Biotin liposome as well as preparation and application thereof

Abstract

The invention relates to the technical field of syphilis detection kits, in particular to a biotin liposome as well as preparation and application thereof. The preparation method comprises the following steps: firstly, uniformly mixing lecithin, cholesterol, cardiolipin and biotinylated lecithin to obtain lipid; dissolving lipid in a solvent, uniformly mixing, adding a buffer solution, uniformly mixing, and carrying out rotary evaporation to remove the solvent; and finally, adding a buffer solution, uniformly mixing, crushing, cleaning precipitate by using a preservation solution, and filtering the product by using a filter membrane to obtain the biotin liposome with the particle size of less than or equal to 200nm and the PI of less than or equal to 0.2. The biotin liposome provided by the invention is fused with a main antigen substance of a syphilis NTT antibody: cardiolipin; the lecithin and cholesterol are added to stabilize the liposome, and the biotinylated lecithin is convenient to be further tightly linked with the streptavidin in the presence of the biotinylated lecithin, so that the streptavidin immobilized liposome is obtained. The streptavidin immobilized liposome can be further used for preparing a syphilis antibody detection reagent capable of simultaneously detecting syphilis NTT and TT antibodies.

Description

Technical Field

[0001] This invention relates to the field of syphilis test kit technology, and in particular to a biotinylate liposome and its preparation and application. Background Technology

[0002] Syphilis is a chronic, systemic sexually transmitted disease caused by Treponema pallidum. It can be divided into acquired syphilis and congenital syphilis. Acquired syphilis is further divided into early and late syphilis. Early syphilis refers to syphilis within two years of infection with Treponema pallidum, including primary, secondary, and early latent syphilis (also known as early latent syphilis). Late syphilis has a course of ≥2 years and includes late benign syphilis, cardiovascular syphilis, and late latent syphilis (also known as late latent syphilis).

[0003] After infection with syphilis, patients will produce two types of antibodies: syphilis-specific antibodies and syphilis-nonspecific antibodies. Clinical diagnosis of syphilis is mainly based on the information provided by serological tests of these two types of antibodies, namely syphilis-specific antibody test (TT) and syphilis-nonspecific antibody test (NTT).

[0004] The nonspecific titration test (NTT) primarily uses a lipid antigen prepared from a mixture of cardiolipin, lecithin, and cholesterol in a specific ratio. It determines the presence of syphilis nonspecific antibodies in a blood sample by observing the agglutination reaction between the antibody and the antigen. Commonly used NTT methods include the Venereal Disease Research Laboratory Test (VDRL), the Rapid Plasma Reagin (RPR) test, and the Toluidine Red Unheated Serum Test (TRUST). Because current NTT methods rely on visual observation of the agglutination reaction, they suffer from drawbacks such as high dependence on manual operation, difficulty in automating and mass production, and difficulty in tracing and comparing test results from different laboratories.

[0005] Triple-test (TT) primarily uses antigens extracted from or recombinantly synthesized from Treponema pallidum. Commonly used methods include: Treponema pallidum gelatin particle test (TPPA), Treponema pallidum hemagglutination test (TPHA), fluorescent Treponema pallidum antibody absorption test (FTA-ABS), Treponema pallidum chemiluminescence assay (TP-CLIA), and rapid test kits (TP-RT). Due to its high sensitivity and specificity, TT is widely used in clinical practice not only for syphilis diagnosis but also for screening in preoperative examinations and maternal health care. However, generally, once a syphilis-specific serological test is positive, the patient will remain positive for life even after regular and effective treatment. Therefore, TT results cannot distinguish between past and current infections, nor can they be used as an indicator for monitoring treatment efficacy; they must be differentiated in conjunction with NTT and clinical manifestations.

[0006] As early as 1982, the WHO proposed (Treponemal Infections, Technical Report Series 674, Geneva: WHO; 1982.) that using a combined NTT and TT test kit in the screening and diagnosis of syphilis would help further improve the efficiency of syphilis detection. A combined test kit using rapid detection technology can more effectively confirm syphilis infection status in a short time, especially in high-risk groups for sexually transmitted infections, and enable timely medical decisions. In its 2023 Point-of-care tests for sexually transmitted infections: Targetproduct profiles, Geneva: WHO; 2023, the WHO further outlined performance expectations for the NTT / TT combined syphilis test kit. However, because the lipid antigen used in NTT is difficult to modify onto solid surfaces, it is difficult to apply it to commonly used clinical testing methodologies, such as lateral chromatography, latex agglutination, and chemiluminescence. Therefore, developing new NTT reagents is very difficult, and it is challenging to achieve reagent simplification and automation.

[0007] Therefore, it is crucial to provide a technical solution to enable rapid and batch detection of NTT's immune assay. Summary of the Invention

[0008] To address the aforementioned problems, the purpose of this invention is to provide a biotinylated liposome and its preparation and application. This invention is also an immunologically based non-specific antibody detection reagent for syphilis, capable of being used in conjunction with syphilis-specific reagents for rapid syphilis screening, facilitating the rapid advancement of medical strategies during treatment. Furthermore, this invention provides a rapid test strip that can simultaneously detect TT and NTT antibodies within 15-30 minutes through simple operation.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] The first objective of this invention is to provide a method for preparing biotinylated liposomes, comprising the following steps:

[0011] (S1) Lipids are obtained by mixing lecithin, cholesterol, cardiolipin and biotinylated lecithin;

[0012] (S2) Dissolve the lipids obtained in step (S1) in a solvent and mix well, then add a buffer solution and mix well, and remove the solvent by rotary evaporation;

[0013] (S3) After step (S2) is completed, a buffer solution is added, the mixture is stirred and then crushed. The precipitate is washed with a preservation solution and then filtered through a filter membrane to obtain biotinylated liposomes with a particle size ≤200nm and PI ≤0.2.

[0014] In one embodiment of the present invention, in step (S1), the molar ratio of lecithin, cholesterol, cardiolipin and biotinylated lecithin is 7-10:5-25:13-20:1-2.5;

[0015] Preferably, the molar ratio of lecithin, cholesterol, cardiolipin and biotinylated lecithin is 10:25:15:2.5.

[0016] In one embodiment of the present invention, in step (S2), the solvent is a mixed solution of methanol and chloroform;

[0017] The mass ratio of the lipid to the buffer solution is 1:25 to 100.

[0018] In one embodiment of the present invention, in step (S3), the product is filtered sequentially through filter membranes of 1.0 μm, 0.8 μm, 0.4 μm, and 0.2 μm.

[0019] A second objective of this invention is to provide a biotinylated liposome prepared by the above method.

[0020] A third objective of this invention is to provide a method for preparing streptavidin-fixed liposomes, comprising the following steps:

[0021] The above biotin liposomes were mixed with streptavidin to obtain streptavidin-fixed liposomes.

[0022] In one embodiment of the present invention, the ratio of biotin liposomes to streptavidin is 20 mg: 0.5-3 mL.

[0023] The fourth objective of this invention is to provide a streptavidin-fixed liposome, which is prepared by the above method.

[0024] The fifth objective of this invention is to provide an application of streptavidin-immobilized liposomes in the preparation of a rapid syphilis antibody detection reagent.

[0025] In one embodiment of the present invention, the rapid syphilis antibody detection reagent is selected from one of the following: chromatography reagent, enzyme-linked immunosorbent assay (ELISA) reagent, chemiluminescent immunoassay reagent, immunoblotting reagent, rapid plasma reagin ring card test reagent, serological test reagent, dot immunochromatographic reagent, or fluorescent immunoassay reagent.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The biotinylated liposomes provided by the present invention are infused with the main antigenic substance of syphilis NTT antibody: cardiolipin; the addition of lecithin and cholesterol makes it stable and more controllable and easier to obtain in industrial production; biotinylated lecithin is added to the liposomes to link with streptavidin;

[0028] (2) In the prior art, after the preparation of liposomes, the drying and fixation of liposomes becomes a difficult point. Liposomes are usually stored in liquid form under cold storage and added when used. The prior art discloses the fixation of cardiolipin micelles, using anticardiolipin antigen to link with cardiolipin and fix it. The difficulty of this fixation method lies in the difficulty of obtaining and the price of anticardiolipin antigen, while in this application, streptavidin and biotin are tightly linked and the two substances are easier to obtain.

[0029] (3) In the existing NTT detection technology, VDRL detection is time-consuming, requires operators, and is difficult to obtain and preserve raw materials, and there is no alternative solution for the time being. The present invention provides a solid phase detection reagent for NTT, which is more convenient, the signal observation is convenient and immediate, and it can be interpreted within 15 minutes, providing a possibility of automation. At the same time, the present invention can detect syphilis NTT and TT antibodies at the same time, and can detect whether a patient is in the syphilis infection stage with a small number of samples at once. Attached Figure Description

[0030] Figure 1 Characterization diagram of the biotinylated liposomes prepared in Example 1;

[0031] Figure 2 The image shows the characterization of the biotinylated liposomes prepared in Comparative Example 1.

[0032] Figure 3 The characterization diagram of the biotinylated liposomes prepared in Comparative Example 4 is shown.

[0033] Figure 4 The image shows the characterization of the biotinylated liposomes prepared in Comparative Example 5.

[0034] Figure 5 A schematic diagram of the structure of streptavidin-immobilized liposomes;

[0035] Figure 6 This is a schematic diagram of the streaking pattern on the antigen plate;

[0036] Figure 7 This is a schematic diagram of the assembly of chromatography reagents;

[0037] Figure 8 This is a schematic diagram of the color development of the chromatography reagent. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0039] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0040] Example 1

[0041] This embodiment provides a method for preparing biotinylated liposomes, including the following steps:

[0042] (S1) The lipids were obtained by mixing lecithin, cholesterol, 14:0 cardiolipin and biotinylated lecithin (the molar ratio of lecithin, cholesterol, 14:0 cardiolipin and biotinylated lecithin was 2:5:3:0.5, and the specific raw material manufacturers' product numbers are shown in Table 1).

[0043] Table 1 Summary of Raw Material Manufacturer Codes

[0044]

[0045]

[0046] (S2) Dissolve 39.5 mg of lipid obtained in step (S1) in 3.5 mL of solvent (a mixture of chloroform and methanol with a volume ratio of 1:6) and mix well (sonicate in a water bath for 1 min). Then add 1 mL of HEPES solution at 45 °C and mix well (sonicate for 1 min). Remove the solvent by rotary evaporation at 60 °C. Observe that the milky white liquid gradually becomes transparent.

[0047] (S3) After step (S2), add 1 mL of HEPES solution for hydration, mix by ultrasonication in a water bath, and then break down the precipitate (60℃, 10 min). After centrifugation (10000 rpm, 15 min), wash the precipitate with a preservation solution (a mixture of casein, trehalose, sucrose, and 0.02 M HEPES (pH 7.5); the concentration of casein in the mixture is 0.1 wt%, the concentration of trehalose is 10 wt%, the concentration of sucrose is 2.5 wt%, and the concentration of 0.02 M HEPES is 0.2 wt%) (repeat 3 times until the supernatant is relatively clear). Filter the product sequentially at 60℃ using 1.0 μm, 0.8 μm, 0.4 μm, and 0.2 μm filter membranes (filter 21 times for each size) to obtain biotin liposomes.

[0048] The biotinylated liposomes prepared in this embodiment were subjected to performance analysis:

[0049] 1) Particle size measurement using a particle size analyzer revealed that the biotinylated liposomes prepared in this embodiment had a particle size ≤200nm and a PI ≤0.2 (e.g., ...). Figure 1(as shown);

[0050] 2) Testing the antigenicity of biotinylated liposomes in this embodiment using NTT quality control samples: When the volume ratio of NTT strong / weak positive quality control samples to biotinylated liposomes is 10:1, biotinylated liposomes can quickly produce flocculation with the strong / weak positive quality control samples.

[0051] 3) Testing the biotin liposomes in this embodiment using streptavidin: When the concentration ratio of streptavidin to biotin liposomes is 20:0.5, the liquid becomes cloudy and milky white after shaking.

[0052] Comparative Example 1

[0053] This comparative example provides a method for preparing biotinylated liposomes, including the following steps:

[0054] (S1) Lipids are obtained by mixing lecithin, cholesterol, 16:0-18:1 cardiolipin and biotinylated lecithin (the molar ratio of lecithin, cholesterol, 16:0-18:1 cardiolipin and biotinylated lecithin is 2:5:3:0.5).

[0055] (S2) Dissolve 39.5 mg of lipid obtained in step (S1) in 3.5 mL of solvent (a mixture of chloroform and methanol with a volume ratio of 1:6) and mix well (sonicate in a water bath for 1 min). Then add 1 mL of HEPES solution at 45 °C and mix well (sonicate for 1 min). Remove the solvent by rotary evaporation at 60 °C. Observe that the milky white liquid gradually becomes transparent.

[0056] (S3) After step (S2), add 1 mL of HEPES solution for hydration, mix by ultrasonication in a water bath, and then break down the precipitate (60℃, 10 min). After centrifugation (10000 rpm, 15 min), wash the precipitate with a preservation solution (a mixture of casein, trehalose, sucrose, and 0.02 M HEPES (pH 7.5), in which the concentration of casein is 0.1 wt%, the concentration of trehalose is 10 wt%, the concentration of sucrose is 2.5 wt%, and the concentration of 0.02 M HEPES is 0.2 wt%) (repeat 3 times until the supernatant is relatively clear). Filter the product sequentially at 60℃ using 1.0 μm, 0.8 μm, 0.4 μm, and 0.2 μm filter membranes (filter 21 times for each size) to obtain biotin liposomes.

[0057] The performance of the biotinylated liposomes prepared in this comparative example was analyzed:

[0058] 1) Particle size measurement using a particle size analyzer revealed that the biotinylated liposomes prepared in this comparative example had a particle size ≤200 nm and a PI ≤0.2 (e.g., ...). Figure 2 (as shown);

[0059] 2) The antigenicity of biotinylated liposomes in this comparative example was tested using NTT quality control samples: When the volume ratio of NTT weak positive quality control to biotinylated liposomes was 10:1, flocculation could be slowly observed with strong positive quality control samples, while no flocculation occurred with weak positive quality control samples.

[0060] 3) Test the biotin liposomes using streptavidin: When the concentration ratio of streptavidin to biotin liposomes is 20:0.5, the liquid will appear cloudy and milky after shaking.

[0061] Comparative Example 2

[0062] This comparative example provides a method for preparing biotinylated liposomes, which is the same as in Example 1 except that lecithin is not added.

[0063] The results showed that without the addition of lecithin, it was impossible to prepare well-formed liposomes.

[0064] Comparative Example 3

[0065] This comparative example provides a method for preparing biotinylated liposomes, which is the same as in Example 1 except that cholesterol is not added.

[0066] The results showed that the liposomes prepared without the addition of cholesterol were unstable and easily broken.

[0067] Comparative Example 4

[0068] This comparative example provides a method for preparing biotinylated liposomes, which is the same as in Example 1 except that biotinylated lecithin is not added.

[0069] The performance of the liposomes prepared in this comparative example was analyzed:

[0070] 1) Particle size measurement using a particle size analyzer revealed that the biotinylated liposomes prepared in this comparative example had a particle size >200 nm and a PI ≤0.2 (e.g., ...). Figure 3 (as shown);

[0071] 2) The antigenicity of biotinylated liposomes in this comparative example was tested using NTT quality control samples: When the volume ratio of NTT weak positive quality control to biotinylated liposomes was 10:1, flocculation could be slowly observed with strong positive quality control samples, while no flocculation occurred with weak positive quality control samples.

[0072] 3) Testing the biotin liposomes with streptavidin: The liposomes do not react with streptavidin and cannot be used for subsequent fixation operations.

[0073] Comparative Example 5

[0074] This comparative example provides a method for preparing biotinylated liposomes, with the ratio of lecithin, cholesterol, 14:0 cardiolipin and biotinylated lecithin changed to a molar ratio of 27:35:3:2.5, and all other aspects are the same as in Example 1.

[0075] The performance of the liposomes prepared in this comparative example was analyzed:

[0076] 1) Particle size measurement using a particle size analyzer revealed that the biotinylated liposomes prepared in this comparative example had a particle size ≤200 nm and a PI ≤0.3 (e.g., ...). Figure 4 (as shown);

[0077] 2) The biotinylated liposomes in this comparative sample were tested for antigenicity using NTT quality control samples: they did not react with the NTT positive quality control samples, therefore they were not antigenic and could not be used as a subsequent raw material.

[0078] Example 2

[0079] This embodiment provides a method for preparing streptavidin-immobilized liposomes, including the following steps:

[0080] The streptavidin was diluted to 1.5 mg / mL using the biotin liposomes prepared in Example 1 (this concentration can be adjusted to 1.0–2.0 mg / mL as needed), and the mixture was shaken to ensure thorough binding of biotin and streptavidin to the liposomes, resulting in streptavidin-fixed liposomes (see schematic diagram). Figure 5 Show).

[0081] Example 3

[0082] This embodiment provides a method for preparing a chromatography reagent, including the following steps:

[0083] (A1) Preparation of antigen plates:

[0084] (A101) Dilute the recombinant syphilis specific antigen to 0.6 mg / mL using antibody diluent (which can be adjusted to 0.5-0.8 mg / mL as needed) to obtain the recombinant syphilis specific antigen diluent.

[0085] Dilute the goat anti-rabbit antibody to 1.2 mg / mL using antibody diluent (adjust to 0.8–1.5 mg / mL as needed) to obtain the goat anti-rabbit antibody diluent.

[0086] (A102) Liposomes immobilized with streptavidin prepared in Example 2, syphilis recombinant specific antigen diluent, and goat anti-rabbit antibody diluent were respectively streaked onto nitrocellulose membranes (streaking volume: 30 μL). Figure 6 (As shown), dry at 37℃ for 3 hours to obtain antigen plates.

[0087] (A2) Preparation of the gold pad:

[0088] Adjust the colloidal gold to approximately pH 6.5, mix it with the syphilis recombinant specific antigen dilution solution by shaking, and seal it to obtain the first solution.

[0089] Adjust the colloidal gold to approximately pH 6.5, mix it with mouse anti-human solution by shaking, and seal it to prepare the second solution.

[0090] Adjust the colloidal gold to approximately pH 6.5, mix it with rabbit IgG by shaking, block it, and use it as the third solution;

[0091] The solutions obtained above were mixed thoroughly and then evenly coated onto glass fibers. Taking a 0.6cm*30cm glass fiber strip as an example, 100μL of the first solution was used (which can be adjusted to 80-120μL according to the actual situation), 167μL of the second solution was used (which can be adjusted to 150-180μL according to the actual situation), and 167μL of the third solution was used (which can be adjusted to 100-170μL according to the actual situation). The total volume was brought up to 1mL with diluent, and the mixture was dried at 37℃ for 3h to obtain the gold pad (the manufacturer's product number of the raw material is shown in Table 2).

[0092] Table 2 Summary of Raw Material Manufacturer Codes

[0093] brand Item number Syphilis recombinant specific antigen (antigen plate) Feipeng Bio TP-AG-G5-026 Goat anti-rabbit antibody (antigen plate) ARista ABGAR-0500 Recombinant syphilis specific antigen (gold pad) Feipeng Bio TP-AG-G5-027 Mouse anti-human antibody (gold pad) Boyue Biotechnology H-IgG102 Rabbit IgG antibody (gold pad) ARista AGRIG-0100

[0094] (A3) Assembly of antigen plates:

[0095] according to Figure 7 The structure shown assembles the antigen plate, wherein the sample pad, gold pad, antigen plate and absorbent paper are stacked at the joints.

[0096] The testing and interpretation methods for the chromatography reagents prepared in this embodiment are as follows:

[0097] Add 20 μL of sample (whole blood, plasma or serum) to the sample pad, then add 60 μL of buffer solution, let stand for 15 min, and wait for the liquid to flow towards the absorbent paper;

[0098] When the sample contains NTT antibody, the colloidal gold-labeled mouse anti-human antibody will bind to NTT, and the NTT antibody will be captured by the liposomes on the NTT detection line, thus displaying the color of the colloidal gold carried by the mouse anti-human antibody.

[0099] When a sample contains TT antibody, the colloidal gold-labeled TT antigen will bind to the TT antibody. The TT antibody will be captured by the TT antigen on the TT detection line and will show the color of the colloidal gold carried by the TT antigen.

[0100] When a sufficient amount of sample flows through the control line, the colloidal gold-labeled rabbit IgG will be captured by the goat anti-rabbit antibody on the control line, displaying the color of the colloidal gold carried by the rabbit IgG (e.g., ...). Figure 8 (As shown).

[0101] Example 4

[0102] This embodiment provides a method for preparing immunoturbidimetric reagents, including the following steps:

[0103] (A1) Reagent preparation:

[0104] Prepare reagent 1 according to the table below:

[0105] Table 3 Reagent Preparation Table

[0106]

[0107] Prepare reagent 2: R2 dilution solution according to the table below:

[0108] Table 4 Reagent Preparation Table

[0109]

[0110] (A2) Preparation of biotinylated liposome concentrate (R2 concentrate) (A201) Activation of microspheres

[0111] Prepare the activation solution according to the table below:

[0112] Table 5 Reagent Preparation Table

[0113]

[0114] Activate the system according to the proportions shown in the table below:

[0115] Table 6 Reagent Preparation Table

[0116]

[0117] Mix all the reagents in Table 6 thoroughly. After mixing, place the mixture on a mixer and rotate it for 30 minutes at 50 rpm to fully activate the microspheres and obtain the activated system.

[0118] Preparation of (A202) latex microspheres coupled with streptavidin

[0119] a. Coupling: Under stirring, the activated system and streptavidin were mixed, wherein the volume ratio of the activated system to streptavidin was 10:1. The mixture was stirred on a magnetic stirrer at 400 rpm for 2 hours at room temperature to obtain the coupling system.

[0120] b. Closure: Under stirring, add 5% sodium caseinate to the coupling system, wherein the volume ratio of the coupling system to the 5% sodium caseinate is 10:1, to obtain a closed system;

[0121] c. Homogenization: The closed system was homogenized at 600 Bar pressure using a homogenizer. After homogenization, the system was stirred on a magnetic stirrer for 30 minutes to obtain latex microspheres coupled with streptavidin.

[0122] (A203) Washing and filtration of latex microspheres coupled with streptavidin: After washing and filtration with 5 times the amount of latex microspheres coupled with streptavidin at a pH of 7.40±0.10 and a concentration of 40mM Tris-HCl, the latex microspheres were redispersed to the original volume using R2-1 to obtain the washed and filtered latex microsphere dispersion.

[0123] (A204) Re-sealing: Add biotinylated liposomes to the washed and filtered latex microsphere dispersion (the volume ratio of the washed and filtered latex microsphere dispersion to the biotinylated liposomes is 10:1), mix thoroughly, and stir thoroughly to obtain a biotinylated liposome-labeled latex microsphere concentrate (R2 concentrate).

[0124] (A3) Preparation of R2 reagent

[0125] The R2 reagent is obtained by diluting the R2 concentrate with R2-1 (the volume ratio of R2-1 to the R2 concentrate is 4:1).

[0126] The testing and interpretation methods for the chromatography reagents prepared in this embodiment are as follows:

[0127] Using the Fosun Diagnostics F-C800p / F-C800M fully automated biochemical analyzer, set the following program:

[0128]

[0129] A standard curve was established using quality control materials, and the NTT antibody concentration was determined. A concentration >0.625 IU / ml was considered positive.

[0130] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for preparing biotinylated liposomes, characterized in that, Includes the following steps: (S1) Lipids are obtained by mixing lecithin, cholesterol, cardiolipin and biotinylated lecithin; (S2) Dissolve the lipids obtained in step (S1) in a solvent and mix well, then add a buffer solution and mix well, and remove the solvent by rotary evaporation; (S3) After step (S2) is completed, a buffer solution is added, the mixture is stirred and then crushed. The precipitate is washed with a preservation solution and then filtered through a filter membrane to obtain biotinylated liposomes with a particle size ≤200nm and PI ≤0.

2.

2. The method for preparing biotinylated liposomes according to claim 1, characterized in that, In step (S1), the molar ratio of lecithin, cholesterol, cardiolipin and biotinylated lecithin is 7-10:5-25:13-20:1-2.

5.

3. The method for preparing biotinylated liposomes according to claim 1, characterized in that, In step (S2), the solvent is a mixed solution of methanol and chloroform; The mass ratio of the lipid to the buffer solution is 1:25 to 100.

4. The method for preparing biotinylated liposomes according to claim 1, characterized in that, In step (S3), the product is filtered sequentially through filter membranes of 1.0 μm, 0.8 μm, 0.4 μm, and 0.2 μm.

5. A biotinylated liposome, characterized in that, It is prepared by any of the methods described in claims 1 to 4.

6. A method for preparing streptavidin-immobilized liposomes, characterized in that, Includes the following steps: The biotin liposomes described in claim 5 were mixed with streptavidin to obtain streptavidin-fixed liposomes.

7. The method for preparing streptavidin-immobilized liposomes according to claim 6, characterized in that, The ratio of biotin liposomes to streptavidin is 20 mg: 0.5–3 mL.

8. A streptavidin-immobilized liposome, characterized in that, It is prepared by the method described in claim 7.

9. The use of streptavidin-immobilized liposomes as described in claim 8 in the preparation of syphilis antibody detection reagents.

10. The application according to claim 9, characterized in that, The syphilis antibody detection reagent is selected from one of the following: chromatography reagent, enzyme-linked immunosorbent assay (ELISA) reagent, chemiluminescent immunoassay reagent, immunoblotting reagent, dot immunochromatographic reagent, or fluorescent immunoassay reagent.

Images