WO2011057475A1

WO2011057475A1 - Test strip for liver screen test based on gold immunochromatographic assay and method of preparing the same

Info

Publication number: WO2011057475A1
Application number: PCT/CN2010/001784
Authority: WO
Inventors: 张玥; 韩永俊; 高成秀; 杨超文; 孙宏彬; 孙潇; 张宇婷; 钱杰
Original assignee: 上海科新生物技术股份有限公司
Priority date: 2009-11-12
Filing date: 2010-11-08
Publication date: 2011-05-19
Also published as: CN102062777B; CN102062777A

Abstract

A test strip for liver screen test based on gold immunochromatographic assay and a method of preparing the same are provided. The test strip for liver screen test based on gold immunochromatographic assay includes: a cellulose nitrate film (3), a conjugate pad (2), a sample pad (1) and a wicking pad (6), which are stuck on a PVC substrate (7) and overlap each other in sequence. A detection line (4) and a control line (5) are provided on the cellulose nitrate film (3). Mitochondrial M2 type antigen is coated on the detection line (4), and antibody (c) is coated on the control line (5). Antibody (a) and antibody (b) labeled by colloidal gold are coated on the conjugate pad (2).

Description

Colloidal gold chromatography liver disease test strip and preparation method thereof

The invention belongs to the field of medical immunology, and particularly relates to a test paper for detecting liver diseases by colloidal gold immunochromatography and a preparation method thereof. Background technique

Primary bi l iary cirrhosis (PBC) is an organ-specific autoimmune liver disease that destroys small and medium-sized bile ducts in the liver, which is characterized by inflammation of the hepatic portal vein leading to fibrosis. Hardening or even functional failure. Hepatitis and cirrhosis patients with various pathogen detection indicators are negative, need to consider primary biliary cirrhosis or autoimmune hepatitis. The early use of drug therapy can control the progress of the disease, and the key to the treatment of PBC is early treatment, and the early treatment is based on early diagnosis, and early diagnosis has become the focus of attention.

A variety of autoantibodies can be detected in patients with PBC, such as anti-nuclear antibody (ANA), anti-mitochondrial antibody (AMA), anti-smooth muscle antibody (SMA anti-liver kidney microsomal antibody (LM), etc. The most important antibody is AMA. PBC is often accompanied by high titer AA, which is characteristic of this disease in the early stage of the disease. AMA was first discovered by indirect immunofluorescence in the serum of PBC patients by WalRer et al in 1965, and subsequent studies showed that PBC patients The AMA positive rate can be as high as 90%, and this test has become the main inspection item for PBC diagnosis. (Neberger J, Thomson R. PBC and AMA - a what is the connection [J] . Hepatology, 1999, 29 (1) 265 -271 )

In recent years, it has been found that there are several subtypes of AMA. There are 9 subtypes (M1 ~ M9) found so far, and 4 related to PBC, namely M2, M4, M8, M9, among which M2 subtype antibody (AMA- M2) is a specific antibody to PBC and has a diagnostic significance for this disease. AMA-M2 mitochondrial antibodies have a sensitivity of more than 93% for PBC detection and a specificity of almost 95%. (Leung PS, Van de water J , Coppel R L. et al. Molecular aspects and the pathoh) gi ( al bas is of primary bi l iary cirrhos is [J] Autoimnlun, 1996, 9 (2) : 119-128. )

M2 anti-mitochondrial antibodies can occur in PBC patients several years or even decades before clinical symptoms and histological changes occur (Jiang Xiaohua, Zhong Renqian, Kong Xiantao. Pathogenesis of primary biliary cirrhosis Chinese Journal of Immunology, 2002, 18: 586-588). Therefore, the immediate detection of M2 anti-mitochondrial antibodies is very important for early detection and diagnosis of PBC. In the published "PBC Diagnostic Guide" by the American Association of Liver Diseases (AASLD) in 2000, one of the most important ones is the M2 subtype anti-mitochondrial antibody. (AMA-M2) is positive.

With the study of the M2 target antigen, it was identified that the target antigen of M2 is a component of the 2-oxo 3⁄4^ hydrogenase complex [2-0ADC] on the mitochondria: pyruvate dehydrogenase (PDC-E2), 2-oxo acid dehydrogenase (BC0ADC-E2), 2-oxopentane hydrogenase (0GDC-E2), X protein, etc., and the epitope of PDC is mainly located in the lactone region and part of the ester group The positive rate in the serum of PBC patients was 95%. BC0ADC, the epitope of 0GDC is located in the lactone region, the positive rate is 53% ~ 55%, 39% - 88%. There is no cross-reaction between the three antigens, and the M2 anti-mitochondrial antibody in the serum of PBC patients can be combined with one kind or More than one antigen reaction. Combined detection of three target antigens, the positive rate can reach 95% ~ 99%, and the specificity is also very high, the normal person is only 0. 53⁄4 (Jia Jidong autoantibody detection in the diagnosis of autoimmune liver disease [J] Chinese Journal of Laboratory Medicine, 2003, 26 (2) 116-120).

This product uses genetic engineering methods (Dingkang, Chen Yan and other human M2 doublet target antigen PO-E2 fusion protein clone expression and identification. [J] Clinical Military Medical Journal. 2007,6 36(3):323: 325 . ) Successfully cloned and prokaryotically expressed three target antigens all of which are human, namely pyruvate dehydrogenase (PDC), 2-oxoacid dehydrogenase (BC0ADC), 2-oxoglutarate dehydrogenase (0GDC), And successfully connected to form an antigenic protein triplet is the detection antigen of the product.

The current M2 anti-mitochondrial antibodies are detected by indirect immunofluorescence (Indirectmmunof luorescence, IIF) and Enzyme-l inked Immunosorbent Assay (ESA).

The commercially available M2 anti-mitochondrial antibody detection kits are mainly ELISA kits.

The ELISA procedure is cumbersome. It takes about three hours to complete the whole experiment. It also requires professional immunology technicians to carry out the experiment in the laboratory, and the results of the experiment need to be read by the microplate reader. This is the result of the primary medical institution. It is difficult to achieve the experimental test of the project in the laboratory and small clinics. At the same time, the ELISA method is susceptible to various environmental factors such as temperature and incubation time, which brings many inconveniences to the test.

Indirect immunofluorescence, observation results require professional operators to operate special instruments and equipment. Indirect immunofluorescence method has long detection time but must have fluorescence microscope observation results, and has certain subjective human error. It is also susceptible to other interferences and produces false positives. Difficulties, not suitable for the detection of high-throughput specimens.

In order to overcome the deficiencies of the above detection methods, we applied colloidal gold chromatography to the detection of M2 anti-mitochondrial antibodies. The gold-immunochromatography assay (GICA) is a colloidal gold labeling technique. The colloidal gold is used as the tracer, and the strip fiber chromatography material is used as the solid phase. The sample solution is chromatographed by capillary effect. Swim on the strip, so that the test object in the sample and the bond pad are targeted The receptor (such as an antigen or an antibody) of the analyte is immunoreactive and is immunoreactive with the antigen (or antibody) on the strip fiber chromatography material to be trapped, thereby forming a magenta band visible to the naked eye, which is intuitive The results of the experiment, for the purpose of rapid detection (Sikowicz G et al. One-step chromatographic immunoassay for qualitative determination of choricogonadotropin in urine. Clin Chem. 1990 (36) 1579-1586.). Only use the sample to sample to the sample On the pad, the negative-positive result is judged according to the occurrence of the purple-red band on the test line in a few minutes. Compared with other detection methods, the detection time is short, only 5 ~ 10 minutes, the operator does not need training, the operation is simple and fast, no need to cry storage, convenient storage and transportation.

In terms of autoimmune diseases, some test strip products for detecting autoimmune diseases have appeared on the foreign market, but the colloidal gold immunoassay for M2 anti-mitochondrial antibodies has not been published in the domestic and foreign markets. The invention first introduces the antigen protein expressed by the genetic engineering bacteria into the test strip, and realizes the detection performance with high specificity, high sensitivity and high accuracy. Commercialized ELISA kits have also appeared on the market. However, there are still many differences between the gold standard chromatographic test papers and the target. The detection time is short, that is, 5-10 minutes, and the interpretation results are simple. In addition, the test paper has high specificity, high sensitivity and high accuracy, which can meet the rapid screening of PBC in the market, and provide conditions for early diagnosis and treatment of disease 5L. At the same time, it can also meet the needs of grassroots laboratory, instant detection and bedside detection. . Summary of the invention

In order to overcome the deficiencies of the above methodologies, the present invention applies colloidal gold chromatography to the detection of Μ2 type anti-mitochondrial antibodies, and at the same time applies Μ2 type mitochondrial antigen protein to colloidal gold chromatography for the first time, and uses indirect method to realize blood. The detection of Μ2 anti-mitochondrial antibody in the sputum enables high specificity, high sensitivity and high accuracy of detection performance, and rapid screening of positive samples of Μ2 anti-mitochondrial antibodies can quickly and conveniently assist in the diagnosis of primary biliary cirrhosis. .

One of the objects of the present invention is to provide a colloidal gold chromatography liver disease test strip.

The second object of the present invention is to provide a method for preparing a colloidal gold chromatography liver disease test strip. A colloidal gold chromatography liver disease test strip comprising a nitrocellulose coating (3), a bonding pad (2), a sample pad (1), and an absorbent pad (6), which are sequentially attached to a substrate, wherein :

The binding pad is coated with a gold standard antibody (a) and a gold standard antibody (b).

The nitrocellulose coating film (3) has a detection line (4) and a quality control line (5),

The detection line is coated with M2 type mitochondrial antigen protein, wherein the quality control line (5) is coated with antibody (c). Further, the antibody (a) is an anti-human IgG monoclonal antibody or an anti-human IgG polyclonal antibody, or one or more of Staphylococcal Protein A (SPA) or Streptococcal Protein G (Protein G). The polyclonal antibody in the antibody (a) is one of a mouse source, a horse source, a sheep source, a rabbit source or a guinea pig source, and the monoclonal antibody in the antibody (a) is one of a mouse source or a rabbit source. Kind.

The antibody (b) and the antibody (c) are each one of a monoclonal antibody or a polyclonal antibody, wherein (b) and (c) can specifically bind to form an immune complex.

The polyclonal antibody in the antibody (b) and the antibody (c) is one of a mouse source, a horse source, a sheep source, a rabbit source or a guinea pig source, and the monoclonal antibody is one of a mouse source or a rabbit source. .

The protein coated on the detection line is an M2 type mitochondrial antigen protein obtained by expressing a cloned gene.

The test is a qualitative test for M2-type anti-mitochondrial antibodies in human serum to aid in the diagnosis of early primary biliary hepatitis.

The M2 type mitochondrial antigen protein is a component of the mitochondrial 2-oxoacid dehydrogenase complex [2-0ADC] targeting the target antigen of M2: pyruvate dehydrogenase (PDC-E2), 2-oxo acid Dehydrogenase complex E2 (BCOADC-E2), 2-oxoglutarate dehydrogenase complex E2 (OGDC-B2), constructs recombinant gene by gene cloning technology, and then successfully expresses human M2 by prokaryotic expression technology. Three target antigen proteins were successfully ligated to form an antigenic protein triplet (Jiang Xiaohua, Zhong Renqian et al. Cloning, expression and preliminary identification of M2 autoantigen and its triplet. Chinese Journal of Digestion, 2001 (21): 530-533).

The samples were obtained from human serum, plasma, whole blood samples.

The monoclonal antibody to be used according to the present invention can be hybridized as described first by Kohler et al. (Cont in continuous culs of fused cel ls secret ing ant ibody of predefined specificity [J]. Nature, 1975 (256): 495-497). The tumor method is prepared or can be prepared by recombinant DM (see U.S. Patent 4,816,567). "Monoclonal antibodies" by Clackson et al. ( Making ant ibody fragments using phage display l ibraries [J] . Nature, 1991: 624-628 ) and Marks et al ( By-pass ing immunization: Human ant ibodies from V- gene l ibraries displayed On phage [J] . Journal of Molecular Biology, 1991: 581-597 ) The technique is isolated from a phage antibody library.

The polyclonal antibody to be used according to the present invention can be produced by immunizing an animal by Chen Xueqing et al. (Urtra Experimental Method for Immunology. [M], 2000: 15-26).

The SPA, Protein G of the present invention expresses a cloned gene by prokaryotic expression of a cloned recombinant gene, and is expressed by J. Sambrook et al. (Molecular Cloning Experimental Guide. [M], 2002: 1228-1232).

Method for preparing colloidal gold chromatography liver disease test strip, which comprises sample pad, bonding pad and bag The film, the absorbent pad and the bottom plate are combined, and the method comprises the following steps:

Step 1. Preparation of the antigen, the M2 mitochondrial antigen protein is obtained by prokaryotic expression of the cloned gene, and the preparation of the coating membrane is performed by diluting the antigen protein with a coating buffer to a concentration of 1.0-1.5 mg/mL, and the antibody (c Diluted to 0.8 ~ 1.5mg / mL, placed on a 1 ~ ΙΟμΙ / cm nitrocellulose membrane, placed in 37 Ό dry,

And gold standard antibody preparation, is to adjust the colloidal gold pH 7.0~9.0 with 0.1M potassium carbonate, slowly add 4 - 25μ ₈ protein per ml of colloidal gold solution, stir for 10 ~ 30min, then add BSA to the final concentration of 0.5 - 5% Stir for 10~30min, centrifuge, discard the supernatant, wash the precipitate with washing solution for 2~3 times, and resuspend the pellet with the first one volume of the initial volume of the preservation solution.

And the preparation of the bonding pad is a polyester film impregnated with the treatment liquid. After drying, the gold standard antibody (a) and the gold standard antibody (b) are mixed, and then sprayed at a dosage of 0.5 to 4 μl /cm. On the polyester film, after drying at 25 °C~30TC, put it in the environment of 2 °C~8 °C for use.

And the preparation of the sample pad is a glass fiber membrane impregnated with the treatment liquid, and is prepared after drying at 37"C;

Step 2, sequentially bonding the nitrocellulose coating film, the bonding pad, the sample pad, and the absorbent pad prepared by the foregoing step 1 on the bottom plate;

Step 3: Cut the finished material in step 2 into a test strip.

In the step 1, the gold standard antibody is prepared by adjusting the pH of the colloidal gold to 8.0-9.0 with 0.1 M carbonic acid clock, and slowly adding _8-12 μ8 goat anti-human IgG to the colloidal gold solution, stirring for 10-30 min. Then, add BSA to a final concentration of 0.5 to 1%, stir for 10 to 30 minutes, centrifuge, discard the supernatant, wash the precipitate with the washing solution 2-3 times, and resuspend the pellet with one tenth of the initial volume of the preservation solution. Set 4 to reserve.

In the step 1, the method for preparing the gold standard antibody is to adjust the pH of the colloidal gold to 7.0-8.0 with 0.1 M potassium carbonate, ■ ^ ml colloidal gold solution, slowly add 8 ~ 12 pg rabbit I _g G, and stir for 10 to 30 minutes. Then add BSA to a final concentration of 0.5 ~ 1%, stir for 10~30min, centrifuge, discard the supernatant, wash the precipitate with the washing solution 2~3 times, and resuspend the pellet with the initial solution of one tenth of the initial volume. 4 Ό spare.

According to Step 1, preparation of gold-labeled colloidal gold SPA to adjust the pH to 5.0-6.5 with 0.1M potassium carbonate, ml colloidal gold solution was slowly added 10 ~ 15μ ₈ SPA, was stirred for 10 ~ 30min, then was added to the BSA The final concentration is 0.5 ~ 154, stirred for 10~30min, centrifuged, the supernatant is discarded, and the precipitate is washed 2~3 times with the washing liquid. The sediment is resuspended in the last one tenth of the initial volume of the preservation solution, and placed in a buffer for 4 minutes. Then, the gold standard SPA 0D 20 2~4μΐΛ; πι is sprayed on the bonding pad, dried and used.

In the step 1 described, the method for preparing the gold standard SPA is to adjust the colloidal gold H value to 0.1 M carbonic acid clock to 7. 0 ~ 8. 0, ML colloidal gold solution was slowly added to 8 - 12μ Β mouse IgG, stirred for 10 ~ 30min, then added BSA to a final concentration of 0.5 ~ 1%, stirred for 10 ~ 30min, centrifuged, discarded, The precipitate was washed 2 to 3 times with the washing solution, and the pellet was resuspended at the last one tenth of the initial volume of the preservation solution, and stored at 4 ° C until use. Then apply the gold standard SPA0D 30 2 ~ 4μ1/ η to the bonding pad, dry and set aside.

In the step 1, the prepared gold standard antibody (a) and the gold standard rabbit IgG are mixed in a ratio of 20 to 40/15 - 20 , and sprayed with a BIO-Dot spray film machine at 2. 0 ~ 4μ1/οπι The pretreated polyester film is dried at 25 °C - 37'C, sealed, and placed at 2 °C ~ 8 °C for assembly of sticky sheets.

In the step 3 described above, the width of the cut test paper is preferably 4 mm and 3 mm.

The detection principle of the present invention specifically uses affinity chromatography to purify the M2 antigen protein, the gold standard antibody (a) and the gold standard rabbit IgG as a colloidal gold labeling complex, sprayed on the binding pad, and indirectly detect whether the serum sample is in the serum sample. Contains M2 anti-mitochondrial antibodies. At the time of detection, the sample migrates to the binding pad and infiltrates the gold-labeled complex, wherein the human IgG and the gold-labeled antibody (a) combine to form a human IgG-gold standard antibody (a) complex, due to capillary effect, The complex moves forward along the coating membrane. If the serum sample contains M2 anti-mitochondrial antibody, the complex specifically binds to the antigen protein coated on the nitrocellulose membrane to form a gold-labeled antibody (a). The human IgG-M2 antigenic protein triplet complex is trapped on the detection line and gradually enriched to form a deeper purple-red band; as the capillary effect continues to move forward, the gold-labeled rabbit IgG is coated on the quality control line. The specific immune response of the goat anti-rabbit IgG is trapped and gradually enriched to form a deep purple-red band on the quality control line. The excess unbound material continues to be chromatographed onto the absorbent pad, so the detection line and quality control If the serum sample does not contain the M2 anti-mitochondrial antibody, the gold antibody (a) to the 3⁄4^ line does not immunoreact with the antigenic protein coated on the detection line. Therefore, there is no purple-red band at the detection line, and the gold-labeled antibody continues to move forward to reach the absorbent pad, while the gold-labeled rabbit IgG continues to move forward and specifically immunizes with the goat anti-rabbit IgG coated on the quality control line. The reaction was trapped and gradually enriched to form a purplish red band on the quality control line, so that only the band on the quality control was judged to be a negative result.

Compared with the existing detection methods, the advantages of the invention:

1. The uniqueness of this assay is that the recombinant antigen-expressing triplet antigen protein is firstly applied to colloidal gold chromatography test paper, which greatly improves the detection sensitivity. The whole body gold test strip can quickly screen out all positive samples of M2 anti-mitochondrial antibody. A sample larger than 25RU/ml was detected).

2. The advantage of the invention is that the production cost is low. The core reagent required for the M2-type anti-mitochondrial antibody test strip provided by the present invention is an antibody (a), that is, an anti-human IgG or SPA or PROTEIN G, an antibody (c), an antibody (b), an antigen protein, and a reagent for a single test strip. Small amount, and can be purchased by commercial reagents or Self-made, antigenic proteins are derived from commercially available purified genetically engineered antigenic proteins, or prepared by themselves.

3. Compared with other disclosed methods for M2 type anti-mitochondrial antibody detection, the test paper of the present invention has advantages that many other methods cannot match, such as short detection time ( ⁵ ~ 10 min); no special equipment is required. It can realize bedside detection and instant detection of outpatient clinic; It is easy to operate, only one step is required, the operator does not need training, and the detection cost is low; there is no special requirement for temperature, no need to freeze, storage and transportation is convenient, room temperature can be 24 months. DRAWINGS

Figure 1 is a schematic view showing the side structure of the present invention. As shown in Fig. 1, the test paper is sequentially attached to the support rubber sheet (7) with a nitrocellulose coating film (3), a bonding pad (2), a sample pad (1), and a water absorbing pad (6).

The binding pad (2) is coated with a gold standard antibody (a) and a gold standard antibody (b); the nitrocellulose coating film (3) has a detection line (4) and a quality control line (5), wherein the detection line ( 4) coated with M2 type mitochondrial antigen protein, and quality control line (5) coated with antibody (c).

Figure 2 is a schematic diagram of the detection results of the present invention.

Figure 2a shows that after the addition of the sample, the reaction zone 3 ( 5 ) and the control zone 5 ( C ) show a purple-red band at the corresponding position within 3 - 5 minutes;

Figure 2b shows: When both the C region 5 and the T region 4 have a purple-red band, the result is positive, indicating that the serum contains M2 anti-mitochondrial antibodies;

Figure 2c shows: If only a purple-red band appears in the C region 5, the T-zone 4 does not show a purple-red band, and the result is negative, indicating that the serum does not contain the M2-type anti-mitochondrial antibody;

Figure 2d, 2e shows: If there is no purple-red band in the C area 5, no matter whether there is a strip in the T area 4, the test paper fails. detailed description

The colloidal gold immunochromatographic liver disease test strip according to the present invention is shown in Fig. 1. The test paper is sequentially attached to the support rubber sheet (7) with a nitrocellulose coating film (3) and a bonding pad. (2), sample pad (1), absorbent pad (6). The binding pad (2) is coated with a gold standard antibody (a) and a gold standard rabbit IgG. The nitrocellulose coating film (3) has a detection line (4) and a quality control line (5), the detection line (4) is coated with an M2 type mitochondrial antigen protein, and the quality control line (5) is coated with a goat anti- Rabbit IgG.

As shown in Figure 2, after loading, the reaction zone 4 (T) and control zone 5 (C) can be seen after 3 - 5 minutes of reaction. A purple-red band appeared at the corresponding position, as shown in Figure 2a. When both the C-zone 5 and the T-zone 4 showed a purple-red band (as shown in Figure 2b), the result was positive, indicating that the serum contained M2-type anti-mitochondrial antibody. If only a purple-red band appears in area C, and no purple-red band appears in T-zone 4 (as shown in Figure 2c), the result is negative, indicating that the serum does not contain M2-type anti-mitochondrial antibodies; No purple-red bands appear, no matter whether there is a band in the T-zone 4 (as shown in Figures 2d and 2e), the test paper fails.

The invention is further illustrated below in conjunction with specific embodiments.

Example 1 antigen composition

The antigenic protein applied to the test paper is a component of the 2-oxo-hydrogenase complex [2-0ADC] targeting the target antigen of M2, mitochondria: pyruvate dehydrogenase (PDC-E2), 2-oxo acid The dehydrogenase complex E2 (BC0ADC-E2) and 2-oxoglutarate dehydrogenase complex E2 (0GDC-E2) were constructed by gene cloning technology, and then all of them were successfully expressed by prokaryotic expression technology. M2 three target antigen proteins, and successfully joined to form an antigenic protein triplet.

Example 2 Antibody Preparation

Antibody (a) and antibody (c), antibody (b) The preparation of a colloidal gold chromatography liver disease test strip was carried out by the following method. Among them, anti-human IgG, antibody (c) and antibody (b) can generally be produced by multiple subcutaneous (sc) or «intra (ip) injection-free adjuvants and adjuvants on i1⁄2 animals.

By injecting 0.05 mg to 1 mg of the immunological preparation (for goats or mice, respectively) with 3 volumes of Freund, s complete adjuvant, the solution was injected in multiple parts of the skin, and the animal was used 1/5 after one month. 1/10 of the original amount of Frecl's complete adjuvant mixture of human IgG was boosted by subcutaneous injection at multiple sites. After 7 to 14 days, the animals were bled and serum anti-human IgG titers were determined. The animals are boosted until the titer reaches the plateau. Methods for producing polyclonal antibodies are described in a number of immunology textbooks, for example, Chen Xueqing et al., Common Experimental Methods for Immunology. By recovering spleen cells from immunized animals and immortalizing the cells, for example by fusion with myeloma cells or by Epstein-Barr virus, and screening for monoclonal antibodies that express the antibody of interest ( Kohler, Mi lstein. Derivat ion of specif Ic ant ibody-ducing tissue culture and tumor l ines by cel l fus ion [J] . European Journal of Immunology, 1976: 501-511 ).

The recombinant Staphylococcal Protein A and Streptococcus G protein can be prepared by prokaryotic expression of the cloned gene in E. coli. For specific methods, see J. Sambrook et al. (Molecular Cloning Experimental Guide [M], 2002: 1228-1232), or Purchase a commercial SPA or Protein G.

Example 3 Sheep anti-human IgG labeling

Labeling of goat anti-human IgG: Adjusting colloidal gold pH 0. 0 ~ 9. 0 with 0·1M potassium carbonate, per ml of colloid Slowly add 8 ~ 12 g goat anti-human IgG to the gold solution, stir for 10~30min, then add BSA to the final concentration of 0.5-1%, stir for 10~30min, centrifuge, discard the supernatant, and wash the precipitate with washing solution for 2~3 times. The pellet was resuspended at the last time with one tenth of the initial volume of the preservation solution.

Example 4 Rabbit IgG labeling

Labeling of rabbit IgG: Adjust the pH of colloidal gold to 7.0 ~ 8.0 with 0.1M carbonic acid clock, slowly add 8 ~ 12μ _ε rabbit IgG per ml of colloidal gold solution, stir for 10 ~ 30min, then add BSA to the final concentration of 0.5 ~ 1% Stir for 10~30min, centrifuge, discard the supernatant, wash the precipitate with the washing solution for 2~3 times, and resuspend the pellet with the initial solution of one tenth of the initial volume, and set it at 4 °C for use.

Example 5 Labeling of Staphylococcal Protein A (SPA)

SPA protein labeling: adjust the pH of colloidal gold to 5.0 ~ 6.5 with 0.1M potassium carbonate, slowly add 10 - 15 g SPA protein to ^ ml colloidal gold solution, stir for 10 ~ 30min, then add BSA to the final concentration of 0.5-1%, stir 10~30min, centrifuge, discard the supernatant, wash the precipitate with the washing solution 2~3 times, resuspend the pellet with the first one volume of the initial volume of the preservation solution, set 4 times for use.

Example 6 Rat anti-human IgG labeling

Labeling of mouse anti-human IgG: Adjust the pH of colloidal gold to 7.0 ~ 8.0 with 0.1M potassium carbonate, slowly add 8 ~ 12μ ₈ mouse anti-human IgG per ml of colloidal gold solution, stir for 10 - 30min, then add BSA to the final concentration 0.5-1%, stir for 10~30min, centrifuge, discard the supernatant, wash the precipitate with the washing solution 2~3 times, re-suspend the pellet with the first one volume of the initial volume of the preservation solution, set 4'C for use.

Example 7

The preparation of the colloidal gold chromatography liver test test paper of the invention is as follows:

Step 1, preparation of coated film

Dilute the M2 protein to 1.0-1.5 mg/mL with coating buffer, adjust the BIO-Dot instrument, spray the detection line (T), near the end of the bond pad, about 9.5 hidden from the end of the bond pad; with coating buffer Dilute the goat anti-rabbit IgG to 0.8 ~ 1.5mg/mL, and spray the goat anti-rabbit IgG on the quality control line (C) with the BIO-Dot instrument near the absorbent pad, and wake up about 9 from the absorbent pad. The distance between the two lines is about 5-8 瞧, and the spray line should be uniform in thickness. 37Ό drying, packaged for use.

The coating buffer may be borate, carbonate, phosphate, Tris-HC1 or Tris-phosphate, acetate, barbital, etc., the purpose of which is to provide a certain pH. And the ionic strength allows the protein to be coated and firmly coated on the NC membrane, the buffer pH of which is generally in the range of about 6 to 9.5, preferably in the neutral buffer range of 6.5 to 7.5, and most preferably the buffer The pH is in the range of 7.0 to 7.4. The buffer is preferably phosphate. The NC film may be any commercially available nitrocellulose membrane, S&S AB99, Whatman 8μιη, millipore M135 sartorius CN140, and the like. The specific NC film used is not critical to the invention, but several of the above NC films may be preferred in each measurement. Different buffers treated with different surfactants used by different manufacturers have different degrees of affinity with the detection solution antibody solution, which will cause uneven lines, tow or dispersion, so The test paper was assembled to select a preferred NC film.

2. Preparation of colloidal gold solution

Heat 0.01% of the HAuCl ₄ solution to boiling, quickly add every 100 mL of HAuCl ₄ solution to the reducing agent solution, start to have some blue color, then light blue, blue, then heat to appear red, boil for 7 to 10 minutes, appear transparent orange red. It is then filtered through an ultrafiltration or microfiltration membrane (0.45 μM) to remove the polymer and other impurities that may be mixed therein. The prepared colloidal gold should be pure, translucent, free of sediment and floating matter, and discarded when oily substances and a large amount of black granular precipitate appear on the liquid surface.

The reducing agent used therein may be trisodium citrate (Frens 1973), trisodium citrate monosodium citrate (Slot and Gueeze 1985), white phosphorus, preferably trisodium citrate, more preferably 1% citric acid tris. sodium.

The glass container used should be absolutely clean and must be pickled and silicified before use. The water should be deionized ultrapure water with a resistivity of 18.2 ΜΩ.

3. Preparation of gold standard antibody

See Example 3, Example 4, Example 5, and Example 6.

Wherein the washing liquid, that is, the preservation liquid is borate, carbonate, phosphate, Tris-HC1 or Tris-phosphate, acetate, barbital, etc. containing 0.2-1% macromolecular protein, The macromolecular protein may be bovine serum albumin, PEG 20000, casein or the like, and the buffer is preferably a phosphate buffer, more preferably 0.2% BSA pH 7.2 phosphate buffer.

4. Pretreatment of the bonding pad

The polyester film was immersed in a buffer for 30 minutes and dried at 37 Torr.

Buffers that can be used for this purpose include

(a) containing 1% PVA, 0.71% Na ₃ P0 ₄ , 1% BSA, 0.05% NaN ₃ , 0.1% Triton X-100;

(b) 1% PVA, 1% BSA, 0.05% PROCLINTM 300, 0.1% Triton X-100, pH 7.0 PBS;

(c) 1% PVA, 1% BSA, 0.05% PROCLINTM 300, pH 7.0 PBS;

(d) containing 1% PVA, 0.71% Na ₃ P0 ₄ , 13⁄4 BSA, 0.05% NaN ₃ , 0.1% Tween-20;

(e) contains 1% PVA, 0.71% Na ₃ P0 ₄ , 1% BSA, 0.05% NaN ₃ , 0.1% Tween-20, pH 7. 0 PBS; The preferred buffer for the assay described herein is buffer (a) because it has the maximum resolution that distinguishes between negative-positive samples. PROCLINTM 300 and NaN ₃ act as preservatives, while Tween-20 has decontamination and hydrophilic effects.

5. Preparation of bonding pads:

The gold standard anti-human IgG ( a ) and the gold standard antibody ( b ) were mixed in a certain ratio, and sprayed on the pretreated polyester film with a BIO-Dot instrument 0.5 to 4 μl / αη, 25 ° C ~ 30 'C Dry, after drying, seal the bag and set at 2 °C ~ 8 °C for use.

6. Processing of the sample pad

The sample pad was evenly spread on the sample pad with a treatment solution of 45 mL/piece, and dried at 37 ° C, and then packaged in an aluminum foil bag for use.

Buffers which can be used for this purpose include (a) 0. 05M Borax, 0.01 MPBS (H 7. 0 ), 0. 1% Sodium Casein, 1% PEG 20000, 23⁄4 BSA, 0. 05% NaN ₃ ; 0. 05M Borax, 0. 01MPBS ( H 7. 0 ), 0. l%Sodium Casein, 1%PEG20000, 2%Casein, 0. 05%NaN ₃ ; ( c ) 0. 05M Tri s-cl ( pH 7. 0), 0. 01 MPBS, 0. 1% Sodium Casein, 1% PEG 20000, 2% Casein, 0. 05% NaN ₃ „ The preferred buffer for the assay described herein is buffer (a) because It has the highest resolution that distinguishes between positive and negative samples, and NaN ₃ acts as a preservative.

7. Pre-cutting of raw materials

The glass fiber membrane is cut and cut: The glass fiber membrane is cut into a length of 28 cm and a width of 2.4 cm by a slitting machine, and placed in a dry room for use.

Cutting of absorbent paper: Use a paper cutter to cut the absorbent paper into a length equal to that of the PVC plate, ie 28 cm long and 3 cm wide.

Cutting of the polyester film: Cut it into a length equal to the length of the PVC plate by a paper cutter, that is, 28 cm in length and lcm in width, and set it in a dry room for use.

The nitrocellulose membrane, the absorbent paper, the polyester film, and the glass fiber membrane are sequentially laminated on the PVC plastic base plate as shown in Fig. 1 to form a large plate. The temperature of the assembly workshop should be controlled at 25Ό ~ 37Ό and humidity 20% ~ 30%.

8. Cut strips

Use a slitter to cut the large plate into a single serving. The width of each person is cut into a width of 2. 5mm ~ 4mm according to certain requirements. With the detection, the sensitivity can detect the indoor quality control sample (ie weakly positive sample). The degree of color development of the strip is as shown in Fig. 2d, and there is no non-specific strip, then the product is qualified as a qualified product through quality control.

9. Assembly, packaging

Assembling the test paper of one person in the prepared test paper card so that the sample window corresponds to the sample of the test paper Pad, the result display window corresponds to the detection area and control area, and the assembly workshop temperature should be controlled at 25Ό~37Ό, humidity 20%~30%. Then pack it in a bag with a pack of desiccant, instructions, and sample applicator, and store it in the dark at 4~25Ό.

Example 8 determines the mixing ratio of the gold standard antibody (a) and the gold standard antibody (b)

Taking gold-labeled goat anti-human IgG and rabbit IgG as examples to illustrate the determination of ratios, other antibodies such as mouse anti-human IgG and rabbit IgG, staphylococcal protein A and rabbit IgG, streptococcal G protein and rabbit IgG can also be used according to this method. determine.

1. In the step 3 of the seventh embodiment, the mixing ratio of the two is determined as follows: The 0D20 of the gold-labeled rabbit IgG is basically determined by a preliminary experiment, and sprayed on the binding pad with a BIO-Dot spray amount of ΐμΐ/cm, using 0.01 M PBS. To load the buffer, that is, to obtain the band of the desired color intensity, it is determined that the application OD of the rabbit IgG is 1 μl.

2. Gradiently dilute the gold-labeled goat anti-human IgG to a final concentration of 0D 100, 80, 60, 40, 20, then dilute the gold-labeled rabbit IgG to a final concentration of 0D 20, and use the BIO-Dot to treat the above-mentioned gold-standard goat anti-human The sprayed amount of IgG and gold-labeled rabbit IgG was sprayed on the treated polyester film by 3μ1/αη, and the prepared coating film, bonding pad, sample pad and squeegee were sequentially attached to the plastic substrate, with positive serum and critical reference value. Serum and negative serum were used for debugging. Judgment basis: The positive color of the test line (Τ) and the quality control line (C) are consistent with the intensity of the band color. The critical reference value serum can appear, and the 0D value of the negative serum without banding is the The amount of application of the batch. Through this test, OD20-40 is more suitable.

Example 9 test paper composition and reagent preparation

The invention provides a composition of a colloidal gold chromatography liver disease test strip: a nitrocellulose coating film (3), a bonding pad (2), a sample are sequentially grounded on a supporting plastic PVC board (7). Pad (1), absorbent pad (4). The binding pad (2) is coated with a gold standard antibody (a) and a gold standard antibody (b), the nitrocellulose coating film (3) has a detection line and a quality control line, and the detection line (T) package The antibody (c) is coated with the M2 type mitochondrial antigen protein (5) and the quality control line (C), wherein the M2 type mitochondrial antigen protein is derived from a commercially available purified genetically engineered antigen protein, or is prepared by itself.

The recombinantly expressed M2 mitochondrial antigen protein is introduced into the colloidal gold chromatography liver disease test strip, which can detect the M2 anti-mitochondrial antibody in the blood sample, and can quickly and conveniently assist in the diagnosis of primary biliary cirrhosis.

The colloidal gold chromatography liver test test paper of the present invention is prepared by the following reagents:

1. Preparation of coating buffer: 9gNacK 1.15gNa ₂ HP0 ₄ , 0.23g NaH ₂ P0 ₄ » 10gSucrose, 0.5g EDTA dissolved in 1L ultrapure water, filtered and placed in 4"C for use. 2. 11 (1 ₄ preparation: dissolve chloroauric acid with ultrapure water, prepare 1% solution, set 4 Ό for use, valid for four months. 1000 mL 1% HAuC solution formula: 10g HAuCl ₄ , ultrapure water volumetric Up to 1000 mL.

3. Preparation of 1% sodium citrate (Sodium Citrate): Dissolve Sodium Ci trate in ultrapure water, prepare a 1% solution, filter with 0.22 μm η, and use it now.

4. 0. Preparation of 1MK ₂ C0 ₃ : Prepared with ultrapure water, filtered through 0. 22μιη membrane, set at 4 ° C for use, valid for one month. 1000 mL 0. 1M K ₂ C0 ₃ solution formulation: 13. 8g ₂ C0 ₃ ; ultrapure water to a volume of 1000 mL.

5. Preparation of 101⁄2 BSA: Prepared with ultrapure water, 0. 05% sodium azide (NaN ₃ ), 0.22 μιη membrane filtered, set aside, valid for two weeks. lOOOOmL 10% BSA solution formulation: 100g BSA, 0. 5g NaN ₃ ; ultrapure water to a volume of 1000 mL.

6. Preparation of the washing solution, ie the preservation solution:

2% BSA, 0.05% (NaN ₃ ), 0.01 M pH 7. 2 PBS, 0.22 μM membrane filtered, set to 4 Ό, valid for two weeks. 1000 mL labeled wash preservation solution formulation: 20 g BSA, 0.5 g NaN ₃ , 0.01 M pH 7. 2 PBS to 1000 mL.

7. Preparation of gold standard dilution:

Preparation of lOOOOmL dilution: 2. 423g tris, lOgBSA, 0. 2gNaN ₃ dissolved in ultrapure water, adjusted to pH 8. 0, to a volume of 1000mL _o

After diluting the gold standard to the working concentration with a diluent, 20% Sucrose and 5% trehalose were added. Example 10 Sample Processing

Take whole blood l ~ 5ml, natural agglutination for 5 minutes, 3000 ~ 5000g / 5min ~ 10min, take the supernatant to get the sample solution to be tested, at least ΙΟΟμΙ above the sample solution to be tested.

A sample of 50 ~ 70μ1 of the above serum was taken from the sample pad with a micro-applicator, and the sample was gently sifted. Or use a pipette to slowly add 3 ~ 5 drops of serum to the sample pad, observe the results of 5 - lOmin, and judge the positive result according to the appearance of the band.

Example 11 Detection and clinical performance evaluation of the kit

Stability test

1. 1 37 °C acceleration stability

The test paper was placed at 37 ° C for accelerated test, and the indoor control product was taken out for testing every day, and the stability of the test paper was judged by the intra-assay precision of the negative-positive coincidence rate of the negative-positive reference product (10 parts each). After 4 months, the results showed that the quality control products were in line with expectations, and the positive-positive rate of each positive-positive reference product was 100%. _ Yin-positive reference products were 10 M2 anti-mitochondrial antibody-positive serum and 10 M2-type anti-drugs. Mitochondrial antibody negative serum. 1. 2 4 ° C stability test

The test paper was placed at 4 ° C for routine stability test, and the indoor quality control product was taken out every month. The stability of the test paper was also judged by the intra-assay precision of the negative-positive coincidence rate of the negative-positive reference products (10 each). .

After 12 months, the results showed that the quality control test results were in line with expectations, and the positive-positive compliance rate of each negative-positive reference product was 100%. After 18 months, the results showed that the positive-yellow coincidence rate of each negative-positive reference product was still 1003⁄4. After 24 months, the test results showed that there was a false negative. Based on the above results, the test paper is stored at 2 ~ 8X and is stable within 2 years.

2. Diagnostic sensitivity

Sera from 100 patients diagnosed with primary biliary hepatitis (PBC) were collected from the clinic, and the serum of PBC patients collected above was tested using self-made colloidal gold test paper according to the instructions in the instructions.

After 5 minutes, the statistical results are as follows:

Based on the results of the above statistics, based on the detection of 100 confirmed PBC patient sera, the number of M2 anti-mitochondrial antibody positive samples was 91 and the negative samples were 9 cases. Therefore, by calculation:

Diagnostic sensitivity (%) =91/ ( 91+9 ) xl00%=91%

3. Diagnostic specificity

Collect 300 serum samples from healthy blood donors and non-PBC patients (including other autoimmune diseases such as rheumatoid arthritis and self-protected liver patients) from the clinic. Use the self-made colloidal gold test paper according to the instructions in the instructions. The collected serum of healthy blood donors and non-PBC patients were tested. After 5 minutes, the statistical results are as follows:

Based on the statistical analysis of 300 sera of non-PBC patients and healthy blood donors, it was found that the number of M2 anti-mitochondrial antibody-negative samples measured in healthy blood donors was 296, while the non-PBC patient serum M2 anti-mitochondria. The number of antibody-negative samples was 297. So by calculation:

(Healthy blood donors) diagnostic specificity (%) = 296/300xl00 = 98. 6% (non-PBC patient serum) diagnostic specificity (%) = 297/300 = 99%

600 control groups (including healthy blood donors and non-PBC patients)

Diagnostic specificity (%) = 593/600 = 98. 8%

4. Diagnostic accuracy

Based on the above statistics of diagnostic specificity and diagnostic sensitivity, the following summary table can be obtained:

According to the above statistical table, the number of positive samples detected in the serum of patients who confirmed PBC was 98. For the serum of 600 healthy donors and non-PBC patients, 593 negative samples were detected.

Ie: Diagnostic accuracy = ( 91+593 ) /700=97. 7%

5. Intra-assay inaccuracy

Select one batch of test paper produced, and select four different concentrations of serum (strong, medium, weak, negative), and make 10 replicates per serum. Observe the results after 5 minutes. Different sero-negative results, positive results, strong yang serum 0 10 zhongyang serum 0 10 weak yang serum 0 10

Negative serum 10 0

According to the above results, the same batch of colloidal gold chromatography liver disease test strips were produced, and four samples of different concentrations of serum were tested. Each sample was repeated 10 times, and all of them were accurately separated and positively obtained. Yin positive results.

Therefore, the following conclusions were drawn: There was no intra-assay imprecision in the colloidal gold chromatography liver disease test strip.

6. Inter-assay inaccuracy

Three different batches of colloidal gold chromatography liver test strips were selected, and four different concentrations of serum (strong, medium, weak, and negative) were selected, and each serum was repeated 10 times. The results were observed after ⁵ minutes. First - batch second batch of third batch of different serum

Negative positive negative positive negative positive strong positive serum 0 10 0 10 0 10 Zhongyang serum 0 10 0 10 0 10 Weak positive serum 0 10 0 10 0 10 Negative serum 10 0 10 0 10 0 According to the above results, the production Three batches of colloidal gold chromatography liver test strips from different batches were tested for four different serum tests, each of which was repeated 10 times, and all of them were accurately positive for yin, resulting in consistent positive results.

Therefore, the following conclusions were drawn: There was no inter-batch imprecision in the colloidal gold chromatography liver disease test strip.

7. Products with different methodologies of similar testing items - Comparative test of M2 anti-mitochondrial antibody detection kit (ELISA method) produced by Ou Meng Company of Germany. 100 samples of random patient samples were collected from clinical samples, respectively, using Euromonal ELISA reagents. The box and the self-made colloidal gold chromatography liver test strip were tested and the following results were obtained:

Positive coincidence rate: 56/57xl00%=98. 2%

Negative coincidence rate:

6%

Overall coincidence rate: ( 56+42 ) /100=98%

According to the above statistics, the total coincidence rate of the self-made colloidal gold chromatography liver disease test strip and the Eugene ELISA kit can reach 98%. The above description of the present invention is not intended to be limiting, and other embodiments based on the inventive concept are within the scope of the present invention.

Claims

Rights request

1. A colloidal gold chromatography method for detecting liver disease, comprising a nitrocellulose coating film, a bonding pad, a sample pad, and an absorbent pad, which are sequentially attached to the bottom plate, and are characterized in that:

The nitrocellulose coating film has a detection line (T) and a quality control line (C).

The test line is coated with an M2 type mitochondrial antigen protein, wherein the quality control line (C) is coated with an antibody (c).

The colloidal gold chromatography liver disease test strip according to claim 1, wherein the antibody (a) is an anti-human IgG monoclonal antibody or an anti-human IgG polyclonal antibody, or a staphylococcal protein A (SPA) or One or more of the Streptococcus G protein (Protein G).

3. The colloidal gold chromatography liver disease test strip according to claim 2, wherein: the antibody

The polyclonal antibody in (a) is one of a murine source, a horse source, a sheep source, a rabbit source or a guinea pig source, and the monoclonal antibody in the antibody is one of a mouse source or a rabbit source.

4. The colloidal gold chromatography liver disease test strip according to claim 1, wherein: the antibody

(b) and antibody (c) are both monoclonal antibodies or polyclonal antibodies, wherein (b) and (c) can specifically bind to form an immune complex.

The colloidal gold chromatography liver disease test strip according to claim 4, wherein the polyclonal antibodies in the antibody (b) and the antibody (c) are a mouse source, a horse source, a sheep source, a rabbit source or a guinea pig. One of the sources, the monoclonal antibody is one of a murine or a rabbit source.

The colloidal gold chromatography liver disease test strip according to claim 1, wherein the protein coated on the detection line is an M2 type mitochondrial antigen protein obtained by expressing a cloning gene.

A method for preparing a colloidal gold chromatography liver test strip, which is composed of a nitrocellulose coating film, a bonding pad, a sample pad, an absorbent pad and a bottom plate, as described in the above claims 1-6. , characterized in that the method comprises the following steps:

Step 1. Preparation of the antigen, the M2 mitochondrial antigen protein is obtained by prokaryotic expression of the cloned gene, and the preparation of the coating membrane is carried out by diluting the antigenic protein with a coating buffer to a concentration of 1.0-

1.5mg/mL, the antibody (c) was diluted to 0.8 ~ 1.5mg / mL, placed on a 1 ~ ΙΟμΙ / cm nitrocellulose membrane, placed at 37 ° C for drying,

And gold standard antibody preparation, is to adjust the colloidal gold pH 7.0-9.0 with 0.1M potassium carbonate, ^: ml of colloidal gold solution slowly added 4 - 25μ ₈ protein, stir for 10-30 min, then add BSA to the final concentration

0. 5 ~ 5%, stir for 10 ~ 30min, centrifuge, discard the supernatant, wash the precipitate with washing solution for 2 ~ 3 times, and resuspend the pellet with one tenth of the initial volume of the preservation solution.

And the preparation of the bonding pad is a polyester film impregnated with the treatment liquid. After the drying, the gold standard antibody (a) and the gold standard antibody (b) are mixed, and then sprayed at a dose of 0.5 to 4 μl/αη. The pretreated polyester film is dried at 25 °C ~ 30O and placed in an environment of 2 °C ~ 8 °C for use.

And the preparation of the sample pad is a glass fiber membrane impregnated with the treatment liquid, and is prepared after drying at 37 ;;

Step 3: Cut the finished material in step 2 into a test strip.

The method for preparing a colloidal gold chromatography liver test strip according to claim 7, wherein: in the step 1, the gold standard antibody is prepared by adjusting the pH of the colloidal gold with 0.1 M potassium carbonate. To 8. 0 ~ 9. 0, slowly add 8 ~ 12μ _β goat anti-human IgG per ml of colloidal gold solution, stir for 10 ~ 30min, then add BSA to the final concentration of 0.5 ~ 1%, stir for 10 ~ 30min, centrifuge Discard the supernatant, wash the pellet with the washing solution 2 to 3 times, and resuspend the pellet with the first one volume of the initial volume of the preservation solution.

The method for preparing a gold-labeled antibody is to adjust the colloidal gold with 0.1 M potassium carbonate according to the method of the present invention. The pH value is 7. 0 ~ 8. 0, ■ ^ ml colloidal gold solution is slowly added to 8 ~ 12μ ₈ rabbit IgG, stirred for 10 ~ 30min, then BSA is added to the final concentration of 0. 5 ~ 1%, stirred for 10 ~ 30min Centrifuge, discard the supernatant, and wash the pellet with the washing solution for 2 to 3 times. The pellet is resuspended in the last one tenth of the initial volume of the preservation solution.

The method for preparing a colloidal gold chromatography liver test strip according to claim 7, wherein: in the step 1, the method for preparing the gold standard SPA is to adjust the colloidal gold H value with 0.1 M carbonate clock. To 5.0 to 6.5, cc of colloidal gold solution was slowly added to 10 ~ 15μ § SPA, stirred for 10 ~ 30min, then added BSA to a final concentration of 0.5 ~ 1%, stirred for 10 ~ 30min, centrifuged, discarded, The precipitate was washed 2 to 3 times with the washing solution, and the pellet was resuspended at the last time with one tenth of the initial volume of the preservation solution, and placed at 4 Torr for use. Then apply the gold standard SPA 0D 20 2 ~ 4 l/cm to the bonding pad, dry and set aside.

The method for preparing a colloidal gold chromatography liver disease test strip according to claim 7, wherein: in the step 1, the gold standard SPA is prepared by adjusting the colloidal gold H value with 0.1 M potassium carbonate. To 7. 0 ~ 8. 0, ^^ ml colloidal gold solution was slowly added to 8 ~ 12μ ₈ mouse anti-human IgG, stirred for 10 ~ 30min, then added BSA to a final concentration of 0.5 ~ 1%, stirred for 10 ~ 30min, centrifuged Abandon the supernatant, will The precipitate is washed 2 - 3 times with the washing solution, and the pellet is resuspended at the last time with a titration of the initial volume of the preservation solution.

4 ° C spare. Then apply the gold standard SPA 0D 30 2 ~ 4μ1/ η to the bonding pad, dry and set aside.

The method for preparing a colloidal gold chromatography liver disease test strip according to claim 7, wherein: in the step 1, the prepared gold standard antibody (a) and the gold standard rabbit IgG are 20~ Mix 40/15-20, use BIO-Dot spray machine to spray on the pretreated polyester film with 2. 0 ~ 4μ1/αη, dry at 25Ό ~ 37Ό, seal the bag, set 2'C ~ 8°C Assemble the adhesive board.

The method for preparing a colloidal gold chromatography liver disease test strip according to claim 7, wherein in the step 3, the width of the cut test strip is preferably 4 mm and 3 mm.