阅读说明：本技术 一种基于均相化学发光免疫竞争法检测犬c-反应蛋白的试剂盒 (Kit for detecting C-reactive protein of dog based on homogeneous chemiluminescence immune competition method ) 是由 曹丹 于 2019-12-12 设计创作，主要内容包括：本发明公开了一种基于均相化学发光免疫竞争法检测犬C-反应蛋白的试剂盒,其中,所述试剂盒包括DNA1-cCRP抗原偶联物、DNA2-cCRP抗体偶联物、标记吖啶酯(AE)的DNA3和氧化石墨烯(GO)结合抗氧化剂(AOD)。利用本发明的试剂盒进行检测,其操作简单、结果准确、耗时短、精密度高,并且能够定量检测；样本无需稀释,不存在HOOK效应。(The invention discloses a kit for detecting canine C-reactive protein based on a homogeneous chemiluminescence immune competition method, wherein the kit comprises a DNA1-cCRP antigen conjugate, a DNA2-cCRP antibody conjugate, DNA3 for marking Acridinium Ester (AE) and Graphene Oxide (GO) combined Antioxidant (AOD). The kit disclosed by the invention is used for detection, is simple to operate, accurate in result, short in time consumption and high in precision, and can be used for quantitative detection; the sample did not need to be diluted and there was no HOOK effect.)

1. A kit for detecting C-reactive protein of a dog based on a homogeneous chemiluminescence immune competition method, wherein the kit comprises a DNA1-cCRP antigen conjugate, a DNA2-cCRP antibody conjugate, a DNA3 labeled with Acridinium Ester (AE) and a Graphene Oxide (GO) conjugated Antioxidant (AOD).

2. The kit for detecting the C-reactive protein of the dog based on the homogeneous chemiluminescence immune competition method of claim 1, wherein the concentration of the DNA1-cCRP antigen conjugate is 1-5 nM; the concentration of the DNA2-cCRP antibody conjugate is 5-20 nM; the concentration of the DNA3 for marking Acridinium Ester (AE) is 0.05-0.2 mu M; and the Graphene Oxide (GO) bound Antioxidant (AOD) concentration is 20 μ g/ml.

3. The kit for detecting canine C-reactive protein based on the homogeneous chemiluminescent immunocompetence according to claim 1, wherein the concentration of the DNA 1-crp antigen conjugate is 1 nM; the concentration of the DNA2-cCRP antibody conjugate was 10 nM; the concentration of the Acridinium Ester (AE) labeled DNA3 was 0.1. mu.M.

4. The kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method of claim 1, wherein the DNA1 comprises 54 bases, the 3' end is modified by NH2C7, and the DNA1-cCRP antigen conjugate is formed by covalent binding of bis (sulfosuccinic) suberic acid (BS3) as a coupling agent and amino groups on cCRP antigen.

5. The kit for detecting the C-reactive protein of the dog based on the homogeneous chemiluminescence immune competition method of claim 1, wherein the DNA2 contains 52 bases, the 5' end is modified by NH2C6, and the DNA2-cCRP antibody conjugate is formed by covalently binding a coupling agent BS3 and an amino group on the cCRP antibody.

6. The kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method according to claim 1, wherein said DNA3 comprises 21-base, 5' -modified acridinium ester derivative (AE); 3-10 bases from the 5 'end of the DNA3 are complementary to the 3-10 base sites from the 3' end of the DNA 2; 5-12 bases from the 3 'end of the DNA3 are complementary to the bases at the 3-10 base positions from the 5' end of the DNA 1.

7. The kit for detecting canine C-reactive protein based on homogeneous chemiluminescent competition method of claim 1 wherein the DNA1 is 7 bases complementary to the DNA 2.

8. The kit for detecting canine C-reactive protein based on the homogeneous chemiluminescent immunocompetence method of claim 1, wherein the DNA3 has 8 bases complementary to the DNA1 and the DNA2, respectively.

9. The kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method of claim 1, wherein the sequence (5 '-3') of the DNA1 is as follows: ACGCTGAGTTATCAACGAC TTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C 7;

the DNA2 sequence (5 '-3') is as follows: NH2C6-TACGTCCAGAACTTTACC AAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC;

the DNA3 sequence (5 '-3') is as follows: AE-NH2C6-CGATCTCAGCAACTCAG CAGCG.

10. A method for detecting canine C-reactive protein using the kit of any one of claims 1 to 9, wherein the method comprises the steps of:

(1) mixing calibration solutions with different concentrations or a whole blood sample containing the cCRP with a detection solution in the kit according to the volume ratio of 1:10, placing the mixed solution in an HSCL-10000 chemiluminescence apparatus, and incubating for 5min at 37 ℃;

(2) after incubation, 200 μ L of chemiluminescent substrate was added by HSCL-10000 chemiluminescence apparatus and the chemiluminescent signal of the solution was immediately detected by a photomultiplier tube (PMT) for 3s, based on the recorded chemiluminescence values (RLU), to obtain a calibration curve for crp and the concentration of crp in the sample to be tested.

Technical Field

The invention relates to the technical field of chemiluminescence immunoassay, in particular to a kit for detecting canine C-reactive protein based on a homogeneous chemiluminescence immune competition method.

Background

In recent years, as human CRP has a very important role in clinical disease diagnosis, disease course detection and prognosis, research on CRP has become a hot spot of current human clinical medical research. Although the application of CRP in human medicine is very extensive and intensive and the detection technology is very mature, CRP is not widely applied to routine detection in veterinary clinical, and the research on the change of CRP concentration in diseases is only in the laboratory research stage. The research on canine clinical CRP has been carried out abroad in the seventies of the last century, but few documents are recorded at home.

Canine CRP is documented as an acute phase protein in canine serum, as well as human CRP. The serum CRP content of normal dogs is very low and is generally lower than 5mg/L, and the serum CRP content is increased by more than one hundred times by inflammatory cytokines, interleukin 6 and tumor necrosis factor secreted by giant saliva cells during the inflammatory reaction and pathological changes of organisms. The CRP concentration is elevated in dogs suffering from inflammation, infection and tissue damage, such as leptospirosis, babesiosis, parvovirus infection, surgical trauma, lymphoma and angiosarcoma malignancies, pyometra, acute pancreatitis, immune-mediated hemolytic anemia, arthritis, glomerulonephritis, experimental inflammation. Because of the nature of canine CRP in the acute phase protein, it not only provides an aid to differential diagnosis, but it is also a marker of inflammation in veterinary medicine, and is also a test model for human CRP in the study of human acute phase proteins. Therefore, the method has important significance for the research of canine CRP.

A plurality of dog CRP detection methods, such as immunoturbidimetry, enzyme-linked immunosorbent assay, colloidal gold immunochromatography, fluorescence immunochromatography, a dog-specific laser turbidimetric immunoassay (LNIA) developed in Japan, and the like, have been developed in many laboratories at home and abroad. Immunoturbidimetry is greatly affected by interference factors; ELISA is only suitable for laboratory research and is not suitable for clinical diagnosis because the detection method is time-consuming and labor-consuming; colloidal gold and fluorescence immunochromatography belong to qualitative and semi-quantitative detection, and result accuracy is not high. There is an urgent need for accurate, rapid and quantitative canine CRP detection products, with chemiluminescence being the best choice, especially homogeneous chemiluminescence.

Therefore, it is clear that the above-mentioned existing canine C-reactive protein (crp) detection method and use still have inconvenience and defects, and further improvement is needed. In order to solve the problems in the method for detecting canine CRP, the relevant manufacturers have tried to solve the problems without diligent attention, but there has been no suitable design developed and completed for a long time, and no suitable method for solving the problems has been provided in the general methods, which is obviously a problem that the relevant manufacturers want to solve.

In view of the above-mentioned drawbacks of the existing methods for detecting canine CRP, the present inventors have made active research and innovation based on practical experience and professional knowledge that are abundant over many years in the design and manufacture of such products, and with the application of theory, in order to create a new kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method, which can improve the existing detection methods and make them more practical. After continuous research and design and repeated trial and improvement, the invention with practical value is finally created.

Disclosure of Invention

The invention aims to overcome the defects of the existing detection method and provide a novel kit for detecting the C-reactive protein of a dog based on a homogeneous chemiluminescence immune competition method, and the technical problem to be solved is to ensure that the detection result is accurate, rapid and quantitative, so that the kit is more practical and has industrial utilization value.

The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme.

The kit for detecting the C-reactive protein of the dog based on the homogeneous chemiluminescence immune competition method comprises a DNA1-cCRP antigen conjugate, a DNA2-cCRP antibody conjugate, DNA3 labeled with Acridinium Ester (AE) and Graphene Oxide (GO) combined Antioxidant (AOD).

The kit for detecting the canine C-reactive protein based on the homogeneous chemiluminescence immune competition method is characterized in that the concentration of the DNA1-cCRP antigen conjugate is 1-5 nM; the concentration of the DNA2-cCRP antibody conjugate is 5-20 nM; the concentration of the DNA3 for marking Acridinium Ester (AE) is 0.05-0.2 mu M; and the Graphene Oxide (GO) bound Antioxidant (AOD) concentration is 20 μ g/ml.

The aforementioned kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method, wherein the concentration of the DNA 1-crp antigen conjugate is 1 nM; the concentration of the DNA2-cCRP antibody conjugate was 10 nM; the concentration of the Acridinium Ester (AE) labeled DNA3 was 0.1. mu.M.

The kit for detecting the canine C-reactive protein based on the homogeneous chemiluminescence immune competition method is characterized in that the DNA1 contains 54 bases, the 3' end of the DNA is modified by NH2C7, and the DNA1-cCRP antigen conjugate is formed by covalent binding of a coupling agent bis (sulfosuccinic) suberic acid (BS3) and an amino group on the cCRP antigen.

The kit for detecting the canine C-reactive protein based on the homogeneous chemiluminescence immune competition method is characterized in that the DNA2 contains 52 bases, the 5' end of the DNA is modified by NH2C6, and the DNA2-cCRP antibody conjugate is formed by covalently bonding a coupling agent BS3 and an amino group on the cCRP antibody.

The aforementioned kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method, wherein the DNA3 contains 21 bases, and acridinium ester derivative (AE) is modified at 5' end; 3-10 bases from the 5 'end of the DNA3 are complementary to the 3-10 base sites from the 3' end of the DNA 2; 5-12 bases from the 3 'end of the DNA3 are complementary to the bases at the 3-10 base positions from the 5' end of the DNA 1.

The aforementioned kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method, wherein the DNA1 is 7 bases complementary to the DNA 2.

The kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method is described in the specification, wherein the DNA3 has 8 bases complementary to the DNA1 and the DNA2 respectively.

The aforementioned kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method, wherein the sequence (5 '-3') of the DNA1 is as follows: ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C 7;

the DNA2 sequence (5 '-3') is as follows: NH2C 6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC;

the DNA3 sequence (5 '-3') is as follows: AE-NH2C 6-CGATCTCAGCAACTCAGCAGCG.

The object of the present invention and the technical problem to be solved are also achieved by the following technical means.

According to the invention, the method for detecting the canine C-reactive protein by using the kit is provided, wherein the method comprises the following steps:

(1) mixing calibration solutions with different concentrations or a whole blood sample containing the cCRP with a detection solution in the kit according to the volume ratio of 1:10, placing the mixed solution in an HSCL-10000 chemiluminescence apparatus, and incubating for 5min at 37 ℃;

(2) after incubation, 200 μ L of chemiluminescent substrate was added by HSCL-10000 chemiluminescence apparatus and the chemiluminescent signal of the solution was immediately detected by a photomultiplier tube (PMT) for 3s, based on the recorded chemiluminescence values (RLU), to obtain a calibration curve for crp and the concentration of crp in the sample to be tested.

By the technical scheme, the invention (name) at least has the following advantages: the kit disclosed by the invention is simple to operate, can be used for loading whole blood, and outputting a detection report within about 5 minutes, so that the turnover time (TAT) of a clinical examination specimen is greatly shortened, and the kit is suitable for the requirements of veterinary emergency detection. The kit provided by the invention is used for detecting the canine C-reactive protein on the basis of a homogeneous chemiluminescence immune competition method, can realize quantitative detection of a sample, and has the advantages of accurate result, high precision and short time consumption. When the sample is detected, the sample does not need to be diluted, and the HOOK effect does not exist.

In conclusion, the special kit for detecting the canine C-reactive protein based on the homogeneous chemiluminescence immune competition method can effectively solve the problems of low accuracy and low sensitivity of the detection result in the method for detecting the canine C-reactive protein in the prior art. The method has the advantages and practical value, does not have similar design publication or use but is really innovative in the similar method, has great improvement on the method or the function, has great technical progress, produces good and practical effects, and has multiple enhanced effects compared with the prior art, thereby being more suitable for practical use, having industrial wide utilization value and being a novel, improved and practical new design.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

The specific methods and compositions of the present invention are detailed in the following examples and figures thereof.

Drawings

The invention is further illustrated with reference to the following figures and examples.

FIG. 1 is a schematic diagram showing the detection of canine C-reactive protein by the kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method of the invention;

FIG. 2 shows a complementary pairing diagram of DNA3, DNA1 and DNA2 in the present invention.

Detailed Description

To further illustrate the technical means and effects of the present invention for achieving the predetermined objects, the following detailed description of the embodiments, methods, steps, structures, features and effects of the kit for detecting canine C-reactive protein based on homogeneous chemiluminescence immune competition method according to the present invention is provided with the accompanying drawings and preferred embodiments.

As described herein, the kit for detecting canine C-reactive protein based on the homogeneous chemiluminescence immune competition method of the present invention comprises a DNA1-cCRP antigen conjugate, a DNA2-cCRP antibody conjugate, a DNA3 labeled with Acridinium Ester (AE), and Graphene Oxide (GO) conjugated Antioxidant (AOD).

Wherein, the DNA1 contains 54 bases, the 3' end is modified with NH2C7, and the amino group on the cCRP antigen is covalently combined through a coupling agent bis (sulfosuccinic) suberic acid (BS3) to form a DNA1-cCRP antigen conjugate; the DNA2 contains 52 bases, is modified by NH2C6 at the 5' end and is covalently bonded with amino on the cCRP antibody through a coupling agent BS3 to form a DNA2-cCRP antibody conjugate; DNA3 contains 21 bases, and acridinium ester derivatives (AE) are modified at the 5' end; 3-10 bases of the DNA3 from the 5 'end are completely complementary with bases of the DNA2 from the 3-10 base sites from the 3' end; 5-12 bases from the 3 'end of the DNA3 are completely complementary to the bases at the 3-10 base positions from the 5' end of the DNA 1. DNA1 is 7 bases complementary to DNA 2; DNA3 has 8 bases complementary to DNA1 and DNA2, respectively. Antioxidants (AODs) include, but are not limited to, cannabidiol, vitamin C, vitamin E, tea polyphenols, glutathione, and the like. Graphene oxide coupled AOD: carboxyl on the graphene oxide is combined with hydroxyl on the AOD through a sulfoxide oxide condensing agent; carboxyl on the graphene oxide is activated by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to be combined with amino on the AOD.

Specifically, the sequence (5 '-3') of DNA1 is as follows: ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C 7; the DNA2 sequence (5 '-3') is as follows: NH2C 6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC; the DNA3 sequence (5 '-3') is as follows: AE-NH2C 6-CGATCTCAGCAACTCAGCAGCG.

The specific detection method is as follows:

1. mixing whole blood// serum/plasma/other sample and detection reagent and incubating at 37 ℃ for 5-10 minutes;

2. adding a chemiluminescence substrate, and collecting optical signals through a PMT detection module in a chemiluminescence detector;

3. the instrument automatically calls the standard curve to report the concentration of the substance in the sample.