 culture to the negative control wells, 3 replicate wells per condition well, taking care of the sterile procedure. Put in 5% CO at 37 DEG C₂The incubator is used for 16-24 h.

Example 5 detection of human peripheral blood samples Using the ELISPOT detection kit of example 1

The detection method of the embodiment comprises the following steps:

2.8 remove the blocking solution, add 90ul of the cell suspension of step 2.7 per well, and add specific stimulator (OMP 2a/2b and L-OMP19, 10 ug/ml) to the stimulation wells, specific inhibitor (polypeptide RSPIAIALFMSLA, 10ug/ml of L-OMP 16) to the inhibition wells, phytohemagglutinin (PHA, 10 ug/ml) to the positive control wells, 10%1640 medium to the negative control wells, 3 replicate wells per condition well, taking care of sterile handling, respectively. Put in 5% CO at 37 DEG C₂The incubator is used for 16-24 h.

Example 6 detection of human peripheral blood samples Using the ELISPOT assay kit of example 1

The detection method of the embodiment comprises the following steps:

2.8 removal of blocking solution, addition of 90ul per well of the cell suspension of step 2.7, addition of specific stimulators (LPS and L-OMP10, 10 ug/ml) to the stimulated wells, specific inhibitors (polypeptide SLAVAGCASKKNLP of L-OMP16, 10 ug/ml) to the inhibited wells, plant agglutination, respectively at 10 ul/wellElement (PHA, 10 ug/ml) to positive control wells, 10%1640 culture medium to negative control wells, 3 replicate wells per condition well, taking care of the sterile procedure. Put in 5% CO at 37 DEG C₂The incubator is used for 16-24 h.

The results of testing 126 clinical samples by the methods of examples 2-6 and the bacterial culture method, respectively, are shown in Table 1, and the results of testing 25 blood donor samples are shown in Table 2, which proves that the ELISPOT method has a much higher detection rate than the bacterial culture method. And demonstrates that the specificity and sensitivity of the detection by both the front and back are higher than the one-directional detection as shown in table 3.

TABLE 1 examination of 126 clinical patient specimens by ELISPOT and bacterial culture methods of examples 2-6, respectively

TABLE 2 detection of 25 donor samples by ELISPOT and bacterial culture methods of examples 2-6, respectively

TABLE 3 comparison of the results of the test with the combination of the single stimulus and the combined inhibitors

The ELISA is a detection technology developed on the basis of ELISA, and can quantitatively detect low-frequency cell subsets at the single cell level. Since ELISPOT visualizes a single cell, the sensitivity is improved by 2-3 orders of magnitude compared to traditional ELISA. Therefore, the ELISPOT detection technology greatly improves the sensitivity and specificity of brucellosis detection. The method is applied to the detection of Peripheral Blood Mononuclear Cell (PBMCs) samples of patients with brucella by an ELISPOT test method, and the ELISPOT detection is carried out on 126 PBMCs of patients diagnosed with brucella, and the results show that the positive rate of brucella diagnosis reaches 98.4% (124/126, example 2), 96.8% (122/126, example 3), 99.2% (125/126, example 4), 97.6% (123/126, example 5) and 95.2% (120/126, example 6), while the positive rate of bacterial culture is only 6% (8/126) (Table 1) in the traditional method, the ELISPOT is not detected in blood donors (Table 2), and the detection by adopting a forward specific stimulant and a reverse specific inhibitor (namely, a specific stimulant (stimulation group) + a specific inhibitor (inhibition group)) remarkably reduces the probability of false positive (only the specific stimulant is 8-24) compared with that by adopting a stimulation group only % false positive) (table 3), namely, the detection sensitivity is improved, panic (caused by brucellosis infection, infertility and arthralgia, and great harm to human bodies due to the fact that antibiotics are needed for treatment) is avoided being brought to patients due to the detection of the false positive, and the self immunity can be preliminarily and better evaluated, so that the specificity and the sensitivity of brucellosis detection are greatly improved, and the method is a quick and efficient detection means. The invisible bacteria carriers are detected through a specific stimulant and a specific inhibitor in a bidirectional way, namely the fact that Brucella still exists in a chronic asymptomatic patient, an immune escape phenomenon occurs, and a corresponding immune response cannot be generated is proved, and the immunosuppressive capacity of the L-OMP16 for PBMCs of Brucella patients is further proved in clinical aspects.

The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.

Claims

Translated fromChinese

1.一种用于检测布鲁氏菌病的ELISPOT检测试剂盒，所述ELISPOT检测试剂盒包括：支持介质、捕获抗体、检测抗体、阴性对照和阳性对照和特异性刺激剂，其特征在于，所述ELISPOT检测试剂盒还包括特异性抑制剂，以通过正向的特异性刺激剂和反向的特异性抑制剂来测定是否存在布鲁氏菌感染；其中，所述特异性抑制剂包括布鲁氏菌脂化型外膜蛋白16或者该蛋白的多肽。1. a kind of ELISPOT detection kit for detecting brucellosis, described ELISPOT detection kit comprises: support medium, capture antibody, detection antibody, negative control and positive control and specific stimulator, it is characterized in that, The ELISPOT detection kit also includes a specific inhibitor, to measure whether there is Brucella infection by a forward specific stimulator and a reverse specific inhibitor; wherein, the specific inhibitor includes a cloth Routella lipidated outer membrane protein 16 or a polypeptide of the protein.

2.根据权利要求1所述的用于检测布鲁氏菌病的ELISPOT检测试剂盒，其特征在于，所述特异性刺激剂包括布鲁氏菌脂多糖、特异性蛋白及该特异性蛋白的多肽的一种或几种。2. the ELISPOT detection kit for detecting brucellosis according to claim 1, is characterized in that, described specificity stimulant comprises brucella lipopolysaccharide, specific protein and this specific protein. one or more of the polypeptides.

4.根据权利要求1所述的用于检测布鲁氏菌病的ELISPOT检测试剂盒，其特征在于，所述捕获抗体为IFN-γ捕获抗体。4. the ELISPOT detection kit for detecting brucellosis according to claim 1, is characterized in that, described capture antibody is IFN-γ capture antibody.

5.根据权利要求4所述的用于检测布鲁氏菌病的ELISPOT检测试剂盒，其特征在于，所述检测抗体为生物素标记或碱性磷酸酶标记的IFN-γ检测抗体。5. the ELISPOT detection kit for detecting brucellosis according to claim 4, is characterized in that, described detection antibody is the IFN-γ detection antibody of biotin labeling or alkaline phosphatase labeling.

6.布鲁氏菌脂化型外膜蛋白16作为特异性抑制剂的以下任一应用：6. Any of the following applications of Brucella lipidated outer membrane protein 16 as a specific inhibitor:

(1)在制备用于检测布鲁氏菌病的ELISPOT检测试剂盒中应用；(1) Application in the preparation of an ELISPOT detection kit for the detection of brucellosis;

(2)在制备诊断、预防或治疗布鲁氏菌引起的疾病的试剂或药物中的应用。(2) Application in the preparation of reagents or medicines for diagnosing, preventing or treating diseases caused by Brucella.

7.布鲁氏菌脂化型外膜蛋白16的多肽作为特异性抑制剂的以下任一应用：7. Any of the following applications of the polypeptide of Brucella lipidated outer membrane protein 16 as a specific inhibitor: