Diagnostic method based on mouse Lyme disease serology indirect ELISA laboratory

Abstract

The invention discloses a diagnostic method based on mouse lyme disease serology indirect ELISA laboratory, and relates to the technical field of immunoserology detection.The diagnostic method comprises the steps that experimental conditions are optimized through a system, and the optimal reaction system is determined as follows: the antigen coating concentration is 0.2 microgram / microliter, the serum dilution ratio is 1: 200, and the enzyme-labeled secondary antibody concentration is 1: 2000. 0.8), and meanwhile, the antigen dosage is obviously reduced. Dynamic analysis of the antibody shows that the level of the antibody infecting mouse serum reaches a peak 12-25 days after inoculation, and the threshold value of the specific antibody ratio (ArB%) is 71.43%. Researches prove that the method has the characteristics of simplicity and convenience in operation, economy and high efficiency, and is suitable for primary laboratories and large-scale screening. The method provides reliable standardized technical support for lyme disease laboratory diagnosis and epidemiological monitoring.

Description

Technical Field

[0001] This invention relates to the field of immunoserological detection technology, specifically to a laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA. Background Art

[0002] Lyme disease is a tick-borne zoonotic disease caused by Borrelia burgdorferi, posing a serious threat to public health and livestock. The clinical manifestations of Lyme disease are complex and diverse, varying considerably between humans and different animal species, and often involve organs and tissues such as the heart, skin, joints, and nervous system. It frequently causes multi-system and multi-organ damage in humans, leading to lifelong disability or even death in severe cases. The disease is widely distributed, now found in more than 70 countries and regions. Borrelia garinii is one of the main pathogenic genotypes in northern my country.

[0003] Currently, serological testing (such as ELISA) is the core method for diagnosing Lyme disease. However, existing methods suffer from insufficient antigen standardization and cross-reactivity interference, limiting their sensitivity and specificity. Other methods include indirect immunofluorescence (IFA) and Western blot (WB) to detect specific antibodies (IgM, IgG) in Lyme disease patients. IFA was the earliest method used for serological diagnosis; it is simple and easy to master, but it is prone to false positives or false negatives and is time-consuming, making it unsuitable for large-scale sample testing. ELISA has better sensitivity and repeatability than IFA, but it suffers from non-specific reactions. In recent years, due to the better sensitivity and specificity of WB, combined with preliminary results from ELISA or IFA, a laboratory diagnosis can generally be made. Therefore, developing a highly sensitive, highly specific, and cost-effective standardized ELISA method is crucial for the early diagnosis and epidemiological surveillance of Lyme disease.

[0004] However, limitations in antigen design remain a problem that needs to be overcome. These include defects in natural antigens, the presence of non-specific lipoproteins in whole-cell lysis antigens leading to false positives, and insufficient coverage by recombinant antigens, with a single recombinant antigen (such as OspC) failing to identify all genotypes of infection, resulting in a false negative rate as high as 30% for European isolates. Summary of the Invention

[0005] To address the aforementioned technical problems, a laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA is provided. This technical solution overcomes the limitations of the aforementioned antigen design.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA, comprising:

[0008] Step 1: Cultivation of the bacterial strain and preparation of mouse serum;

[0009] Step 2: Preparation of positive serum;

[0010] Step 3: Serological indirect ELISA method;

[0011] Step 4: Determination of the optimal working concentration of whole-cell antigen;

[0012] Step 5: Determination of the optimal working concentration of serum;

[0013] Step 6: Determination of the optimal working concentration of enzyme-labeled secondary antibody;

[0014] Furthermore, when the whole-cell antigen concentration of the B. garinii standard strain (ATCC, ATCC51383) was 1.07 μg / μL, the serum concentration was set at 1:100, and the enzyme-labeled secondary antibody concentration was set at 1:5000, the antigen concentrations in the same serum sample were 0.2 μg / μL, 0.4 μg / μL, 0.8 μg / μL, and 1.6 μg / μL, respectively. In order to save antigen, this invention screened 0.2 μg / μL as the optimal working concentration of antigen.

[0015] Preferably, the present invention relates to the application of a dedicated mouse Lyme disease serological indirect ELISA laboratory diagnostic method.

[0016] Preferably, the coating step specifically includes:

[0017] The natural whole-cell antigen of the B. garinii standard strain was coated onto 96-well polystyrene microplates at a concentration of 0.20 ± 0.02 μg / μL using carbonate buffer at pH 9.6.

[0018] Preferably, the serum reaction step specifically includes:

[0019] Add mouse serum samples diluted 1:100±5 and incubate at 37°C for 45 minutes.

[0020] Preferably, the signal amplification step specifically includes:

[0021] Add horseradish peroxidase (HRP)-labeled anti-mouse IgG secondary antibody diluted 1:5000±500.

[0022] Preferably, the determination of the critical value specifically includes:

[0023] A sample is considered positive when its OD450nm value is ≥ 2.5 times the mean of the negative control and ≥ 0.6 times the OD value of the positive control.

[0024] Preferably, the antigen concentration specifically includes:

[0025] Gradient screening experiments determined that, under the same serum and secondary antibody concentrations, a concentration of 0.2 μg / μL saves more than 80% of antigen usage compared to 0.4 μg / μL, 0.8 μg / μL, and 1.6 μg / μL, while maintaining sensitivity ≥86% and specificity ≥94%.

[0026] Preferably, the optimal working concentration for screening serum in this invention is 1:200.

[0027] Preferably, the optimal working concentration of the enzyme-labeled secondary antibody selected by the present invention is 1:2000.

[0028] Preferably, the present invention has determined the ELISA reaction threshold for the B. garinii strain antibody test to be 71.43%.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] This invention significantly reduces the amount of antigen used while maintaining sensitivity, making it highly economical. Furthermore, the optimization of conditions such as the optimal serum dilution ratio and the optimal concentration of enzyme-labeled secondary antibody not only improves the sensitivity and specificity of the detection, but also achieves a dual breakthrough in sensitivity and economy, making it particularly suitable for resource-limited primary laboratories and large-scale laboratory screening.

[0031] With its ease of operation (no special equipment required) and significant cost advantages (60% reduction in consumption per test), this invention precisely fills the gap in standardized diagnostic tools for primary laboratories. It can be used as a primary screening method to support the construction of large-scale epidemiological surveillance networks, and can also be extended to the field of vaccine efficacy evaluation, providing a key technical support for the integrated "diagnosis-prevention-control" system for Lyme disease. Attached Figure Description

[0032] Figure 1 A flowchart of a laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA;

[0033] Figure 2 This is a graph showing the dynamic changes in antibodies in mice infected with B. garinii strain. Detailed Implementation

[0034] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0035] Reference Figure 1 As shown, a laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA includes:

[0036] Step 1: Cultivation of the bacterial strain and preparation of mouse serum;

[0037] Step 2: Preparation of positive serum;

[0038] Step 3: Serological indirect ELISA method;

[0039] Step 4: Determination of the optimal working concentration of whole-cell antigen;

[0040] Step 5: Determination of the optimal working concentration of serum;

[0041] Step 6: Determination of the optimal working concentration of enzyme-labeled secondary antibody;

[0042] Furthermore, when the whole-cell antigen concentration of the B. garinii standard strain was 1.07 μg / μL, the serum concentration was set at 1:100, and the enzyme-labeled secondary antibody concentration was set at 1:5000, the antigen concentrations for the same serum sample were 0.2 μg / μL, 0.4 μg / μL, 0.8 μg / μL, and 1.6 μg / μL, respectively. In order to save antigen, this invention screened 0.2 μg / μL as the optimal working concentration of antigen.

[0043] It should be noted that the basic layer of pathogens (steps 1-2) is as follows:

[0044] Enrichment culture was performed using the standard strain of B. garinii (ATCC51383), and natural whole-cell antigen (concentration baseline value 1.07 μg / μL) was obtained by ultrasonic disruption.

[0045] The preparation of positive serum innovatively adopts a multi-round immunization-affinity purification strategy: mice are immunized three times with inactivated bacteria and CpG adjuvant, and then high-affinity IgG (titer ≥1:12,800) is screened through Protein G column to ensure the reliability of the detection standard.

[0046] Parameter optimization layer (steps 4-6):

[0047] Antigen concentration (0.2 μg / μL): The initial concentration of 1.07 μg / μL was used for serial dilution screening. The saturated adsorption experiment confirmed that the antigen coverage of the microplate surface at this concentration was 68.5% (close to the optimal value of 70% for monolayer), avoiding the steric hindrance effect caused by high concentration.

[0048] Serum dilution (1:100): determined based on the inflection point of the antibody dilution curve. This dilution enables the detection limit of low-affinity antibodies (early infection IgM) to reach 0.1 ng / mL, while suppressing non-specific background signals to OD<0.12.

[0049] Secondary antibody concentration (1:5000): Matched to the catalytic efficiency threshold of horseradish peroxidase (HRP), while ensuring a signal-to-noise ratio >20, the consumption of enzyme-labeled reagent is only 20% of that of the traditional method (1:1000).

[0050] Detect the execution layer (step 3):

[0051] The unique dual-condition interpretation rule requires that the positive result be determined simultaneously by an OD value ≥ 2.5 times the mean negative value and ≥ 0.6 times the positive control value, thus eliminating the risk of false positives caused by batch-to-batch differences in reagents.

[0052] Reference Figure 2 As shown, in the mouse group infected with *B. garinii*, serum samples were collected at 20 time points, and three samples with higher OD values ​​at each time point were selected for antibody dynamic detection. The specific antibody ratio (ArB%) was 71.43%, meaning that an ELISA reaction threshold less than 71.43% was considered negative, while a threshold greater than 71.43% indicated that the sample was infected with or had been infected with *B. garinii*. The antibody levels in this experiment varied considerably, showing an initial increase followed by a decrease within one week. This may be related to the bacterial inoculation amount and the mice's own immunity.

[0053] Referring to Table 1, the whole-cell antigen concentration of B. garinii strain prepared in this invention was 1.07 μg / μL. When the serum concentration was set at 1:100 and the enzyme-labeled secondary antibody concentration was set at 1:5000, the OD values ​​of antigen concentrations of 0.2 μg / μL, 0.4 μg / μL, 0.8 μg / μL, and 1.6 μg / μL in the same serum sample were found to be not significantly different. In order to save antigen, 0.2 μg / μL was selected as the optimal working concentration of antigen.

[0054]

[0055]

[0056] Table 1

[0057] Table 1. OD values ​​of the three strains at different antigen concentrations:

[0058] B. garinii strain (F3 generation) was inoculated into 5 mL of BSK-H medium, 300 μL / tube, and incubated at 33°C for 7–14 days until the bacterial cells reached the logarithmic growth phase (10⁻⁶). 7 ~10 8Collect bacterial culture (F4) at / mL, take 200μL of culture medium and inoculate it into a 4mL spirochete culture tube, with an inoculation volume of 5%. Add the remaining bacterial culture to a 1.5mL centrifuge tube, centrifuge at 12000rpm for 10min, discard the supernatant, add 500μL PBS, pipette the pellet, and transfer it to a new 1.5mL centrifuge tube. Wash twice with PBS, centrifuge at 12000rpm for 5min, discard the supernatant, dilute with 200μL PBS, and count using a Peyronie's indexing plate. The whole-cell antigen concentrations of B. garinii strain were 0.2μg / μL, 0.4μg / μL, 0.8μg / μL, and 1.6μg / μL, respectively. The serum concentration was set at 1:100, and the enzyme-labeled secondary antibody concentration was set at 1:5000. Serum was collected on day 7 post-infection, and three sera were taken from each strain for OD value determination. Bg-7, Bg-8, and Bg-9 are the infection group numbers of the strains.

[0059]

[0060] Table 2

[0061] Referring to Table 2, when the enzyme-labeled secondary antibody concentration was set at 1:5000, comparison of the OD values ​​of the *B. garinii* strain groups revealed that, with a fixed serum concentration, the OD values ​​did not differ significantly when the antigen concentrations were 0.2 μg / μL, 0.8 μg / μL, and 1.6 μg / μL. Therefore, the optimal working concentration of the antigen was again determined to be 0.2 μg / μL. When the serum concentration was 1:200, the OD value remained relatively high, around 1.9. Therefore, the optimal working serum concentration was selected as 1:200 in this experiment. Serum (Bg-8) from day 7 post-infection with *B. garinii* was randomly selected and serially diluted to 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200. The enzyme-labeled secondary antibody concentration was set at 1:5000, and the OD values ​​were measured.

[0062]

[0063] Table 3

[0064] Referring to Table 3, when the antigen concentration was fixed at 0.2 μg / μL and the serum concentration was 1:200, the OD value of the enzyme-labeled secondary antibody at a concentration of 1:2000 was 1.9605, which was relatively high compared to other enzyme-labeled secondary antibody concentrations. Therefore, the optimal working concentration of the enzyme-labeled secondary antibody was selected as 1:2000 in this experiment. After determining the optimal working concentrations of serum and antigen, serum (Bg-9) from day 7 post-infection with *B. garinii* was randomly selected, and the enzyme-labeled secondary antibody concentrations were set to 1:1000, 1:2000, 1:5000, 1:8000, 1:10000, and 1:50000, respectively. OD values ​​were then measured.

[0065] Implementation steps:

[0066] Step 1: Experimental Preparation

[0067] Core reagents:

[0068] B. garinii natural whole bacterial antigen (original concentration 1.07 μg / μL), mouse serum sample to be tested, HRP-labeled anti-mouse IgG secondary antibody, coating buffer (pH 9.6 carbonate buffer), TMB chromogenic solution, and stop solution (2M H2SO4);

[0069] Step 2: Antigen coating

[0070] Antigen dilution: Dilute the original antigen (1.07 μg / μL) with coating buffer to 0.20 ± 0.02 μg / μL;

[0071] Coating procedure: Add 100 μL of diluted antigen to each well of a 96-well polystyrene plate; after coating, incubate overnight at 4°C (16–18 hours);

[0072] Washing: Discard the liquid in the wells and wash 3 times with PBST (PBS containing 0.05% Tween-20), soaking for 1 minute each time;

[0073] Critical control point: The coating concentration must be strictly controlled within 0.18–0.22 μg / μL. If it exceeds the range, it must be prepared again.

[0074] Step 3: Seal

[0075] Blocking solution preparation: 5% skim milk powder-PBST solution;

[0076] Sealing procedure: Add 200 μL of sealing solution to each well; incubate at 37°C for 2 hours;

[0077] Washing: Same as step 1, wash 3 times;

[0078] Step 4: Serum reaction

[0079] Serum dilution: The serum to be tested was diluted with PBST at a ratio of 1:100±5 (i.e., 1:95 to 1:105); the positive control serum was diluted at a ratio of 1:200 (preferred method);

[0080] Sample loading and incubation: Add 100 μL of diluted serum to each well; incubate at 37°C for 45 minutes;

[0081] Washing: Same as step 1, wash 5 times;

[0082] Step 5: Secondary antibody reaction

[0083] Secondary antibody dilution: HRP-labeled secondary antibody was diluted with PBST at a ratio of 1:5000±500 (i.e., 1:4500 to 1:5500);

[0084] Sample loading and incubation: Add 100 μL of diluted secondary antibody to each well; incubate at 37°C in the dark for 45 minutes;

[0085] Washing: Same as step 1, wash 5 times;

[0086] Step 6: Signal Color Display

[0087] Addition of colorimetric reagent: Add 100 μL TMB substrate solution to each well;

[0088] Incubation in the dark at room temperature (25°C) for 15 minutes;

[0089] To terminate the reaction, add 50 μL of 2M H2SO4 solution to each well.

[0090] Step 7: Result Interpretation

[0091] OD value determination: Read the absorbance at 450nm wavelength using an ELISA reader (reference wavelength 630nm);

[0092] Positive result determination: Both of the following conditions must be met:

[0093] Condition 1: OD 450 ≥ 2.5 times the mean of negative controls;

[0094] Condition 2: OD 450 ≥0.6 × positive control OD value;

[0095] Threshold verification: If the sample OD value is ≥ 71.43% × positive control OD value (preferred scheme), it is directly judged as positive;

[0096] Step 8: Waste Disposal

[0097] All consumables that come into contact with pathogens must be autoclaved at 121°C for 30 minutes;

[0098] Acidic waste liquid is neutralized with 10% NaOH before being discharged.

[0099] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA, characterized in that, include: Step 1: Cultivation of the bacterial strain and preparation of mouse serum; Step 2: Preparation of positive serum; Step 3: Serological indirect ELISA method; Step 4: Determination of the optimal working concentration of whole-cell antigen; Step 5: Determination of the optimal working concentration of serum; Step 6: Determination of the optimal working concentration of enzyme-labeled secondary antibody; Furthermore, when the whole-cell antigen concentration of B. garinii strain was 1.07 μg / μL, the serum concentration was set at 1:100, and the enzyme-labeled secondary antibody concentration was set at 1:5000, the antigen concentrations for the same serum sample were 0.2 μg / μL, 0.4 μg / μL, 0.8 μg / μL, and 1.6 μg / μL, respectively. In order to save antigen, this invention screened 0.2 μg / μL as the optimal working concentration of antigen.

2. The laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, This invention relates to the application of a dedicated mouse Lyme disease serological indirect ELISA laboratory diagnostic method.

3. The laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, The coating step specifically includes: The natural whole-cell antigen of B. garinii strain was coated onto 96-well polystyrene microplates at a concentration of 0.20 ± 0.02 μg / μL using carbonate buffer at pH 9.

6.

4. The laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, The serum reaction steps specifically include: Add mouse serum samples diluted 1:100±5 and incubate at 37°C for 45 minutes.

5. The laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, The signal amplification step specifically includes: Add horseradish peroxidase (HRP)-labeled anti-mouse IgG secondary antibody diluted 1:5000±500.

6. The laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, The determination of the critical value specifically includes: A sample is considered positive when its OD450nm value is ≥ 2.5 times the mean of the negative control and ≥ 0.6 times the OD value of the positive control.

7. The laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, The antigen concentration specifically includes: Gradient screening experiments determined that, under the same serum and secondary antibody concentrations, a concentration of 0.2 μg / μL saves more than 80% of antigen usage compared to 0.4 μg / μL, 0.8 μg / μL, and 1.6 μg / μL, while maintaining sensitivity ≥86% and specificity ≥94%.

8. The laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, The optimal working concentration for screening serum in this invention is selected as 1:

200.

9. A laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, The optimal working concentration of the enzyme-labeled secondary antibody was determined to be 1:2000.

10. A laboratory diagnostic method based on mouse Lyme disease serological indirect ELISA according to claim 1, characterized in that, This invention determined the ELISA reaction threshold of B. garinii strain antibody assay to be 71.43%.

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