CN103149357B - A kind of Test paper card utilizing competition law to detect Brucella abortus antibody - Google Patents

Abstract

The invention discloses a detection test paper card for detecting Brucella bovis antibody by using a competition method, which belongs to the technical field of immune detection. The detection test paper card is composed of a casing and a test strip inside it. The casing is composed of an upper plate and a lower plate. The upper plate has a detection window and a sampling hole. Pad, colloidal gold marker pad, detection reaction area, the sample absorption pad is facing the sample hole, and the detection reaction area is facing the detection window. The invention also discloses a method for detecting the Brucella bovis antibody by using the test strip. The on-site rapid detection test paper card for Brucella bovis antibody of the present invention has high sensitivity and repeat rate, strong specificity, good stability, low false positive rate and false negative rate, convenient use, easy observation and distinction, and is suitable for rapid clinical diagnosis and treatment. Large-scale application of epidemiological surveys in grassroots and rural areas.

Description

A kind of detection test paper card that utilizes competition method to detect Brucella bovis antibody

technical field

The invention belongs to the technical field of immune detection, and in particular relates to a detection test paper card for detecting Brucella bovis antibody by using a competition method.

Background technique

Brucella is a Gram-negative short bacillus. When it is first isolated, it is mostly spherical, club-shaped and oval. After subculture, the bacteria gradually become short rod-shaped. The bacteria have no flagella and do not form spores. In 1985, the WHO Expert Committee on Brucellosis divided the genus Brucella into 6 species and 19 biotypes, namely sheep species (biotype 1-3), bovine species (biotype 1-7.9), pig species (biotype 1-7.9), and pig species ( Biotypes 1 to 5) and sheep-type epididymis species, sarira species, and dog species (one biotype each). Clinically, sheep, cattle, and pigs have the greatest significance, and the sheep breed has the strongest pathogenicity. Human disease caused by Brucella is also known by several names: Maltese fever, Mediterranean relaxation fever, wave fever, or undulating fever. Bovine brucellosis exists widely in my country, and its pathogenicity is also very strong. Especially in the Northeast, North China, and Northwest areas where animal husbandry is the mainstay, there are often reports of outbreaks, which directly endanger human health and seriously plague the development of animal husbandry. Import and export trade with animals and their products.

Currently, diagnostic methods for bovine brucellosis mainly include isolation and identification of pathogen, tiger bengal plate agglutination test (RBT), test tube agglutination test (SAT), complement fixation test (CFT), enzyme-linked immunosorbent assay (ELISA), PCR, etc. The isolation and identification of pathogens takes a long time and the operation steps are cumbersome, which is only suitable for the research needs of the laboratory and has no practical value; the complement fixation test is easy to cause hemolysis, and its application is very limited. False positive reaction, and it is difficult to detect the content of the antibody contained in the sample; Tiger red plate agglutination test also has a high false positive reaction, which needs to be improved; ELISA has high sensitivity and good quantitative effect, and is a widely used However, this method is cumbersome to operate, and the qualitative and quantitative analysis can only be determined by professionals, so it is not suitable for large-scale detection in grassroots and rural areas.

Since the advent of immunocolloidal gold technology, it has been developed rapidly. Widely used in the fields of chemical detection and immunological detection, especially the colloidal gold immunochromatography technology has the advantages of simple operation, fast sensitivity, clear results, easy judgment and storage, and does not require any equipment. It is more suitable for rapid clinical diagnosis and large-scale application of epidemiological investigations in grassroots and rural areas.

Contents of the invention

The purpose of the present invention is to provide a rapid on-site detection test paper card for detecting Brucella bovis antibody by a competitive method and a detection method thereof with high sensitivity, good specificity, simple operation and low cost.

A detection test paper card for detecting Brucella bovis antibody by using a competition method, the detection test paper card is composed of a casing 1 and a test strip 2 inside it, the casing 1 is composed of an upper plate 3 and a lower plate 4, and the upper plate 3 There is a detection window 5 and a sample injection hole 6. The test strip 2 is composed of a base plate 7 and a sample absorption pad 8, a colloidal gold marker pad 9, and a detection reaction area 10 pasted on the base plate 7 in sequence. The sample absorption pad 8 is directly opposite to the sample loading The hole 6 and the detection reaction area 10 are facing the detection window 5 .

The bottom plate 7 is made of nitrocellulose membrane.

The colloidal gold marker pad 9 is coated with the Brucella bovis antigen-colloidal gold marker.

The preparation method of described brucella bovis antigen-colloidal gold marker is:

(1) Dialyze the Brucella bovis antigen in 0.005M/L pH7.0 NaCl solution at 4°C overnight, centrifuge at 10000-12000rpm for 1h at 4°C;

(2) the pH of the colloidal gold solution of OD526 is adjusted to 4.0-9.0;

(3) The brucella bovis antigen obtained by step (1) centrifugation is diluted to 5 μg/ml～50 μg/ml with the borate buffer solution of 0.005M/LpH9.0, and the brucella bovis after getting 1ml dilution The antigen was added to 1ml of colloidal gold solution, oscillated and mixed, and left to stand for 5 minutes, then 0.1ml of 10% NaCl solution was added, mixed and left to stand for 2 hours, and centrifuged to obtain the colloidal gold-labeled Brucella bovis antigen.

The detection reaction area 10 is composed of a test area T coated with an antibody against Brucella bovis and a quality control area C coated with a secondary antibody against Brucella bovis.

The sample absorbent pad contains lectin or anti-erythrocyte antibody.

The anti-erythrocyte antibody is an anti-erythrocyte monoclonal antibody conventionally prepared by immunizing mice, sheep or rabbits with human erythrocytes.

The detection method of the detection test paper card of above-mentioned detection brucella bovis antibody is characterized in that, carries out according to the following steps:

(1) Blood sample processing: blood samples are collected in a clean and dry container, and the collected fresh blood is added with a concentration of 2.5% anticoagulant;

(2) Take 100 μL of the processed blood sample and drop it into the sample hole;

(3) After the blood sample flows through the test area and the quality control area, determine whether the sample contains Brucella bovis antibody. The determination rules are as follows:

Positive (-): Only a purple-red band appears in the quality control area, and no purple-red band appears in the test area;

Negative (+): Two purple-red bands appear, one in the test area and the other in the quality control area;

Invalid: When the quality control area does not display a purple-red band, no matter whether the test area shows a purple-red band or not, the test strip is judged invalid.

The detection principle of the present invention: there are letters "T" and "C" on the surface of the Brucella antibody rapid detection test strip as detection lines and quality control lines. The conjugate of Brucella antigen and colloidal gold particles is used to coat on the cellulose acetate membrane; the Brucella antibody and the secondary antibody are respectively coated on the test area T and the quality control area C on the nitrocellulose membrane. Test and control lines are not displayed in the result window until any sample is provided. When the serum sample is added to the sample hole, the Brucella antibody in the sample and the antigen-colloidal gold conjugate chromatographically migrate to the detection area to compete with the Brucella antibody. When the concentration of Brucella antibody in the sample exceeds a certain amount, the Brucella antigen-colloidal gold conjugate cannot combine with the Brucella antibody on the detection line, and no purple line appears in the detection area at this time; When there is no Brucella antibody or the concentration of Brucella antibody in the sample is lower than a certain value, the colloidal gold conjugate will combine with the Brucella antibody on the detection line, thus displaying a purple line in the detection area; regardless of the sample Whether the test contains Brucella antibodies, a purple line will appear in the quality control area to show that the detection is effective.

Beneficial effects of the present invention: the on-site rapid detection of Brucella bovis antibody test paper card of the present invention has high sensitivity and repetition rate, strong specificity, good stability, false positive rate and false negative rate are controlled below 1%, and the absorption pad contains The phytohemagglutinin or anti-erythrocyte antibody can effectively agglutinate and intercept red blood cells, preventing red blood cells from penetrating through the absorbent pad and interfering with subsequent detection. Large-scale application of epidemiological surveys in rural areas and rural areas.

Description of drawings

Fig. 1 is the structural representation of test paper card of the present invention;

Fig. 2 is the structural representation of test strip of the present invention;

In the figure, 1-shell, 2-test strip, 3-upper plate, 4-lower plate, 5-detection window, 6-sample injection hole, 7-bottom plate, 8-sample absorption pad, 9-colloidal gold marker pad, 10-detection reaction area, T-test area, C-quality control area.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Embodiment 1 Utilizes the sandwich method to detect the detection test paper card structure of the Brucella bovis antibody

Utilize the sandwich method to detect the detection test paper card of bovine brucella antibody, as shown in Figure 1 and Figure 2, described detection test paper card is made of shell 1 and the test paper strip 2 inside it, and shell 1 is made of upper plate 3 and lower The upper plate 3 has a detection window 5 and a sample injection hole 6. The test strip 2 is composed of a bottom plate 7, a sample absorption pad 8, a colloidal gold marker pad 9, and a detection reaction area 10 sequentially pasted on the bottom plate 7. The sample The absorbent pad 8 is facing the sample injection hole 6 , and the detection reaction area 10 is facing the detection window 5 . Colloidal gold marker pad 9 is coated with Brucella antigen and colloidal gold marker. The detection reaction area 10 is composed of a test area T coated with Brucella antibodies and a quality control area C coated with Brucella secondary antibodies.

The sample absorbent pad contains lectins or anti-erythrocyte antibodies.

The anti-erythrocyte antibody is a conventional anti-erythrocyte monoclonal antibody prepared by immunizing mice, sheep or rabbits with human erythrocytes.

Embodiment 2 detects the method for bovine brucella antibody

1. Preparation of Brucella bovis antigen-colloidal gold marker

(1) Dialyze the Brucella bovis antigen in 0.005M/L pH7.0 NaCl solution at 4°C overnight, centrifuge at 10000-12000rpm for 1h at 4°C;

(2) the pH of the colloidal gold solution of OD526 is adjusted to 4.0-9.0;

(3) The brucella bovis antigen obtained by step (1) centrifugation is diluted to 5 μg/ml～50 μg/ml with the borate buffer solution of 0.005M/LpH9.0, and the brucella bovis after getting 1ml dilution The antigen was added to 1ml of colloidal gold solution, oscillated and mixed, and left to stand for 5 minutes, then 0.1ml of 10% NaCl solution was added, mixed and left to stand for 2 hours, and centrifuged to obtain the colloidal gold-labeled Brucella bovis antigen.

2. Processing of blood samples

Take 100 bovine blood samples immunized with Brucella bovis antigen, collect the blood samples in a clean and dry container, add 2.5% anticoagulant to the collected fresh blood; refrigerate the blood samples at 2°C to 8°C Can be stored for 48 hours.

3. Detection steps

(1) Add 100 μL of 100 processed blood samples, 100 μL of standard Brucella bovis antibody (concentration: 10 μg/ml), and 100 μL of deionized water into the sample well;

(2) After the blood sample, standard Brucella bovis antibody, and deionized water flow through the test area and quality control area of the respective test strips, determine whether the sample contains Brucella bovis antibody. The determination rules are as follows:

Positive (-): Only a purple-red band appears in the quality control area, and no purple-red band appears in the test area;

Negative (+): Two purple-red bands appear, one in the test area and the other in the quality control area;

Invalid: When the quality control area does not display a purple-red band, no matter whether the test area shows a purple-red band or not, the test strip is judged invalid.

The judgment result is: 98 bovine blood samples are positive, 1 is negative, 1 is invalid, and the sensitivity is 99%.

Embodiment 3 detects the test paper card false positive rate and the false negative test experiment of bovine brucella antibody

1. False Positive Tests

(1) Take 100 parts of common bovine blood samples, and use a blood pretreatment device to process the blood samples;

(2) Add 100 μL each of 100 processed common bovine blood samples and 100 μL deionized water into the sample injection hole;

(2) After the blood sample and deionized water flow through the test area and quality control area of the respective test strips, determine whether the sample contains Brucella bovis antibody. The determination rules are as follows:

Positive (-): Only a purple-red band appears in the quality control area, and no purple-red band appears in the test area;

Negative (+): Two purple-red bands appear, one in the test area and the other in the quality control area;

Invalid: When the quality control area does not display a purple-red band, no matter whether the test area shows a purple-red band or not, the test strip is judged invalid.

The judgment result is: 1 bovine blood sample is positive, 99 samples are negative, all are valid, and the false positive rate is 1%.

2. False Negative Tests

(1) Take 100 copies of standard Brucella bovis antibody (concentration: 10 μg/ml) each 100 μL, deionized water 100 μL, drop into the sample hole;

(2) After the standard Brucella bovis antibody and deionized water flow through the test area and the quality control area of the respective test strips, determine whether the sample contains Brucella bovis antibody. The determination rules are as follows:

Positive (-): Only a purple-red band appears in the quality control area, and no purple-red band appears in the test area;

Negative (+): Two purple-red bands appear, one in the test area and the other in the quality control area;

Invalid: When the quality control area does not display a purple-red band, no matter whether the test area shows a purple-red band or not, the test strip is judged invalid.

The judgment result is: 99 bovine blood samples are positive, 1 is negative, all are effective, and the false negative rate is 1%.

Embodiment 4 detects the mensuration of other properties of the test strip of brucella bovis antibody

(1) Specific determination of Brucella antibody on-site rapid detection test paper card

The prepared Brucella antibody test card was used to detect the three kinds of bacterial whole blood that are close to the strain of Brucella, and the results were all negative, indicating that the specificity was good and there was no crossover.

(2) Sensitivity of on-site rapid test strips for Brucella antibodies

Take the bovine positive serum of Brucella, and use ELISA to detect its average titer of about 1:8000. The tiger red plate agglutination antigen is detected as positive when the blood dilution is lower than 50 times; rapid on-site detection of Brucella antibody The blood titer of the positive result detected by the test paper card is 1:6000, and when the blood dilution is ≥6000 times, the test line of the test paper card has unclear or no color development. Therefore, the sensitivity of Brucella antibody on-site rapid detection of real card is significantly higher than that of tiger red plate agglutination test, but its sensitivity is still lower than that of ELISA.

(3) Reproducibility of Brucella antibody on-site rapid detection test card

5 test paper cards were tested in parallel, and the test results were consistent, indicating that the test method was repeatable.

(4) The stability of the on-site rapid detection test card for Brucella antibodies

Place Brucella antibody on-site rapid detection test paper card at 4°C, and regularly test the standard positive serum. The results show that its stability can be stored for 1 year. After 1 year, the gold release rate slows down, and the chromatography speed slows down. The stability of the product is 1 year at 4°C.

Claims (1)

1. a detection test paper card utilizing competition method to detect brucella bovis antibody, it is characterized in that, described detection test paper card is made of shell (1) and its inner test paper strip (2), and shell (1) is made of The upper plate (3) and the lower plate (4) are composed, the upper plate (3) has a detection window (5) and a sample injection hole (6), and the test strip (2) is composed of the bottom plate (7) and the bottom plate (7). The sample absorption pad (8), colloidal gold marking pad (9), and detection reaction area (10) are pasted in sequence. The sample absorption pad (8) is facing the sample injection hole (6), and the detection reaction area (10) is facing the detection reaction area. window(5); The material of the bottom plate (7) is a nitrocellulose membrane; The colloidal gold marker pad (9) is coated with the Brucella bovis antigen-colloidal gold marker; The preparation method of described brucella bovis antigen-colloidal gold marker is: (1) Dialyze the Brucella bovis antigen in 0.005M/L pH7.0 NaCl solution at 4°C overnight, centrifuge at 10000-12000rpm for 1h at 4°C; (2) the pH of the colloidal gold solution of OD526 is adjusted to 4.0-9.0; (3) The brucella bovis antigen obtained by step (1) centrifugation is diluted to 5 μg/ml～50 μg/ml with the borate buffer solution of 0.005M/LpH9.0, and the brucella bovis after getting 1ml dilution The antigen was added to 1ml colloidal gold solution, oscillated and mixed, and left to stand for 5 minutes, then 0.1ml of 10% NaCl solution was added, mixed and left to stand for 2 hours, and centrifuged to obtain the colloidal gold-labeled Brucella bovis antigen; Described detection reaction area (10) is made up of the test area (T) of coating Brucella bovis antibody and the quality control area (C) of coating Brucella bovis primary antibody; The sample absorbent pad contains plant hemagglutinin or anti-erythrocyte antibody; The anti-erythrocyte antibody is an anti-erythrocyte monoclonal antibody conventionally prepared by immunizing mice, sheep or rabbits with human erythrocytes; The detection method of the above-mentioned detection test paper card is carried out according to the following steps: (1) Blood sample processing: blood samples are collected in a clean and dry container, and the collected fresh blood is added with a concentration of 2.5% anticoagulant; (2) Take 100 μL of the processed blood sample and drop it into the sample hole; (3) After the blood sample flows through the test area and the quality control area, determine whether the sample contains Brucella bovis antibody. The determination rules are as follows: Positive (+): Only a purple-red band appears in the quality control area, and no purple-red band appears in the test area; Negative (-): Two purple-red bands appear, one in the test area and the other in the quality control area; Invalid: When the quality control area does not display a purple-red band, no matter whether the test area shows a purple-red band or not, the test strip is judged invalid.