CN202453359U - Dot gold immunofiltration kit of echinococcus multillocularis circulating antigen - Google Patents

Abstract

The utility model provides a dot gold immunofiltration kit of an echinococcus multillocularis circulating antigen, which relates to an echinococcus multillocularis circulating antigen detecting reagent. The dot gold immunofiltration kit is provided with a detecting plate, a gold labeled antibody resistant to the echinococcus multillocularis circulating antigen as well as a washing liquid; the detecting plate is provided with a carrier medium, a microporous filter membrane, a water absorbing medium, an echinococcus multillocularis circulating antigen detecting point and a contrast point; the echinococcus multillocularis circulating antigen detecting point and the contrast point are arranged on the microporous filter membrane in sequence; the echinococcus multillocularis circulating antigens EM2 and EM18 are coated at the contrast point; the microporous filter membrane is fixed on the water absorbing medium and put into the carrier medium; and a through hole opposite to the microporous filter membrane is formed on one side of the carrier medium. The dot gold immunofiltration kit of the echinococcus multillocularis circulating antigen, provided by the utility model, has the advantages of extremely small specimen quantity needed in detection, no need of special instruments, simplicity, convenience and rapidness in detection, strong specificity, high sensitivity, accuracy, reliability, low cost and wide application; and a result is directly judged by naked eyes.

Description

Echinococcus multilocularis circulating antigen dot gold immunofiltration kit

technical field

The utility model relates to a detection reagent for echinococcus multilocularis circulating antigen, in particular to an immunofiltration detection kit for echinococcus multilocularis circulating antigen carried out by colloidal gold immunofiltration technology (immunofiltration).

Background technique

Alveolar echinococcosis (AE) is a zoonotic parasitic disease caused by the larvae of Echinococcus multilocularia (Em) of the genus Echinococcus, mainly distributed in Inner Mongolia and Xinjiang in my country. The vast pastoral areas such as Tibet, Gansu, Ningxia, Qinghai and western Sichuan are one of the main parasitic diseases currently being controlled in my country. The disease is considered the deadliest helminth infection and one of the most dangerous "zoonotic diseases" worldwide. Humans are infected by ingesting Echinococcus eggs, and then the larvae infiltrate, migrate and grow in the patient's liver in a multivesicular, tumor-like manner, rapidly destroying liver parenchymal cells, and eventually leading to liver insufficiency or loss, accompanied by lung and brain damage. Metastasis in important organs, etc., the mortality rate of patients within 10 years after diagnosis is as high as 90% ([1] Filippou D., Tselepis D., Filippou G. and Papadopoulos V. Advances in Liver Echinococcosis: Diagnosis and Treatment. Clinical Gastroenterology and Hepatology. 2007 , 5(2): 152-159.). The effective treatment of this disease is still mainly surgery, and the high recurrence rate after surgery is a major problem in the prevention and treatment of AE. Therefore, while paying attention to early diagnosis and treatment, it is also of great significance to strengthen the monitoring of the curative effect of patients with alveolar echinococcosis, the monitoring of the progression of the disease, and the control of the recurrence of the disease.

At present, imaging examinations, immunological tests such as enzyme-linked immunosorbent assay (ELISA) and Western blotting are mainly used to diagnose and monitor the condition of AE patients ([2] Brunetti E, Kern P, Vuitton D A. Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis inhumans [J]. Acta tropica, 2010, 114(1): 1-16). Imaging examinations often confuse some atypical and small lesions with liver cancer and liver abscess ([3] Zhang Kuan, Wang Huiping, Huang Shan, et al. CT diagnosis of hepatic alveolar echinococcosis[J]. Practical Radiology Journal, 2011, 27(7): 1117-1119), and immunological diagnosis has the advantages of rapidity, sensitivity, specificity and low price, so it is currently widely used in clinical diagnosis and epidemiological investigation of the disease. However, due to the influence of factors such as the type and quality of antigens used in immunological diagnosis, they often lack sensitivity and specificity, and there are cross-reactions with antigens of Echinococcus granulosus, cysticercosis and other parasites, so looking for highly sensitive and specific Detection methods have always been a research hotspot in the diagnosis and prevention of AE. Most of the recombinant antigens currently used in diagnosis are based on the detection of polyclonal antibodies, but the specificity and sensitivity of immunological detection methods based on polyclonal antibodies need to be improved ([4] Carmena D, Benito A, Eraso E. The immunodiagnosis of Echinococcus multilocularis infection. Clin. Microbiol. Infect, 2007, 13: 460-475).

Parasite circulating antigen (CAg) refers to the macromolecular particles discharged by living parasites into the body fluid of the host, mainly substances that have antigenic properties in excreted secretions or sheds, and can be detected by serum immunological tests . Because circulating antibodies can still exist for a long time after the treatment of patients, it cannot distinguish current infection from past infection, so it is not suitable for efficacy evaluation. It is generally believed that the detection of CAg can indicate the existence of active infection, and can be used to judge patients with current symptoms and evaluate the curative effect, so CAg has become a diagnostic target antigen. With the continuous deepening of the research on parasite CAg, and the recognition of its role and position in the occurrence, development and pathophysiology of parasitic diseases, the research content of CAg has been expanded to explore the regularity of growth and decline, outcome, etc. Role in immunopathology and mechanisms of immune regulation is recognized. Early serological methods used crude extracts of Echinococcus multilocularis as antigens, which cross-reacted with other pathogens (such as Echinococcus granulosus), so false positives sometimes occurred. With the popularization of molecular biology techniques, more and more recombinant antigens have been applied to Echinococcus multilocularis experiments. At present, more Echinococcus multilocularis antigens have been studied, including EM2, EM4, EM10, EM13, and EM16. The recombinant antigen prepared by recombinant DNA technology can overcome the shortcomings of the crude extract of Echinococcus multilocularis, and can quickly and economically prepare unlimited specific recombinant Echinococcus multilocularis antigens ([5] Li Wengui, Chen Yatang. Echinococcus multilocularis em18 Research progress [J]. Chinese Journal of Zoonoses (ISTIC, 2009, 25(1): 56-57). Establishing a simple and quick reagent for screening, the detection of CAg in AE patients can not only assist the detection of circulating antigens in the diagnosis of the disease, but also have important significance for the evaluation of the curative effect of disease treatment and disease monitoring.

Contents of the invention

The purpose of the utility model is to provide a kind of Echinococcus multilocularis circulating antigen spotted gold immunofiltration kit.

The utility model is provided with a detection board, a gold-labeled anti-Echinococcus multilocularis circulating antigen antibody and a washing solution, and the detection board is provided with a carrier medium, a microporous filter membrane, a water-absorbing medium, and a detection point for the Echinococcus multilocularis circulating antigen and control points; the Echinococcus multilocularis circulating antigen detection point and the control point are successively set on the microporous membrane; the Echinococcus multilocularis circulating antigen detection point is coated with an antibody against the Echinococcus multilocularis circulating antigen , coated with Echinococcus multilocularis antigens EM2 and EM18 at the control point, fixed the spotted microporous filter membrane on the water-absorbing medium, and then put it into the carrier medium, and one side of the carrier medium was provided with micropores The through hole opposite to the filter membrane; the carrier medium includes a base plate and a cover plate buckled on the base plate, and a through hole facing the microporous filter membrane is opened on the cover plate to facilitate sample addition.

The carrier medium can be PVC board or the like.

The microporous membrane can be nitrocellulose membrane, etc., and the pore size of the microporous membrane can be 0.1-0.5 μm.

As the water-absorbing medium, paper with cellulose as the main component can be used.

The utility model can be prepared by the following methods:

1) Prepare the Echinococcus multilocularis antigens EM2 and EM18, the specific method is: using gene cloning technology, PCR amplifying the DNA encoding the Echinococcus multilocularis antigen, and inserting it into Escherichia coli to express it, Echinococcus multilocularis Recombinant antigens EM2 and EM18.

2) Prepare the monoclonal antibodies of Echinococcus multilocularis recombinant antigen EM2 and EM18, the specific method is as follows:

(1) Prepare the monoclonal antibody of Echinococcus multilocularis recombinant antigen EM2. The specific method is: mix the recombinant antigen EM2 with an equal volume of Freund's complete adjuvant to immunize mice, and immunize the spleen cells of mice and SP2/0 mice Myeloma cells were fused through cell fusion, and the hybridoma cells secreting high-titer antibodies were screened for expanded culture and cryopreservation; the hybridoma cells were cloned by the limiting dilution method, and the positive hybridoma cells were injected intraperitoneally into mice; when the abdomen of the mice was obviously raised, Ascitic fluid was collected, centrifuged to obtain the supernatant, sterile filtered, and the ascites was purified by affinity chromatography to obtain EM2 monoclonal antibody.

(2) Prepare the monoclonal antibody of Echinococcus multilocularis recombinant antigen EM18, the specific method is: mix the recombinant antigen EM18 with an equal volume of Freund's complete adjuvant to immunize mice, and immunize the spleen cells of mice and SP2/0 mice Myeloma cells were fused through cell fusion, and the hybridoma cells secreting high-titer antibodies were screened for expanded culture and cryopreservation; the hybridoma cells were cloned by the limiting dilution method, and the positive hybridoma cells were injected intraperitoneally into mice; when the abdomen of the mice was obviously raised, Collect ascites, centrifuge to get supernatant, filter aseptically, and purify ascites by affinity chromatography to obtain EM18 monoclonal antibody;

(3) Mix the anti-Echinococcus multilocularis circulating antigen EM2 monoclonal antibody and the anti-Echinococcus multilocularis circulating antigen EM18 monoclonal antibody; The cestode worm circulating antigen EM18 monoclonal antibody can be mixed according to the volume ratio 1: (0.2 ~ 5); the final concentration of the goat anti-mouse IgG polyclonal antibody is 1 ~ 4mg/mL; the sample volume of the two is 1 μL/mm ³ ; The concentration of the anti-Echinococcus multilocularis circulating antigen antibody can be 1-4 mg/mL.

3) Spotting on nitrocellulose membrane

Echinococcus multilocularis circulating antigen detection point is coated with anti-Echinococcus multilocularis circulating antigen antibody, Echinococcus multilocularis antigen EM2 and EM18 are coated at the control point, and dried; the drying temperature is 37°C.

4) Preparation of colloidal gold

Prepare 25nm colloidal gold by trisodium citrate reduction method. The specific method is: take 1mL of 1% chloroauric acid and add it to 100mL deionized double-distilled water. Heat to boiling, add 2.0mL of 1% trisodium citrate aqueous solution under magnetic heating and stirring, continue heating until the solution is wine-colored, and place it in a brown bottle after cooling and store it in a 4°C refrigerator for later use; the concentration of the trisodium citrate is 2%.

5) labeling of colloidal gold and anti-Echinococcus multilocularis circulating antigen antibody, the specific method is as follows:

(1) Take 10 mL of colloidal gold, adjust to pH 5.4 with 0.1 mol/L NaOH, add 100 μg of anti-Echinococcus multilocularis circulating antigen EM2 monoclonal antibody, mix well, let stand for 5 min, add 5% BSA 1 mL and mix well, 4 Centrifuge at 10,000 r/min for 1 h at ℃, discard the supernatant, dissolve the precipitate with TBS buffer to 10 mL, centrifuge at 10,000 r/min at 4 °C for 1 h, discard the supernatant, and dilute the precipitate to 1 mL with PBS to obtain colloidal gold-labeled anti-multilocular Echinococcus circulating antigen EM2 monoclonal antibody;

(2) Take 10 mL of colloidal gold, adjust to pH 5.4 with 0.1 mol/L NaOH, add 100 μg of anti-Echinococcus multilocularis circulating antigen EM18 monoclonal antibody, mix well, let stand for 5 min, add 1 mL of 5% BSA and mix well, 4 Centrifuge at 10,000 r/min for 1 h at ℃, discard the supernatant, dissolve the precipitate with TBS buffer to 10 mL, centrifuge at 10,000 r/min at 4 °C for 1 h, discard the supernatant, and dilute the precipitate to 1 mL with PBS to obtain colloidal gold-labeled anti-multilocular Echinococcus circulating antigen EM18 monoclonal antibody;

(3) Colloidal gold-labeled anti-Echinococcus multilocularis circulating antigen EM2 monoclonal antibody and anti-Echinococcus multilocularis circulating antigen EM18 monoclonal antibody are mixed, the anti-Echinococcus multilocularis circulating antigen EM2 monoclonal antibody and The anti-Echinococcus multilocularis circulating antigen EM18 monoclonal antibody was mixed at a volume ratio of 1: (0.2-5).

6) Prepare 10mmol/L pH7.4 phosphate buffered saline solution containing 0.5% Tween20 as washing solution;

7) Prepare Echinococcus multilocularis circulating antigen dot gold immunofiltration kit, the specific method is as follows:

Echinococcus multilocularis circulating antigen detection points and control points are sequentially set on the microporous membrane; the detection points are coated with anti-Echinococcus multilocularis circulating antigen antibodies, and the control points are coated with Echinococcus multilocularis antigens EM2 and EM18; fix the sampled microporous membrane on the water-absorbing medium, and then put it into the carrier medium, one side of the carrier medium has a through hole opposite to the microporous membrane; equipped with a microporous filter The carrier medium of the membrane is a detection plate; the detection plate, the gold-labeled anti-Echinococcus multilocularis circulating antigen antibody and the washing solution together form the Echinococcus multilocularis circulating antigen dot gold immunofiltration kit.

The utility model adopts the colloidal gold immune diafiltration technology to establish the Echinococcus multilocularis circulating antigen spot gold immune diafiltration kit, which can be used for the detection of the Echinococcus multilocularis circulating antigen in samples such as whole blood, serum and plasma. When testing, the required sample volume is extremely small, no special equipment is required, and the results can be directly interpreted with the naked eye. The detection is simple and fast, with strong specificity, high sensitivity, accuracy and reliability, low cost, and wide application.

Description of drawings

Fig. 1 is a schematic diagram of the assembly of the detection board in the embodiment of the present invention.

Fig. 2 is a schematic diagram of the structure and composition of the embodiment of the utility model.

Figure 3 is a schematic diagram of the experimental results model. In Fig. 3, (1) is a schematic diagram before use, (2) is an invalid test (product quality problem), (3) Echinococcus multilocularis circulating antigen is negative, (4) Echinococcus multilocularis circulating antigen is positive.

Detailed ways

The following embodiments will further illustrate the utility model in conjunction with the accompanying drawings.

The embodiment of the utility model is provided with a carrier medium, a microporous filter membrane, a water-absorbing medium, a detection point, a control point, a gold-labeled anti-echinococcus multilocularis circulating antigen antibody, and a washing liquid.

Referring to Figures 1 and 2, the embodiment of the Echinococcus multilocularis circulating antigen dot gold immunofiltration kit is provided with a detection plate 8, a gold-labeled anti-Echinococcus multilocularis circulating antigen antibody 9 and a washing solution 10, the Detection plate 8 is provided with carrier medium (comprising bottom plate 1 and cover plate 2 buckled on the bottom plate), microporous filter membrane 5, water-absorbing medium 6, echinococcus multilocularis circulating antigen detection point 3 and control point 4; Echinococcus multilocularis circulating antigen detection point 3 and control point 4 are successively set on the microporous filter membrane 5; Echinococcus multilocularis circulating antigen detection point 3 is coated with anti-Echinococcus multilocularis circulating antigen antibody, and in the control Point 4 is coated with Echinococcus multilocularis antigens EM2 and EM18, and the microporous filter membrane 5 of the sample is fixed on the water-absorbing medium 6, and then put into the carrier medium, and one side of the carrier medium is provided with micropores. The through hole opposite to the filter membrane 5; the carrier medium includes a base plate 1 and a cover plate 2 buckled on the base plate, and the cover plate 2 is provided with a through hole facing the microporous filter membrane 5, so as to add samples. The detection plate 8 , the gold-labeled anti-Echinococcus multilocularis circulating antigen antibody 9 and the washing solution 10 together constitute the Echinococcus multilocularis circulating antigen dotted gold immunofiltration kit.

The carrier medium can be PVC board or the like.

The microporous membrane can be nitrocellulose membrane, etc., and the pore size of the microporous membrane can be 0.1-0.5 μm.

As the water-absorbing medium, paper with cellulose as the main component can be used.

The preparation method of the echinococcus multilocularis circulating antigen dot gold immunofiltration kit comprises the following steps:

1) Preparation of Echinococcus multilocularis antigens EM2 and EM18

Using gene cloning technology, PCR amplified DNA encoding Echinococcus multilocularis antigens, and inserted into Escherichia coli to express Echinococcus multilocularis recombinant antigens EM2 and EM18.

2) Preparation of monoclonal antibodies against Echinococcus multilocularis recombinant antigens EM2 and EM18

The recombinant antigen EM2 was mixed with an equal volume of Freund's complete adjuvant to immunize mice, the splenocytes of immunized mice and SP2/0 mouse myeloma cells were fused, and the hybridoma cells secreting high-titer antibodies were screened for expanded culture, Freeze. Hybridoma cells were cloned by limiting dilution method. Mice were intraperitoneally injected with positive hybridoma cells. When the abdomen of the mouse was obviously raised, the ascites was collected, centrifuged to obtain the supernatant, sterile filtered, and the ascites was purified by affinity chromatography to obtain EM2 monoclonal antibody.

The preparation of the monoclonal antibody of the Echinococcus multilocularis recombinant antigen EM18 is the same as the above method.

3) Spotting on nitrocellulose membrane

Coat the anti-Echinococcus multilocularis circulating antigen antibody at the detection point, coat the Echinococcus multilocularis antigen EM2 and EM18 at the control point, and dry;

4) Preparation of colloidal gold

Adopt trisodium citrate reduction method to prepare 25nm colloidal gold, get 1% chloroauric acid 1mL and join in 100mL deionized double distilled water, the chloroauric acid concentration that obtains is 0.01%, is placed in the flask with condensing device and is heated to boiling, Add 2.0mL of 1% trisodium citrate aqueous solution under magnetic heating and stirring, continue to heat until the solution turns wine-colored, and put it in a brown bottle after cooling and store it in a 4°C refrigerator for later use;

5) Labeling of colloidal gold and antibodies against circulating antigens of Echinococcus multilocularis

Take 10 mL of colloidal gold, adjust the pH to 5.4 with 0.1 mol/L NaOH, add 100 μg of anti-Echinococcus multilocularis circulating antigen EM2 monoclonal antibody, mix well, let stand for 5 min, add 1 mL of 5% BSA and mix well, 4°C, 10000r Centrifuge for 1 hour at 4°C, discard the supernatant, dissolve the precipitate with TBS buffer to 10 mL, centrifuge at 10,000 r/min at 4°C for 1 hour, discard the supernatant, and dilute the precipitate to 1 mL with PBS to obtain colloidal gold-labeled anti-Echinococcus multilocularis Circulating antigen EM2 monoclonal antibody;

The preparation of the monoclonal antibody against Echinococcus multilocularis circulating antigen EM18 is the same as above;

6) Washing solution preparation

Prepare 10mmol/L pH7.4 phosphate buffered saline solution containing 0.5% Tween20 as washing solution;

7) Preparation of Echinococcus multilocularis circulating antigen dot gold immunofiltration kit

Echinococcus multilocularis circulating antigen detection points and control points are sequentially set on the microporous membrane; the detection points are coated with anti-Echinococcus multilocularis circulating antigen antibodies, and the control points are coated with Echinococcus multilocularis antigens EM2 and EM18. Fix the sampled microporous membrane membrane on the water-absorbing medium, and then put it into the carrier medium, one side of the carrier medium has a through hole opposite to the microporous membrane; the carrier medium equipped with the microporous membrane It is a detection board; the detection board, the gold-labeled anti-Echinococcus multilocularis circulating antigen antibody and the washing solution together form the Echinococcus multilocularis circulating antigen dotted gold immunofiltration kit.

The clinical specimens of the patients detected by the dot gold immunofiltration method are given below:

1) Balance: add 1 drop of washing solution to the reaction well to balance the detection membrane;

2) Adding samples: Take the sample to be tested and add it to the reaction well, so that the antigen in the sample to be tested can be combined with the antibody against the circulating antigen of Echinococcus multilocularis, and wait for the liquid to percolate downward and be absorbed by absorbent paper;

3) Washing: Add 4 drops of washing solution to the reaction well to clear the film, and wait for the liquid to percolate downward and be absorbed by absorbent paper;

4) Add gold-labeled antibody: add 1 drop of gold-labeled anti-Echinococcus multilocularis circulating antigen antibody to the reaction well, so that the label on it can recognize the antigen, and wait for the liquid to percolate downward and be absorbed by absorbent paper ;

5) Washing: Add 4 drops of washing solution to the reaction well to clear the film, and wait for the liquid to percolate downward and be absorbed by absorbent paper;

Judgment of results: See Figure 3, read the test results by visual inspection, if there is only a purple-red band in the control area of the test plate of the Echinococcus multilocularis circulating antigen spot gold immunofiltration kit, it is judged as negative; If there is a purple band in the control area, it is judged as positive; after adding the sample for detection, no purple band appears in the detection area and the control area, which is an invalid result.

The performance test of Echinococcus multilocularis circulating antigen dot gold immunofiltration kit is given below:

1) Appearance inspection: The kit is not damaged, the coated reaction film is smooth, clean, free of pollution spots, and has no cracks.

2) Coincidence rate of positive specimens: 50 positive reference sera with different titers positive for Echinococcus multilocularis were tested with Echinococcus multilocularis circulating antigen dot gold immunofiltration kit, and the positive coincidence rate was calculated. The positive reference serum was determined by clinical specimens determined by ELISA (imported reagents).

3) Negative specimen coincidence rate: use 50 negative reference sera to test, and calculate the negative coincidence rate. Negative reference serum was determined using clinical specimens determined by ELISA (imported reagents).

4) Intra-batch variation: Echinococcus multilocularis Circulating Antigen Dot Gold Immunofiltration Kit of the same batch is tested with characteristic serum. It is required that positive serum test results show that the color of the color band is consistent, and negative serum test results are negative.

5) Batch-to-batch differences: Different batches of Echinococcus multilocularis circulating antigen dot gold immunofiltration kits are tested with characteristic serum, and the positive serum test results are required to show the same color depth of the ribbon, and the negative serum test results are negative.

6) Interference test: The test result is not interfered by specimen hemolysis (n=50), lipemia (n=50) and jaundice (n=50).

7) Cross-reaction: Use this Echinococcus multilocularis circulating antigen dot gold immunofiltration kit to conduct systemic lupus erythematosus (n=30), rheumatoid disease (n=30), immune hepatitis (n=30) No cross-reaction was found in the detection of autoimmune diseases.

8) Stability test: apply the Arrhenius rule, place the Echinococcus multilocularis circulatory antigen spot gold immunofiltration kit at 37°C for 20 days and test it. The above indicators have no significant changes. Ensure that the finished product is stored at room temperature and dry. for 18 months.

Specific examples are given below.

Example 1

Coat the antibody against the circulating antigen of Echinococcus multilocularis at the detection point of the nitrocellulose membrane, coat the antibody against the circulating antigen of Echinococcus multilocularis at the detection point, and fix the spotted microporous membrane membrane on the water-absorbing medium Then put it into a carrier medium to assemble a detection plate, one side of the carrier medium has a through hole opposite to the microporous filter membrane, the prepared colloidal gold immunofiltration detection plate is sealed at room temperature and stored for later use. Wherein, the antibody concentration of anti-Echinococcus multilocularis circulating antigen is 1mg/mL, is anti-Echinococcus multilocularis circulating antigen EM2 monoclonal antibody and anti-Echinococcus multilocularis circulating antigen EM18 monoclonal antibody with volume ratio 1: 1 mixed; the final concentration of goat anti-mouse IgG polyclonal antibody is 1 mg/mL; the sample volume of both is 1 μL/mm ³ ;

Colloidal gold-labeled anti-Echinococcus multilocularis circulating antigen EM2 monoclonal antibody and anti-Echinococcus multilocularis circulating antigen EM18 monoclonal antibody at a concentration of 1 mg/mL were mixed at a volume ratio of 1:1 to prepare gold-labeled Antibodies against circulating antigens of Echinococcus multilocularis, bottled for later use.

Prepare 10mmol/L pH7.4 phosphate-buffered saline solution containing 0.5% Tween20 as washing solution, bottle it for later use.

The colloidal gold immunofiltration detection plate, the gold-labeled anti-Echinococcus multilocularis circulating antigen antibody and the washing solution are combined to form the Echinococcus multilocularis circulating antigen dot gold immunofiltration kit.

Add 1 drop of washing solution to the reaction well to balance the detection membrane; take 50 μL of the serum of the specimen to be tested and add it to the sample area of the Echinococcus multilocularis circulating antigen dot gold immunofiltration detection plate; after the liquid is absorbed, add 4 Drop washing solution; after the liquid is absorbed, add 1 drop of gold-labeled anti-Echinococcus multilocularis circulating antigen antibody to the reaction well; finally add 4 drops of washing solution and observe the results. If only one purple-red band appears in the control area of the Echinococcus multilocularis circulating antigen detection plate, it is judged as negative; if a purple-red band appears in both the detection area and the control area, it is judged as positive; There is no purple band in the area and the control area, which is an invalid result.

Example 2

Similar to Example 1, the difference is that the anti-Echinococcus multilocularis circulating antigen antibody coated at the detection point of the nitrocellulose membrane is only composed of anti-Echinococcus multilocularis circulating antigen EM2 monoclonal antibody, and does not contain anti-Echinococcus multilocularis Monoclonal antibody to circulating antigen EM18. Result judgment is identical with embodiment 1.

Example 3

Similar to Example 1, the difference is that the anti-Echinococcus multilocularis circulating antigen antibody coated at the detection point of the nitrocellulose membrane only consists of anti-Echinococcus multilocularis circulating antigen EM18 monoclonal antibody, and does not contain anti-Echinococcus multilocularis Circulating antigen EM2 monoclonal antibody. Result judgment is identical with embodiment 1.

Example 4

Performance verification test: The Echinococcus multilocularis circulating antigen dot gold immunofiltration kit was prepared according to the scheme in Example 1, and then the performance verification was carried out.

1) Appearance inspection: The kit is not damaged, the coated reaction film is smooth, clean, free of pollution spots, and has no cracks.

2) Coincidence rate of positive specimens: 50 positive reference sera with different titers positive for Echinococcus multilocularis were tested with Echinococcus multilocularis circulating antigen dot gold immunofiltration kit, and the positive coincidence rate was calculated. The positive reference serum was determined by clinical specimens determined by ELISA (imported reagents).

3) Negative specimen coincidence rate: use 50 negative reference sera to test, and calculate the negative coincidence rate. Negative reference serum was determined using clinical specimens determined by ELISA (imported reagents).

4) Intra-batch variation: Echinococcus multilocularis Circulating Antigen Dot Gold Immunofiltration Kit of the same batch is tested with characteristic serum. It is required that positive serum test results show that the color of the color band is consistent, and negative serum test results are negative.

5) Batch-to-batch differences: Different batches of Echinococcus multilocularis circulating antigen dot gold immunofiltration kits are tested with characteristic serum, and the positive serum test results are required to show the same color depth of the ribbon, and the negative serum test results are negative.

6) Interference test: The test result is not interfered by specimen hemolysis (n=50), lipemia (n=50) and jaundice (n=50).

7) Cross-reaction: Use this Echinococcus multilocularis circulating antigen dot gold immunofiltration kit to conduct systemic lupus erythematosus (n=30), rheumatoid disease (n=30), immune hepatitis (n=30) No cross-reaction was found in the detection of autoimmune diseases.

8) Stability test: apply the Arrhenius rule, place the Echinococcus multilocularis circulatory antigen spot gold immunofiltration kit at 37°C for 20 days, and test it. The above indicators have no significant changes. Ensure that the finished product is stored at room temperature and dry. for 18 months.

The Echinococcus multilocularis circulating antigen dot gold immunofiltration kit prepared by the utility model can be used for the detection of the Echinococcus multilocularis circulating antigen in specimens such as whole blood, serum and plasma. The instrument can directly interpret the results with the naked eye. Moreover, the detection method is simple and fast, has strong specificity, high sensitivity, accuracy and reliability, low cost and wide application.

Claims (3)

1. Echinococcus multilocularis CAg spot gold immunity percolation kit; It is characterized in that being provided with the antibody and the cleansing solution of check-out console, the anti-Echinococcus multilocularis CAg of golden mark, said check-out console is provided with mounting medium, miillpore filter, water sucting medium, Echinococcus multilocularis CAg check point and control point; Said Echinococcus multilocularis CAg check point and control point are located on the miillpore filter successively; Encapsulate the antibody of anti-Echinococcus multilocularis CAg at Echinococcus multilocularis CAg check point place; Encapsulate Echinococcus multilocularis antigen EM2 and EM18 at the control point place; The miillpore filter of point sample is fixed on the water sucting medium; Put into mounting medium then, a side of said mounting medium has the through hole relative with miillpore filter; Said mounting medium comprises base plate and is buckled in the cover plate on the base plate, offers the through hole over against miillpore filter on the cover plate.

2. Echinococcus multilocularis CAg spot gold immunity percolation kit as claimed in claim 1 is characterized in that said mounting medium is the PVC plate.

3. Echinococcus multilocularis CAg spot gold immunity percolation kit as claimed in claim 1 is characterized in that it is the nitrocellulose membrane of 0.1～0.5 μ m that said miillpore filter adopts the aperture.

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