CN101858914A - Syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip and preparation method thereof - Google Patents

Abstract

A syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strip and a preparation method thereof relate to a syphilis-specific total antibody detection reagent. It is equipped with a carrier plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane, a syphilis-specific total antibody detection line, a control line and an absorption pad. Preparation of recombinant syphilis antigen TPN17 and TPN47 nitrocellulose membrane spotting; preparation of colloidal gold; labeling of colloidal gold and TPN17, TPN47; preparation of immunochromatographic detection strips. It can be used for the detection of syphilis-specific total antibodies in samples such as whole blood, serum, plasma and cerebrospinal fluid. In the detection, 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

Syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip and preparation method thereof

technical field

The invention relates to a syphilis-specific total antibody detection reagent, in particular to a syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip and a preparation method thereof.

Background technique

Syphilis is a sexually transmitted disease caused by Treponema pallidum (TP), and its pathogen is Treponema pallidum, which belongs to the family Treponemaceae. Treponema pallidum is mainly transmitted through sexual contact, blood transfusion, wound or placenta. Treponema pallidum enters the blood from the lymph nodes near the infected area, spreads throughout the body, and affects almost all tissues and organs of the body. The World Health Organization (WHO) once predicted optimistically: "Due to the availability of highly sensitive detection methods and efficient treatment options, syphilis is a sexually transmitted disease that can be successfully controlled through public health measures." Regrettably, syphilis is still a worldwide public health problem, and there is a lack of effective administrative control measures. There are about 12 million patients worldwide every year, including 600,000 pregnant women. (See: Health Protection Agency Center for Infections. International Encyclopedia of Public Health-Syphilis[M]. London, UK: Health Protection Agency Centre, 2008, 289-297) In fact, the current status of syphilis infection may be worse than imagined pessimistic.

The survey found that syphilis infection has been widely present in the general population.

Treponema pallidum cannot be cultured in vitro, and the diagnosis and epidemiological investigation of syphilis mainly rely on serological tests, including two types of specific antibody and reagin detection. Syphilis-specific antibody IgM (TP-IgM) and IgG (TP-IgG) antibodies are produced after 2 weeks and 4 weeks, respectively, and even if the patient receives adequate treatment, they can still exist for a long time, and even persist for life (see: Luis J F , Felipe U S, Santa G C, et al.Evaluation of a rapid strip and a particle agglutinationtests for syphilis diagnosis[J].Diagnostic Microbiology and Infectious Disease, 2007, 59:123-126); while another antibody substance reacts The hormone is produced late, usually 5 to 7 weeks after infection (see: Lin Yueyuan. The value and clinical related issues of TPPA and TRUST in the diagnosis of syphilis [J]. Journal of Radioimmunology, 2009, 22(3) : 295-297.), and late syphilis, late syphilis treatment and latent syphilis may be negative. Therefore, the positive rate and sensitivity of syphilis-specific antibodies were significantly higher than that of reagin. TP-IgM is the first specific antibody that appears in the body after syphilis infection. As long as there are live Treponema pallidum, its TP-IgM will be maintained at a certain level. Martina H et al (see: Martina H, Daan W N, Mart M, et al. Comparison of a Treponema pallidum IgM immunoblot with a 19S fluorescent treponemal antibody absorption test for the diagnosis of congenital syphilis[J].Diagnostic and Microbiology, 2 59:61-66.) believed that TP-IgM is a serological marker of early infection and activity of syphilis, Li Burong et al. Journal of the Fourth Military Medical University, 2007, 28(16): 1495-1497) believes that TP-IgM, like TP-DNA, represents an index of syphilis infectivity. On the premise of excluding the recent anti-syphilitic treatment, if TP-IgM does not turn negative, it indicates that Treponema pallidum may remain in the body or the treatment is not complete. TP-IgM negative turned positive again during follow-up, indicating re-infection with syphilis (see: Rawstron SA, Mehta S, Bromberg K, et al. syphilis [J]. Sex Transm Dis, 2004, 31(2): 123-126). Although a negative TP-IgM does not completely rule out infectivity, a positive TP-IgM must suggest that the patient is infectious.

Early serological methods used intact Treponema pallidum as an antigen, and TP for research and diagnosis was obtained by infecting rabbit testis with TP. This method was expensive, and the amount of TP obtained was small, impure (mixed with host protein), and other pathogens There is cross-reactivity, so false positives also occur from time to time. With the popularity of molecular biology techniques and the successive cloning of Treponema pallidum antigens, more and more recombinant antigens have been used in syphilis experiments. At present, the TP antigens that have been studied more include TPN17, TPN47, TPN15, TPN44.5, TPN36, TP0453, TP0684 and TPr family. The recombinant antigen prepared by recombinant DNA technology can overcome the shortcomings of the complete TP antigen, and can quickly and economically prepare unlimited specific recombinant TP antigen.

Syphilis-specific antibody detection is a confirmatory test for syphilis, including TPHA, TPPA, ELISA, FTA-ABS and Western-blot, etc., with high specificity. However, in the face of severe forms of prevention and control, not only specific and accurate detection methods are needed, but also a simpler and faster reagent for screening is needed, so as to provide countermeasures for clinical and disease prevention and control.

Contents of the invention

The purpose of the present invention is to provide a syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strip and a preparation method thereof.

The syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip of the present invention is provided with carrier plate, sample pad, colloidal gold pad, nitrocellulose membrane (NC membrane), syphilis-specific total antibody detection line, control line and absorption pad.

The sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad are pasted on the surface of the carrier plate in sequence, one end of the sample pad is set on one end of the colloidal gold pad, and the other end of the colloidal gold pad is set on one end of the nitrocellulose membrane One end of the absorbent pad is set on the other end of the nitrocellulose membrane, and the syphilis-specific total antibody detection line and the control line are sequentially set on the nitrocellulose membrane; the syphilis-specific total antibody detection line is coated with anti-human Ig monoclonal antibody Or syphilis-specific antigen TPN17 and/or syphilis-specific antigen TPN47, coated with IgG antibodies of goat anti-syphilis antigens TPN17 and TPN47 at the control line.

The carrier board can be a PVC board.

The preparation method of the syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip comprises the following steps:

1) Preparation of recombinant syphilis antigens TPN17 and TPN47

Using gene cloning technology, PCR amplified DNA encoding Treponema pallidum antigen, and inserted it into Escherichia coli to express it, and obtained syphilis recombinant antigens TPN17 and TPN47

2) Spotting on nitrocellulose membrane

Anti-human Ig monoclonal antibody is coated on the syphilis-specific total antibody detection line, and sheep anti-syphilis antigen TPN17 and TPN47 IgG antibody are coated on the control line, and dried;

3) 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;

4) Labeling of colloidal gold and TPN17, TPN47

Colloidal gold and syphilis-specific antigen TPN17 labeling: Take 10ml of colloidal gold, adjust to pH5.4 with 0.1mol/L NaOH, add 100μg TPN17, mix well, let stand for 5min, add 1ml of 5% BSA and mix well, 4℃, 10000r Centrifuge for 1 hour at 1/min, 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 TBS to obtain colloidal gold-labeled TPN17 antigen;

The labeling of colloidal gold and syphilis-specific antigen TPN47 is the same as above to obtain the TPN47 antigen labeled with colloidal gold;

Colloidal gold-labeled TPN17 antigen and colloidal gold-labeled TPN47 antigen are mixed, evenly coated on the glass fiber membrane, and dried to prepare a colloidal gold pad;

5) Preparation of immunochromatographic test strips

Paste the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad on the surface of the carrier plate in sequence, set one end of the sample pad on one end of the colloidal gold pad, and set the other end of the colloidal gold pad on one end of the nitrocellulose membrane One end of the absorbent pad is set on the other end of the nitrocellulose membrane, and the syphilis-specific total antibody detection line and the control line are sequentially set on the nitrocellulose membrane; the syphilis-specific total antibody detection line is coated with anti-human Ig monoclonal The antibody is coated with IgG antibodies of goat anti-syphilis antigens TPN17 and TPN47 at the control line, and cut into strips with a strip cutter to obtain colloidal gold immunochromatographic detection reagent strips for syphilis-specific total antibodies.

In step 2), the concentration of the anti-human Ig monoclonal antibody is 1 to 4 mg/mL, and the IgG antibody of goat anti-syphilis antigen TPN17 and TPN47 is composed of anti-TPN17-IgG antibody and anti-TPN47-IgG antibody in a volume ratio of 1: 1 mixed, the final concentration is 1-4 mg/mL; the sample volume of the three is 1 μL/cm.

In step 3), the concentration of trisodium citrate may be 2%.

In step 4), the TPN17 antigen labeled with colloidal gold and the TPN47 antigen labeled with colloidal gold are mixed, preferably the TPN17 antigen labeled with colloidal gold and the TPN47 antigen labeled with colloidal gold are mixed in a volume ratio of 1: (0.2～5) ; The drying temperature may be 37°C.

The invention provides a colloidal gold immunochromatography detection reagent strip for syphilis-specific total antibodies established by colloidal gold immunochromatography technology, which can be used for detection of syphilis-specific total antibodies in samples such as whole blood, serum, plasma, and cerebrospinal fluid. In the detection, 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 structure and composition of an embodiment of the syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strip of the present invention.

Figure 2 is a schematic diagram of the experimental results model. In Figure 2, (1) is a schematic diagram before use, (2) is an invalid test (product quality problem), (3) the virus-specific total antibody is negative, and (4) the virus-specific total antibody is positive.

Detailed ways

The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

Referring to Fig. 1, the colloidal gold immunochromatographic detection reagent strip embodiment of syphilis-specific total antibody of the present invention is provided with carrier plate 1, sample pad 2, colloidal gold pad 3, nitrocellulose membrane (NC membrane) 4, syphilis-specific Total antibody detection line 5, control line 7 and absorbent pad 8.

Sample pad 2, colloidal gold pad 3, nitrocellulose membrane 4 and absorbent pad 8 are pasted on the upper surface of carrier plate 1 in sequence, one end of sample pad 2 is arranged on one end of colloidal gold pad 3, the other end of colloidal gold pad 3 Set on one end of the nitrocellulose membrane 4, one end of the absorbent pad 8 is set on the other end of the nitrocellulose membrane 4, and the syphilis-specific total antibody detection line 5 and the control line 8 are successively set on the nitrocellulose membrane 4; Anti-human Ig monoclonal antibody or syphilis-specific antigen TPN17 and/or syphilis-specific antigen TPN47 was coated at the 5th place of the sex total antibody detection line, and IgG antibody of goat anti-syphilis antigens TPN17 and TPN47 was coated at the 7th place of the control line.

The carrier board 1 is made of PVC board.

The preparation method of the syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip comprises the following steps:

1) Preparation of recombinant syphilis antigens TPN17 and TPN47

Using gene cloning technology, PCR amplified the DNA encoding Treponema pallidum antigen, and inserted it into Escherichia coli to express it, and obtained syphilis recombinant antigens TPN17 and TPN47.

2) Spotting on nitrocellulose membrane

Anti-human Ig monoclonal antibody is coated on the syphilis-specific total antibody detection line, goat anti-syphilis antigen TPN17 and TPN47 IgG antibody are coated on the control line, and dried, and the concentration of the anti-human Ig monoclonal antibody is 1 ~ 4mg/mL, goat anti-syphilis antigens TPN17 and TPN47 IgG antibodies are mixed by anti-TPN17-IgG antibody and anti-TPN47-IgG antibody in a volume ratio of 1:1, and the final concentration is 1-4mg/mL; the sample volume of the three is 1 μL/cm.

3) 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 2.0 mL of 1% trisodium citrate aqueous solution under magnetic heating and stirring, and continue heating until the solution turns wine-colored. After cooling, place it in a brown bottle and store it in a 4°C refrigerator for later use. The concentration of trisodium citrate can be 2%.

4) Labeling of colloidal gold and TPN17, TPN47

Colloidal gold and syphilis-specific antigen TPN17 labeling: Take 10ml of colloidal gold, adjust to pH5.4 with 0.1mol/L NaOH, add 100μg TPN17, mix well, let stand for 5min, add 1ml of 5% BSA and mix well, 4℃, 10000r Centrifuge for 1 hour at 1000 rpm, discard the supernatant, dissolve the precipitate in 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 TBS to obtain colloidal gold-labeled TPN17 antigen.

Colloidal gold and syphilis-specific antigen TPN47 were labeled in the same manner as above to obtain colloidal gold-labeled TPN47 antigen.

The colloidal gold-labeled TPN17 antigen and the colloidal gold-labeled TPN47 antigen are mixed, evenly coated on the glass fiber membrane, and dried to prepare a colloidal gold pad.

The colloidal gold-labeled TPN17 antigen and the colloidal gold-labeled TPN47 antigen are mixed, preferably the colloidal gold-labeled TPN17 antigen and the colloidal gold-labeled TPN47 antigen are mixed in a volume ratio of 1: (0.2～5); The temperature may be 37°C.

5) Preparation of immunochromatographic test strips

Paste the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad on the surface of the carrier plate in sequence, set one end of the sample pad on one end of the colloidal gold pad, and set the other end of the colloidal gold pad on one end of the nitrocellulose membrane One end of the absorbent pad is set on the other end of the nitrocellulose membrane, and the syphilis-specific total antibody detection line and the control line are sequentially set on the nitrocellulose membrane; the syphilis-specific total antibody detection line is coated with anti-human Ig monoclonal The antibody is coated with IgG antibodies of goat anti-syphilis antigens TPN17 and TPN47 at the control line, and cut into strips with a strip cutter to obtain colloidal gold immunochromatographic detection reagent strips for syphilis-specific total antibodies.

The clinical specimens tested by immunochromatography are given below:

Referring to Figure 2, take 5-80 μL of the specimen to be tested (whole blood, serum, plasma, cerebrospinal fluid) and let it stand for 20 minutes to observe the results. If only a purple band appears in the control area C of the test strip, it is judged as negative; if a purple band appears in both the test area T and the control area C, it is judged as positive; There is no purple band in area C, which is an invalid result.

The performance verification of the syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip is given below:

1) Appearance inspection: The white coated reaction film is smooth, clean, free of pollution spots, cracks, adhesive tape without glue opening, and without cutting and beveling.

2) Coincidence rate of positive specimens: 50 copies of positive reference sera with different titers positive for TP-total antibody were tested by syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strips, and the positive coincidence rate was calculated. The positive reference serum was determined by clinical specimens determined by the TPPA (Fuji Corporation, Japan) method.

3) Negative specimen coincidence rate: use 50 negative reference sera to test, and calculate the positive coincidence rate. The negative reference serum was determined by clinical specimens determined by the TPPA (Fuji Corporation, Japan) method.

4) Sensitivity detection: use the indoor quality control serum of the Ministry of Health for detection, the minimum detection limit should be less than or equal to 4NCU/mL, which is equivalent to TPPA (Fuji Corporation, Japan).

5) Intra-batch variation: The same batch of syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strips is tested with characteristic serum, and the positive serum test results are required to show that the color of the color band is consistent, and the negative serum test results are negative.

6) Batch-to-batch differences: Different batches of syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strips are tested with characteristic serum. It is required that the positive serum test results show that the color of the color band is consistent, and the negative serum test results are negative.

7) Interference test: The test result is not interfered by specimen hemolysis (n=50), lipemia (n=50) and jaundice (n=50). Serum (or plasma) comes from the applicant's clinical specimens.

8) Cross-reaction: Use syphilis-specific total antibody colloidal gold immunochromatography detection reagent strips to test for systemic lupus erythematosus (n=30), rheumatoid disease (n=30), immune hepatitis (n=30), etc. In the detection of immune system diseases, no cross-reactivity was found. The sera of autoimmune diseases come from clinically diagnosed patients of the applicant.

9) Stability test: Apply the Arrhenius rule, place the reagent strip at 37°C for 20 days and test it. There is no significant change in the above indicators. Ensure that the finished product is stored under dry conditions at room temperature, and the validity period is 18 months.

Specific examples are given below.

Example 1

Coat the anti-human Ig monoclonal antibody on the nitrocellulose membrane (NC membrane) total detection line, and coat the goat anti-syphilis antigen (TPN17 and TPN47) IgG antibody on the control line, dry at room temperature, seal and store at room temperature for future use. Wherein, the concentration of anti-human Ig monoclonal antibody is 1mg/mL, goat anti-syphilis antigen (TPN17 and TPN47) IgG antibody is mixed by volume ratio 1: 1 by anti-TPN17-IgG antibody and anti-TPN47-IgG antibody, and its final concentration is 1mg/mL; the sample volume of both is 1μL/cm.

The purified gold-labeled recombinant syphilis antigens TPN17 and TPN47 were mixed at a volume ratio of 1:1, evenly spread on glass fiber paper, dried at 37°C, prepared into a gold colloid pad, and sealed for later use. The solid-phase fiber membrane combined with colloidal gold, glass fiber, absorbent paper, etc. are combined in a certain order through the PVC self-adhesive bottom plate, and cut into a certain width of detection strips with a strip cutter. Put the syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strip together with the desiccant into an aluminum foil bag, seal it with the machine, and keep it sealed.

Take 50 μL of the serum of the specimen to be tested, add the sample to the sample area of the syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip, and let it stand for 20 minutes to observe the results. If only a purple-red band appears in the control area of the syphilis-specific total antibody colloidal gold immunochromatography test reagent strip, 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; After the test, no purple-red band appears in the detection area and the control area, which is an invalid result.

Example 2

Similar to Example 1, the difference is that the gold colloid pad is only composed of TPN17 and does not contain TPN47. Result judgment is identical with embodiment 1.

Example 3

Similar to Example 1, the difference is that the gold colloid pad is only composed of TPN47 and does not contain TPN17. Result judgment is identical with embodiment 1.

Example 4

Similar to Example 1, the difference is that the sample to be tested is a cerebrospinal fluid sample, and the result judgment is the same as that of Example 1.

Example 5

Performance verification test: Prepare the rapid detection reagent for syphilis-specific total antibodies according to the scheme in Example 1, and then perform performance verification.

1) Appearance inspection: The white coated reaction film is smooth, clean, free of pollution spots, no cracks, no adhesive tape, the width of the reagent strip is 3±0.1mm, and there is no bevel phenomenon.

2) Conformity rate of positive specimens: 50 TP-total antibody positive reference sera determined by Treponema pallidum-specific antibody gelatin agglutination test (TPPA) (Fuji Corporation, Japan) were detected by syphilis-specific total antibody colloidal gold immunochromatography The reagent strips detected 50 positive samples of TP-total antibody, and the coincidence rate of positive samples was 100%.

3) Negative specimen coincidence rate: 50 copies of treponema pallidum specific antibody gelatin agglutination test (TPPA) (Fuji Co., Ltd., Japan) negative reference sera were not detected positive by colloidal gold immunochromatographic detection reagent strip for syphilis specific total antibody Specimen, the negative specimen coincidence rate was 100%.

4) Sensitivity detection: use the indoor quality control serum of the Ministry of Health for detection, the minimum detection limit should be less than or equal to 4NCU/mL, which is equivalent to TPPA (Fuji Corporation, Japan).

5) Intra-batch differences: the same batch of syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strips, positive clinical samples determined by the detection of characteristic positive serum (TPPA (Fuji Co., Ltd.) Low serum) detection, the same titer detection results show that the color shades of the color bands are consistent, and the negative serum detection results are negative.

6) Batch-to-batch differences: different batches of syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strips, positive clinical samples determined by the detection of characteristic positive serum (TPPA (Fuji Co., Ltd., Japan)) Low serum) detection, the same titer detection results show that the color shades of the color bands are consistent, and the negative serum detection results are negative.

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

8) Cross-reaction: Use syphilis-specific total antibody colloidal gold immunochromatography detection reagent strips to test for systemic lupus erythematosus (n=30), rheumatoid disease (n=38), immune hepatitis (n=40), etc. In the detection of immune system diseases, no cross-reactivity was found.

9) Stability test: Put the syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strip at 37°C for 20 days and then detect it. The above indicators have no significant changes.

The detection of the present invention is carried out on a syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strip, and the detection of the syphilis-specific total antibody is realized in two ways.

Method 1: Using colloidal gold immunochromatography technology, the total antibody detection line and the control line on the nitrocellulose membrane were respectively coated with anti-human Ig monoclonal antibody [or syphilis-specific antigen (TPN17 and/or TPN47)] and sheep IgG antibodies against syphilis antigens (TPN17 and/or TPN47). The purified gold-labeled syphilis recombinant antigens TPN17 and TPN47 are mixed in a certain ratio, pre-coated on glass fiber paper, dried, and prepared into gold colloid pads, supplemented with appropriate sample processing pads, combined reagent strips (assembled The method is shown in Figure 1). When a positive sample is detected, the syphilis-specific total antibody in the sample combines with the colloidal gold-labeled recombinant antigen TPN17 and/or TPN47 to form an immune complex. Due to chromatography, the complex moves forward along the direction of the absorbent paper. When passing through the detection line, ① TP total antibody immune complex combines with pre-coated anti-human Ig monoclonal antibody [or syphilis-specific antigen (TPN17 and/or TPN47)] to form "Au-TPN17 (and/or TPN47)-specific Anti-syphilis total antibody-anti-human Ig monoclonal antibody-solid phase material" or "Au-TPN17 (and/or TPN47)-specific anti-syphilis total antibody-anti-human Ig monoclonal antibody-solid phase material" sandwich Agglomeration and color development; ②The principle of free gold-labeled antibody combined with goat anti-syphilis antigen (TPN17 and TPN47) antibodies at the control line to enrich and develop color. Negative specimens develop color only at the control line.

Method 2: Swap the coated antigen and antibody of method 1: syphilis recombinant antigen (TPN17/TPN47 combination) and human IgG antibody were respectively coated on the total antibody detection line and control line on the nitrocellulose membrane, and the gold-labeled anti- Human Ig monoclonal antibody pre-coated on glass fiber paper.

The syphilis-specific total antibody colloidal gold immunochromatographic detection reagent strip prepared by the two methods can be used for the detection of syphilis-specific total antibody in samples such as whole blood, serum, plasma, and cerebrospinal fluid. The amount of sample is small and no special equipment is required. , and 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 (8)

1. The colloidal gold immunochromatographic detection reagent strip for syphilis-specific total antibody is characterized in that it is provided with carrier plate, sample pad, colloidal gold pad, nitrocellulose membrane, syphilis-specific total antibody detection line, control line and absorption pad; The sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad are pasted on the surface of the carrier plate in sequence, one end of the sample pad is set on one end of the colloidal gold pad, and the other end of the colloidal gold pad is set on one end of the nitrocellulose membrane One end of the absorbent pad is set on the other end of the nitrocellulose membrane, and the syphilis-specific total antibody detection line and the control line are sequentially set on the nitrocellulose membrane; the syphilis-specific total antibody detection line is coated with anti-human Ig monoclonal antibody Or syphilis-specific antigen TPN17 and/or syphilis-specific antigen TPN47, coated with IgG antibodies of goat anti-syphilis antigens TPN17 and TPN47 at the control line. 2. syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip as claimed in claim 1, is characterized in that said carrier plate is a PVC plate. 3. the preparation method of syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip as claimed in claim 1, is characterized in that comprising the following steps: 1) Preparation of recombinant syphilis antigens TPN17 and TPN47 Using gene cloning technology, PCR amplified DNA encoding Treponema pallidum antigen, and inserted it into Escherichia coli to express it, and obtained syphilis recombinant antigens TPN17 and TPN47; 2) Spotting on nitrocellulose membrane Anti-human Ig monoclonal antibody was coated on the syphilis-specific total antibody detection line, and goat anti-syphilis antigen TPN17 and TPN47 IgG antibody were coated on the control line, and dried; 3) 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; 4) Labeling of colloidal gold and TPN17, TPN47 Colloidal gold and syphilis-specific antigen TPN17 labeling: Take 10ml of colloidal gold, adjust to pH5.4 with 0.1mol/L NaOH, add 100μg TPN17, mix well, let stand for 5min, add 1ml of 5% BSA and mix well, 4℃, 10000r Centrifuge for 1 hour at 1/min, 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 TBS to obtain colloidal gold-labeled TPN17 antigen; The labeling of colloidal gold and syphilis-specific antigen TPN47 is the same as above to obtain the TPN47 antigen labeled with colloidal gold; Colloidal gold-labeled TPN17 antigen and colloidal gold-labeled TPN47 antigen are mixed, evenly coated on the glass fiber membrane, and dried to prepare a colloidal gold pad; 5) Preparation of immunochromatographic test strips Paste the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad on the surface of the carrier plate in sequence, set one end of the sample pad on one end of the colloidal gold pad, and set the other end of the colloidal gold pad on one end of the nitrocellulose membrane One end of the absorbent pad is set on the other end of the nitrocellulose membrane, and the syphilis-specific total antibody detection line and the control line are sequentially set on the nitrocellulose membrane; the syphilis-specific total antibody detection line is coated with anti-human Ig monoclonal The antibody is coated with IgG antibodies of goat anti-syphilis antigens TPN17 and TPN47 at the control line, and cut into strips with a strip cutter to obtain colloidal gold immunochromatographic detection reagent strips for syphilis-specific total antibodies. 4. the preparation method of syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip as claimed in claim 3 is characterized in that in step 2) in, the concentration of described anti-human Ig monoclonal antibody is 1～4mg/ mL. 5. the preparation method of syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip as claimed in claim 3 is characterized in that in step 2) in, the IgG antibody of goat anti-syphilis antigen TPN17 and TPN47 is formed by anti-TPN17-IgG The antibody and the anti-TPN47-IgG antibody were mixed at a volume ratio of 1:1, and the final concentration was 1-4 mg/mL; the sample volume of the three was 1 μL/cm. 6. the preparation method of syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip as claimed in claim 3, is characterized in that in step 3), the concentration of described trisodium citrate is 2%. 7. the preparation method of syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip as claimed in claim 3, is characterized in that in step 4) in, described TPN17 antigen of colloidal gold labeling and colloidal gold labeling TPN47 The antigen mixing is that colloidal gold-labeled TPN17 antigen and colloidal gold-labeled TPN47 antigen are mixed at a volume ratio of 1:0.2-5. 8. The preparation method of the syphilis-specific total antibody colloidal gold immunochromatography detection reagent strip as claimed in claim 3, characterized in that in step 4), the drying temperature is 37°C.

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