CN111220808A

CN111220808A - Kit for detecting TP spirochete nonspecific antibody

Info

Publication number: CN111220808A
Application number: CN201911219008.6A
Authority: CN
Inventors: 李博; 王新明; 孙冯博; 娄本光
Original assignee: Autobio Diagnostics Co Ltd
Current assignee: Autobio Diagnostics Co Ltd
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2020-06-02

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to a kit for detecting a TP spirochete non-specific antibody. The invention improves the coupling method of the cardiolipin, the lecithin, the cholesterol and the magnetic particles, so that the magnetic particles can be better combined with a target object in a sample and matched with the enzyme-labeled antibody, thereby obtaining better detection effect. The kit provided by the invention comprises an enzyme-labeled antibody and the magnetic particles coated with the cardiolipin, the lecithin and the cholesterol prepared by the invention, can realize automatic detection of a sample, and has the characteristics of simple and accurate operation and strong anti-interference capability.

Description

Kit for detecting TP spirochete nonspecific antibody

Technical Field

The invention relates to the technical field of in-vitro diagnosis, in particular to a kit for detecting a TP spirochete non-specific antibody.

Background

Syphilis is a Sexually Transmitted Disease (STD) caused by treponema pallidum. More than 10 ten thousand cases of adult syphilis are reported every year around the world. Failure to receive antibiotic treatment in the early stages of the disease can lead to the spread of the disease throughout the body, often resulting in irreversible organ damage, confusion, blindness, or death. The transmission of Human Immunodeficiency Virus (HIV) worldwide has greatly increased the severity of the syphilis problem because genital ulcers, which develop early on in syphilis infection, promote the sexual transmission of HIV.

The course of syphilis is divided into initial stage, second stage, latent stage, neurosyphilis and third (late) stage. The infected person may infect others in the first two stages. Transmission occurs when spirochetes spread from the ulcer of an infected person to the skin of the sexual partner or to the mucous membranes of the genitalia, mouth or anus. Treponema pallidum can also be spread through damaged skin in other parts of the body. In tertiary syphilis and neurosyphilis, the infection is not contagious, but the invasion of organs, tissues and brain by spirochetes can cause serious cardiovascular malformations or neurological diseases and other fatal consequences.

The primary symptom of primary syphilis is ulcers or chancroid. Chancroid can occur within 10 days to 3 months after infection, usually in the body where it is exposed to ulcers of infected partners, such as the penis, vulva, vagina, cervix, rectum, tongue or lips. Chancroid may be overlooked because it lasts only weeks, may be painless or occurs in the body. And the malnutrition disappears with or without treatment. In untreated patients, secondary symptoms will appear after the primary lesion has appeared for about 9 weeks.

Secondary syphilis is often characterized by a rash, characterized by a brown ulcer. Any physical contact, sexual contact or non-sexual contact, with the skin of an infected person, may transmit infection at this stage due to the presence of active pathogens in these ulcers. Other symptoms include mild fever, fatigue, headache, sore throat, hair loss, and swollen glands. These symptoms may be mild, as in chancroid, which is a primary syphilis, and disappear with or without treatment. If left untreated, infected individuals will enter a latent phase.

Latent syphilis is characterized by absence of clinical symptoms or abnormal manifestations of cerebrospinal fluid (CSF), with positive serological tests. Early latent syphilis occurs within one year after infection, is potentially infectious and may recur, while late latent syphilis is associated with immunity to recurrence and resistance to reinfection. In the early stages of syphilis infection, spirochetes may invade the nervous system. Neurosyphilis may develop if left untreated. The disease may take up to 20 years to develop into neurosyphilis, and some people with neurosyphilis cannot show recognizable symptoms, making diagnosis very difficult. Those who present with symptoms that are caused by inflammation of the inner walls of the brain may complain of headache, stiff neck or fever. Although approximately two-thirds of syphilis infected patients are untreated and are not further affected by the disease, approximately one-third of untreated latent syphilis patients develop complications of late stage syphilis or tertiary syphilis.

In the third stage of syphilis, treponema pallidum damages the heart, eyes, brain, nervous system, bones, joints or almost any other part of the body. The third phase may last for years, even decades. Late stage syphilis often leads to cardiovascular disease, mental illness, blindness and even death.

Early diagnosis of syphilis infection is of great importance, since it sometimes has serious and life-threatening consequences and risks transmitting or infecting the hiv virus. However, syphilis is sometimes referred to as a "great imitator" because its early symptoms are similar to many other diseases. Therefore, clinical diagnosis usually does not rely solely on the identification of syphilis symptoms, but on microscopic identification of syphilis and clinical trial results of syphilis biological samples. Microbiological identification of syphilis is generally diagnosed by scraping the surface of an ulcer or chancroid under a special "dark field" microscope to discover microorganisms. The microscope observes the result, and the subjectivity of judgement is big and different institution's result report is big, is difficult to carry out quality control and parallel comparison, needs special technical staff to operate in addition, has the error easily. Therefore, most cases of syphilis are preferably serologically examined using non-specific antibody detection.

Currently used nonspecific antibody detection includes Venereal Disease Research Laboratory (VDRL) assays and rapid plasma reagin (rpr) assays. The VDRL assay detects treponema pallidum antibodies using natural lipids. These antibodies are antibodies to spirochete cardiolipin produced by the immune system of individuals infected with treponema pallidum. The currently available nonspecific antibody detection has the disadvantages of poor specificity and cumbersome operation. Many diseases, such as mycoplasma infection, pneumonia, malaria, acute bacterial and viral infections, can lead to false positives in syphilis tests. For example, intravenous injection of drugs or autoimmune diseases cause tissue damage, resulting in cardiolipin release and production of anti-cardiolipin antibodies. Diagnosis of neurosyphilis is particularly difficult. Furthermore, due to the persistence of anti-treponema pallidum antibodies, specific antibody detection assays cannot be used as a test to monitor the success or failure of antibiotic therapy. Therefore, for the diagnosis of early stage syphilis or of neurosyphilis, sensitive and specific detection of treponema pallidum infection in a sample is required. There is also a need for a simple, high throughput assay that can be used to monitor the efficacy of syphilis treatment.

The chemiluminescence method has the advantages of high sensitivity, wide linear range, high specificity, quick and simple operation, easy automation, no environmental pollution and the like. The chemiluminescence technology is divided into two types according to different reaction carriers: a semi-automatic chemiluminescence technology based on a microporous plate reaction and a full-automatic chemiluminescence technology based on a magnetic particle reaction tend to be homogeneous, faster and easier to automate. Most of the products made in China at present are the former. The majority of the international mainstream large diagnostic companies adopt chemiluminescence technology based on magnetic particle reactions. At present, magnetic particle chemiluminescence detection systems and kits have been developed by several foreign companies. The magnetic particle chemiluminescence detection technology is bound to become one of the main development directions of the detection method, becomes the mainstream technology of TP serological detection, and is widely used for screening and diagnosing syphilis and other diseases.

The magnetic particle chemiluminescence technology is applied to TP detection, has the advantages of high sensitivity, rapidness, accuracy, good repeatability, wide linear range, safety, no toxicity, no radioactive pollution and the like, and is easy to realize automatic detection. Meanwhile, the detection time can be further shortened, and the method is suitable for emergency detection. However, the coupling step between the antigen and the magnetic particle has a large influence on the detection effect, and if the coupling is not adequate, the report value of the positive sample is low, and it is difficult to distinguish the report value from the report value of the negative sample. Therefore, there is no good method for detecting TP helices by magnetic particle chemiluminescence.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a kit for detecting a non-specific antibody of TP spirochete, which comprises a detection reagent of TP spirochete by magnetic particle chemiluminescence. The coupling of the magnetic beads and the antigens (cardiolipin, lecithin and cholesterol) adopts reasonable reagents and good coupling effect, so that the negative and positive report values of the detection have larger difference and are easy to distinguish.

The invention provides a preparation method of magnetic particles coated by cardiolipin, lecithin and cholesterol, which comprises the following steps:

after coupling cardiolipin with BSA, dissolving the coupled cardiolipin with lecithin and cholesterol in absolute ethyl alcohol, then mixing the solution with activated carboxyl magnetic particles, collecting the magnetic particles after coupling, and washing the magnetic particles with PBS buffer solution to prepare the cardiolipin, lecithin and cholesterol coated magnetic particles;

wherein the mass ratio of the cardiolipin to the lecithin to the cholesterol is (0.01-0.05) to (0.11-0.46) to 0.9;

in some embodiments, the mass ratio of cardiolipin to lecithin to cholesterol is 3: 15: 90;

mixing each milliliter of the solution of the cardiolipin, the lecithin and the cholesterol with 0.1-0.5 mu g of the activated carboxyl magnetic particles.

In some embodiments, a solution of cardiolipin, lecithin, and cholesterol is mixed with 0.3 μ g of activated carboxyl magnetic microparticles per ml.

In the invention, after the cardiolipin, the lecithin and the cholesterol are dissolved in the absolute ethyl alcohol, the mass fraction of the cardiolipin is 0.03%, the mass fraction of the lecithin is 0.15% and the mass fraction of the cholesterol is 0.9%.

The diameter of the carboxyl magnetic particles is 0.2-4 μm. The cardiolipin, lecithin and cholesterol are coupled to the magnetic microparticles by covalent cross-linking with a chemical agent.

In the invention, the coupling condition is room temperature oscillation reaction for 30-60 min.

In the invention, the room temperature is 18-30 ℃.

In the present invention, the activation of the magnetic fine particles with carboxyl groups includes: washing the carboxyl magnetic particles by PBS, activating by MES buffer solution containing EDC and MES buffer solution containing NHS, and washing by MES buffer solution to obtain activated carboxyl magnetic particles;

the concentration of the PBS buffer solution is 0.02 mol/L; the concentration of the MES buffer solution is 0.1 mol/L;

in the MES buffer solution containing EDC, the concentration of EDC is 10-70 mg/ml; in the MES buffer solution containing NHS, the concentration of NHS is 10-70 mg/ml.

In some embodiments, the MES buffer containing EDC has a concentration of 40mg/ml EDC; the concentration of NHS in the MES buffer containing NHS was 40 mg/ml.

In the activation, the volume ratio of the MES buffer solution containing EDC to the MES buffer solution containing NHS is 1: 1; the activating condition is shaking reaction at room temperature for 30-60 minutes.

The magnetic particles coated with the cardiolipin, the lecithin and the cholesterol prepared by the preparation method are provided.

The invention also provides a kit for detecting the TP spirochete non-specific antibody, which comprises an enzyme-labeled antibody, and the magnetic particles coated with the cardiolipin, the lecithin and the cholesterol prepared by the preparation method;

the enzyme-labeled antibody is anti-human IgA, IgG and IgM antibodies labeled by horseradish peroxidase.

In the kit, the concentration of the magnetic particles coated by the cardiolipin, the lecithin and the cholesterol is 0.1-0.5 mu g/ml.

In the kit, the preparation method of the enzyme-labeled antibody comprises the following steps: after being activated, horseradish peroxidase is added with anti-human IgA, IgG and IgM antibodies, after the reaction is carried out for 8-12 h at the temperature of 2-8 ℃, sodium borohydride reductase conjugate is added, and after unreacted reagent is removed through dialysis, the horseradish peroxidase is mixed with glycerol with the same volume.

The kit also comprises an enzyme-labeled antibody diluent, wherein the enzyme-labeled antibody diluent consists of water, 20 wt% of calf serum and 0.1 wt% of P300 preservative.

When the enzyme-labeled antibody is used, the enzyme-labeled antibody is diluted by a diluent of the enzyme-labeled antibody according to the proportion of 1: 1000-1: 5000. In a specific embodiment, the dilution ratio is 1: 5000.

After dilution, the concentration of the enzyme-labeled antibody was 0.5. mu.g/ml.

The preservation temperature of the enzyme-labeled antibody is-20 ℃, and the preservation temperature of the enzyme-labeled antibody diluent is 2-8 ℃.

The kit also comprises a chemiluminescent substrate and/or a sample diluent;

the chemiluminescent substrate comprises a substrate A solution and a substrate B solution, wherein the substrate A solution is isoluminol derivative solution, the substrate B solution is hydrogen peroxide,

the sample diluent is PBS buffer.

The sample diluent is PBS buffer solution, and the proportion is 5-200 times when the sample is diluted by the PBS buffer solution.

The kit also comprises a negative control and a positive control;

the negative control is PBS buffer solution;

the positive control is a negative control containing 1/100-1/1000 TP spirochete nonspecific antibody positive substances.

The invention also provides a detection method of the TP spirochete nonspecific antibody, which uses the kit to detect a serum sample.

The invention improves the coupling method of the cardiolipin, the lecithin, the cholesterol and the magnetic particles, so that the magnetic particles can be better combined with a target object in a sample and matched with the enzyme-labeled antibody, thereby obtaining better detection effect. The kit provided by the invention comprises an enzyme-labeled antibody and the magnetic particles coated with the cardiolipin, the lecithin and the cholesterol prepared by the invention, can realize automatic detection of a sample, and has the characteristics of simple and accurate operation and strong anti-interference capability.

Detailed Description

The invention provides a kit for detecting a TP spirochete non-specific antibody, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

Wherein the synthetic cardiolipin in the synthetic antigen is tetramethylpyrimidine cardiolipin purified by silica gel chromatography, the purity is close to 99%, and the synthetic cardiolipin is obtained from polar lipid in a powder form. The final concentration of sodium salt was determined by thin layer chromatography and high pressure liquid chromatography. The samples were stored at-20 ℃ and cardiolipin was originally derived from a semi-synthetic lipid precursor of plant origin. The lecithin powder was purified rapidly by silica gel chromatography with a purity of about 99%. One of the synthetic antigens was an absolute ethanol solution containing 1.2% cholesterol, and filtered by rinsing the filter paper with ethanol. Cholesterol was originally derived from lanolin, purified by re-crvstallization and the crystals were stored at-20 ℃.

The invention is further illustrated by the following examples:

example 1

Preparation of synthetic antigen-coupled magnetic microparticles:

1.1 magnetic particle washing

Putting 200 mul of magnetic particles with carboxyl on the surface in a glass bottle, adsorbing the magnetic particles at the bottom of the glass bottle by using a magnet, and removing the supernatant; 2ml of 0.02M PBS (pH 8.0) was added, and the above operation was repeated 3 times.

1.2 magnetic particle activation

Respectively dissolving EDC and NHS in 0.1M MES (pH 5.0) buffer solution at the concentration of 10-70 mg/ml, and then adding 1ml of each solution into the magnetic particles; lightly shaking for reaction for 30-60 minutes at room temperature; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then 2ml of 0.1M MES (pH 5.0) buffer solution is added, and the magnetic particles are resuspended; the above operation was repeated 2 times.

1.3 magnetic particles coated with synthetic cardiolipin, synthetic lecithin, cholesterol

After the synthetic cardiolipin is coupled with BSA, the synthetic cardiolipin, lecithin and cholesterol are sequentially mixed in absolute ethyl alcohol according to the mass parts of 0.03%, 0.15% and 0.9%, and are uniformly mixed with 1.2 activated magnetic particles according to the concentration of 0.1 mu g/ml, and the mixture is gently shaken at room temperature for reaction for 60 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then adding 1ml of 0.2M PBS (pH 7.3) buffer solution, and resuspending the magnetic particles; the above operation was repeated 5 times.

Finally, the magnetic particles were resuspended in 0.2M PBS (pH 7.3) buffer to a concentration of 0.1. mu.g/ml.

Example 2

Preparation of anti-human IgA + IgG + IgM polyclonal antibody enzyme conjugate

Activating horseradish peroxidase (HRP) by a conventional improved sodium periodate method, adding an anti-human IgA + IgG + IgM polyclonal antibody, reacting overnight at 2-8 ℃, adding a sodium borohydride reductase conjugate, dialyzing to remove unreacted reagents, adding 50% glycerol by volume, and storing at-20 ℃; to a IgA concentration of 0.5. mu.g/ml, an IgG concentration of 0.5. mu.g/ml and an IgM concentration of 0.5. mu.g/ml.

Example 3

The antigen-coupled magnetic microparticles prepared in example 1, the enzyme-labeled antibody solution prepared in example 2, and a sample diluent, a chemiluminescent substrate, a negative control, and a positive control were used together to prepare a kit.

1. Enzyme-labeled antibody diluent:

buffer containing 20% calf serum and 0.1% P300 preservative was prepared with double distilled water.

1. Preparation of the sample dilution

Prepare 1000ml sample dilutions (PBS buffer): 5.80g of disodium hydrogen phosphate dodecahydrate, 0.59g of sodium dihydrogen phosphate dihydrate, 9.00g of sodium chloride and 201 ml of tween-201, wherein the volume is adjusted to 1000ml by using deionized water, and 0.02 percent of sodium azide and 0.1 percent of P300 preservative are added.

2. Negative and positive control preparation

The negative control is a matrix solution, and 1000ml of negative control is prepared: 5.80g of disodium hydrogen phosphate dodecahydrate, 0.59g of sodium dihydrogen phosphate dihydrate and 9.00g of sodium chloride are added with deionized water to reach the constant volume of 1000ml, and 0.02 percent of sodium azide preservative is added.

The positive control is prepared by adding 1/100-1/1000 TP spirochete nonspecific antibody positive substances into a matrix solution.

3. Chemiluminescent substrate solution: the substrate A solution adopts isoluminol derivative solution, the concentration is 0.01-0.1g/L, and the substrate B solution adopts hydrogen peroxide.

Examples of detection

The kit prepared in example 3 is used in combination with an AutoLumo A2000 or AutoLumo A2000 Plus full-automatic chemiluminescence apparatus manufactured by Zhengzhou Antu bioengineering GmbH for detecting TP spirochete nonspecific antibodies:

1. diluting the enzyme-labeled antibody with an enzyme-labeled antibody diluent at a dilution ratio of 1: 5000, and storing at 2-8 ℃.

2. Adding 3 positive control wells (used for determining a Cutoff value), 2 negative control wells and 100 mu L/well into a reaction container (hereinafter referred to as "well") in sequence; adding 0.5 μ L sample (serum) and 100 μ L of the above sample diluent to each of the other wells;

3. adding 20 mu L of magnetic particle suspension into each hole respectively; after mixing, incubating for 15 minutes at 37 ℃; then washing with a cleaning solution for 5 times;

4. adding 100 mu L of enzyme conjugate into each hole; mixing, and incubating at 37 deg.C for 17 min; washing with cleaning solution for 5 times;

5. finally, adding 50 mu L of substrate A liquid and 50 mu L of substrate B liquid into each hole; detecting the luminous intensity 1-5 minutes after mixing;

and (4) calculating a result: the Cut off value is equal to the average luminescence value of the positive control hole multiplied by 0.2;

positive judgment value: S/CO is the luminous value/Cutoff value of the sample to be measured;

when the S/CO is more than or equal to 1.00, the result is judged to be positive; when the S/CO is less than 1.00, the result is judged to be negative.

Secondly, evaluating the performance of the kit

1. Repeatability of

Repeatability means the closeness of each measurement to the mean value, expressed as coefficient of variation CV, determined repeatedly on the same sample.

Selecting 3 samples with different concentrations, detecting for 4 times every day, continuously detecting for 5 days, wherein the coefficient of variation CV is less than 8 percent, and the result is shown in a table 1:

TABLE 1 results of repeated measurements

From the results in table 1 it can be seen that: the coefficient of variation of sample 1 is 5.28%, the coefficient of variation of sample 2 is 6.87%, and the coefficient of variation of sample 3 is 2.79%, which are all less than 10% of the industry requirement, and the repeatability is proved to be good.

2. Specificity of

The positive samples of hepatitis B, hepatitis C, HIV, EB-EA, HSV-1, HSV-2, CMV, VCA-IgG and HAMA are detected, each pathogen is 10 parts, and the cross reactivity is avoided.

12 samples of rheumatoid factor RF positive and antinuclear antibody (ANA) positive samples are detected without interference, and the results are shown in a table 2:

TABLE 2 results of specific detection

As can be seen from the results in table 2: the positive of different detected pathogens does not produce cross reaction with the kit, and the rheumatoid factor RF and the autoantibody ANA do not interfere with the kit.

3. Sample collection and processing

The type of sample for clinical testing is typically serum or plasma. The anticoagulant used for the plasma sample is sodium citrate, EDTA-2Na or heparin sodium and the like. Blood collection tubes of different anticoagulants and common tubes (without additives) need to be checked to determine whether the detection results are interfered. And comparing the positive samples and the negative samples of different blood collection tubes, and detecting whether the interference exists in the detection result. The result shows that the difference of detection signal values of different anticoagulant samples is small, which indicates that the anticoagulant does not have obvious interference on the detection result. Therefore, the type of sample suitable for the kit can be serum or plasma, and the anticoagulant can be EDTA-2Na or K, sodium citrate or heparin, and the results are shown in Table 3:

TABLE 3 comparison of different anticoagulants

4. Interference of endogenous substances in a sample

Endogenous interfering substances in a sample may interfere with the detection result, and the tolerance concentration of the kit to the endogenous interfering substances needs to be assessed. The following pure products were added to 6 positive samples (1 weak positive, 3 medium strong positive, 2 strong positive and 6 negative samples) respectively: 1.0g/L hemoglobin, 0.4g/L bilirubin and 30g/L triglyceride were assayed.

For positive samples, the mean luminescence values of the samples and controls were calculated, and the percentage of interference for each sample luminescence value was calculated: the interference percentage is (sample average luminescence value-reference substance average luminescence value)/reference substance average luminescence value multiplied by 100%, if the interference of the positive sample is within +/-15%, the background of the negative sample has no obvious change, the result judgment is not influenced, and the interference of the kit to endogenous substances in the sample is considered to be acceptable.

The verification shows that the interference rate is less than 15% after the endogenous substances are added, and the interference is acceptable. As the physiological concentration and the pathological concentration of a clinical sample are lower than the evaluation concentration of an endogenous interferent, 0.4g/L bilirubin, 30g/L triglyceride and 1g/L hemoglobin can be considered as endogenous interference tolerance limits of the kit, and the results are shown in a table 4:

TABLE 4 Effect of endogenous interfering substances

5. Reference value determination

Cuo-off values were set between the values of 400-5000 and 5000, and specificity, sensitivity (TPR) and False Positive Rate (FPR) were calculated from the different cut-off values assumed, as shown in Table 5:

TABLE 5 relationship between specificity, sensitivity (TPR) and False Positive Rate (FPR) measured at different cut-off values

Since the sensitivity is 99.40% and the specificity is 99.04% when the cut-off value of the luminescence value is 1200, the sensitivity and the specificity satisfy the national standards, the cut-off value of the kit is set to 1200.

The determination of Cut-off does not mean that the Cut-off is constant for each measurement. Because the cut-off value of each measurement has a certain difference, a cut-off sample, namely a positive control PC is introduced during the calculation of the critical value, the range of the luminous value is 3000-7000, and the average value of the luminous value of the positive control PC is multiplied by a coefficient of 0.2 to form the cut-off value.

Judging the result by adopting an S/CO mode (namely, the Sample luminous value/cut-off value of the Sample to be detected), and judging the result to be positive when the S/CO is more than or equal to 1.00; when the S/CO is less than 1.00, the result is judged to be negative.

Comparative example 1

The steps 1.1 to 1.2 of the preparation of the synthetic antigen-coupled magnetic particles are the same as in example 1.

Mixing natural cardiolipin, lecithin and cholesterol in the proportion of 0.01%, 0.09% and 0.9% in absolute ethyl alcohol, uniformly mixing the mixture with 1.2 activated magnetic particles in the working concentration of 0.1 mu g/ml, and slightly shaking at room temperature for reaction for 60 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then adding 1ml of 0.2M PBS (pH 7.3) buffer solution, and resuspending the magnetic particles; the above operation was repeated 5 times.

Finally, the magnetic particles were resuspended in 0.2M PBS (pH 7.3) buffer to a concentration of 0.1. mu.g/ml and the antigen coupled to the magnetic particles.

The antigen-coupled magnetic particles prepared in the comparative example 1, the enzyme-labeled antibody solution prepared in the example 2, and the sample diluent, the chemiluminescent substrate, the negative control and the positive control provided in the example 3 are prepared together to prepare a kit and detect a sample, but the result shows that the reaction sensitivity is insufficient, the difference between the luminescence values of the negative sample and the positive sample is not large, the ratio of the luminescence values of the positive sample and the negative sample is less than 20, and the negative and the positive samples are difficult to distinguish.

Comparative example 2

The steps 1.1-1.2 of the preparation of the synthetic antigen-coupled magnetic particles are the same as those of example 1,

Coupling synthetic cardiolipin with BSA, mixing the coupled synthetic cardiolipin with natural lecithin and cholesterol in the proportion of 0.03%, 0.01% and 0.9% in absolute ethyl alcohol, uniformly mixing the mixture with 1.2 activated magnetic particles at the working concentration of 0.1 mu g/ml, and carrying out gentle shaking reaction at room temperature for 60 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then adding 1ml of 0.2M PBS (pH 7.3) buffer solution, and resuspending the magnetic particles; the above operation was repeated 5 times. Finally, the magnetic particles were resuspended in 0.2M PBS (pH 7.3) buffer to a concentration of 0.1. mu.g/ml.

The antigen-coupled magnetic particles prepared in the comparative example 2, the enzyme-labeled antibody solution prepared in the example 2, and the sample diluent, the chemiluminescent substrate, the negative control and the positive control provided in the example 3 are prepared into a kit and detected, but the result shows that the specificity is insufficient and a false positive sample appears in clinical detection.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A method for preparing cardiolipin, lecithin and cholesterol coated magnetic microparticles comprising:

2. The method according to claim 1, wherein the mass fraction of cardiolipin, the mass fraction of lecithin, and the mass fraction of cholesterol are 0.03%, 0.15%, and 0.9% after dissolving the cardiolipin, the lecithin, and the cholesterol in absolute ethanol.

3. The preparation method according to claim 1, wherein the coupling condition is shaking reaction at room temperature for 30-60 min.

4. The method according to claim 1, wherein the activation of the magnetic fine carboxyl particles comprises: washing the carboxyl magnetic particles by PBS, activating by MES buffer solution containing EDC and MES buffer solution containing NHS, and washing by MES buffer solution to obtain activated carboxyl magnetic particles;

in the MES buffer solution containing EDC, the concentration of EDC is 10-70 mg/ml;

in the MES buffer solution containing NHS, the concentration of NHS is 10-70 mg/ml;

in the activation, the volume ratio of the MES buffer solution containing EDC to the MES buffer solution containing NHS is 1: 1;

the activating condition is shaking reaction at room temperature for 30-60 minutes.

5. Cardiolipin, lecithin and cholesterol coated magnetic microparticles prepared by the method of any one of claims 1 to 4.

A kit for detecting a TP spirochete non-specific antibody is characterized by comprising an enzyme labeled antibody, and magnetic particles coated with cardiolipin, lecithin and cholesterol prepared by the preparation method of any one of claims 1 to 4;

7. The kit of claim 6, wherein the enzyme-labeled antibody is prepared by the following steps: after being activated, horseradish peroxidase is added with anti-human IgA, IgG and IgM antibodies, after the reaction is carried out for 8-12 h at the temperature of 2-8 ℃, sodium borohydride reductase conjugate is added, and after unreacted reagent is removed through dialysis, the horseradish peroxidase is mixed with glycerol with the same volume.

8. The kit of claim 6 or 7, further comprising an enzyme-labeled antibody diluent consisting of water and 20 wt% calf serum and 0.1 wt% P300 preservative.

9. The kit according to any one of claims 6 to 8, further comprising a chemiluminescent substrate and/or a sample diluent;

the sample diluent is PBS buffer.

10. The kit according to any one of claims 6 to 9, further comprising a negative control and a positive control;

the negative control is PBS buffer solution;