CN118259018B - Preparation of C1s antibody and application thereof in ITP diagnosis - Google Patents

Abstract

The present invention provides an application of C1s antibody in the preparation of ITP diagnostic products, relates to the field of biomedical technology, and includes an application of C1s antibody in the preparation of ITP diagnostic products, wherein the amino acid sequence of the C1s antibody is shown in SEQ ID NO: 1. In this application, a reliable experimental basis is provided for the specific application of C1s antibody in the diagnosis of ITP through specific examples, and a train of thought is provided for further research and development of diagnostic methods for ITP in the later stage.

Description

Preparation of C1s antibody and its application in diagnosis of ITP

Technical Field

The present invention relates to the field of biomedical technology, and in particular to the preparation of C1s antibody and its application in ITP diagnosis.

Background Art

ITP immune thrombocytopenia is a common bleeding disorder that causes a decrease in the number of platelets due to an abnormal immune system. The incidence of ITP varies in both adults and children, with women of childbearing age and people over 60 years old more susceptible to the disease. Globally, the incidence of ITP is approximately 5 to 10 per 100,000.

At present, the diagnosis of ITP is mainly based on the exclusion method [Hou Ming, Hu Yu. Chinese Guidelines for the Diagnosis and Treatment of Primary Immune Thrombocytopenia in Adults (2020 Edition), Chinese Journal of Hematology, 2020, 41 (8): 617-623.]: ① At least 2 consecutive blood routine examinations show a decrease in platelet count (<100×10 ⁹ /L), peripheral blood smear microscopy shows no obvious abnormality in blood cell morphology; ② The spleen is generally not enlarged; ③ Bone marrow examination: The morphological characteristics of bone marrow cells in ITP patients are increased or normal megakaryocytes, accompanied by maturation disorders; ④ Other secondary thrombocytopenias must be excluded: autoimmune diseases, thyroid diseases, lymphoproliferative diseases, myelodysplastic syndrome (MDS), aplastic anemia (AA), various malignant blood diseases, tumor infiltration, chronic liver disease, hypersplenism, common variable immunodeficiency (CVID), secondary thrombocytopenia caused by infection, vaccination, etc., thrombocytopenia caused by platelet consumption, drug-induced thrombocytopenia, alloimmune thrombocytopenia, gestational thrombocytopenia, congenital thrombocytopenia and pseudothrombocytopenia. Another method for clinical diagnosis of ITP is to treat suspected ITP patients with ITP regimens, and if the treatment is effective, it is diagnosed as ITP. It can be seen that there is still a lack of effective diagnostic methods and diagnostic markers for ITP. In addition, additional background knowledge on the possible relationship between ITP and C1s is needed to introduce the significance and clinical application possibilities of the development of this patent.

Summary of the invention

The purpose of the present invention is to solve the technical problems that there is no ITP diagnostic marker in the prior art, and the diagnostic method has low specificity and is easily confused with other diseases.

In order to achieve the above object, the present invention adopts the following technical solutions:

Application of C1s antibody in the preparation of ITP diagnostic products, wherein the amino acid sequence of the C1s antibody is shown in SEQ ID NO: 1-10, wherein SEQ ID NO: 1-3 is the heavy chain gene sequence of the C1s antibody, SEQ ID NO: 4-5 is the heavy chain amino acid sequence of the C1s antibody, SEQ ID NO: 6-8 is the light chain gene sequence of the C1s antibody, and SEQ ID NO: 9-10 is the light chain amino acid sequence of the C1s antibody. Preferably, the diagnostic product includes a kit and a detection reagent, wherein the kit and the detection reagent use the C1s antibody as a specific detection reagent for the target C1s to be detected.

The present application also provides a kit for diagnosing ITP, wherein the kit uses C1s on platelets as a detection target.

Preferably, the kit further comprises other medically acceptable reagents.

The present application also provides a non-diagnostic validation method for the use of C1s antibody in the preparation of ITP products, wherein the validation method comprises the following contents:

S1: Antibody performance verification experiment;

S2: C1s antibody FITC labeling experiment;

S3: Platelet C1s-FITC flow cytometry experiment.

Preferably, the antibody performance verification experiment includes ELISA binding experiment and SDS-PAGE detection.

Preferably, the platelet C1s-FITC flow cytometry experiment includes two groups of experiments, which are respectively a normal human platelet C1s-FITC flow cytometry experiment and a thrombocytopenia C1s-FITC flow cytometry experiment.

Preferably, the platelet C1s-FITC flow cytometry assay comprises the following steps:

A1: Specimen collection and processing;

A2: staining and fixing cells;

Specifically including negative control and sample testing:

Negative control: Take 50ul PBS (pH 7.2) and add it to the labeled A1 test tube, then add 1ul whole blood, and add 5ul of the corresponding isotype control as the negative control of the fluorescent antibody, vortex and incubate in the dark for 15min, centrifuge (1500rpm, 5min), aspirate the supernatant (avoid aspirating red blood cells), add 450ul PBS and mix well, and then test;

Sample detection: Take 50ul PBS and add it to the marked B1 test tube, then add 1ul whole blood, add 5ul CD61-PE and C1s-FITC fluorescent antibodies respectively, incubate in the dark for 15min after vortexing, centrifuge (1500rpm, 5min), aspirate the supernatant (avoid aspirating red blood cells), add 450ul PBS and mix well, and then test;

A3: Test on the computer and analyze the results.

In summary, the specific application of C1s antibody in the diagnosis of ITP through specific examples in this application provides a reliable experimental basis and provides ideas for further research and development of diagnostic methods for ITP in the future.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of SDS-PAGE detection of C1s antibody in one embodiment of the present invention (R: reducing SDS-PAGE; N-R: non-reducing SDS-PAGE);

Figure 2 shows the detection of activated C1s membrane-bound platelets in one embodiment of the present invention, wherein A: platelets are labeled (platelets are in the CD61-positive gate); B: isotype control for platelet detection; C: detection of activated C1s membrane-bound platelets in normal control; D: detection of activated C1s membrane-bound platelets in non-ITP thrombocytopenia; E: detection of activated C1s membrane-bound platelets in ITP; F: statistical analysis of the differences in activated C1s membrane-bound platelets between the normal control group (NC), non-ITP thrombocytopenia group (CC), and ITP group.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments.

The present application provides the preparation of C1s antibody and its application in ITP diagnostic products. The amino acid sequence of the C1s antibody is shown in SEQ ID NO:1-10, wherein SEQ ID NO:1-3 is the heavy chain gene sequence of the C1s antibody, SEQID NO:4-5 is the heavy chain amino acid sequence of the C1s antibody, SEQ ID NO:6-8 is the light chain gene sequence of the C1s antibody, and SEQID NO:9-10 is the light chain amino acid sequence of the C1s antibody.

The diagnostic products include detection reagents and detection kits.

The C1s antibody in the present application was prepared by Shanghai Bio-Tech Co., Ltd. according to the amino acid sequence.

The above contents of the present application are described below in conjunction with specific embodiments.

Example 1: C1s antibody performance verification

S1: ELISA binding assay

Coating: Coating with 2 μg/mL B22750901, 100 μL/well, 37°C for 1 h, washing the plate 6 times, and patting dry;

Blocking: casein blocking solution 200 μL/well, temperature maintained at 25°C, duration 1 hour, wash the plate 6 times, and pat dry;

Binding: 400 nM in the first well, 3-fold dilution with blocking solution, 11 gradients, blank in the last well, 100 μL/well, 25°C for 1 hour, wash 6 times, and pat dry;

HRP: Anti-Mouse Fc specific HRP was diluted with blocking solution at 1:65000, 100 μL/well, 25℃ for 1 hour, washed 6 times, and patted dry;

Color development: 100 μL/well color development solution, color development at room temperature in the dark for 4 minutes;

Stop: 50 μL/well stop solution;

Reading: OD450;

Conclusion: The prepared C1s antibody has a good binding effect with C1s antigen.

Table 1: C1s antibody and C1s antigen binding table

Binding nM 400 133.33 44.44 14.81 4.93 1.64 0.548 0.182 0.060 0.020 0.007 0 EC50 C1s antibody 1.455 1.389 1.391 1.433 1.408 1.245 0.882 0.456 0.219 0.104 0.114 0.044 0.4029 Positive Control 1.434 1.257 1.168 1.157 1.124 0.968 0.68 0.385 0.189 0.102 0.064 0.044 0.5121 Negative control 0.046 0.045 0.046 0.049 0.044 0.066 0.046 0.041 0.042 0.046 0.042 0.046 blank 0.052 0.05 0.045 0.048 0.047 0.06 0.044 0.045 0.044 0.045 0.044 0.039

S2: SDS-PAGE detection

Sample denaturation treatment: reducing heating at 99°C for 6 min; non-reducing heating at 99°C for 3 min;

Sample loading: 10µL/well Electrophoresis (ve180): 180V voltage, set time 40min, end electrophoresis when bromophenol blue runs to the red gel strip;

Dyeing and bleaching: Set the dyeing and bleaching time to 13 minutes;

Use a gel imager, set the parameters to white light and take pictures.

Please refer to Figure 1. The results show that the recombinant C1s antibody is two bands on reducing SDS-PAGE and one band on non-reducing SDS-PAGE.

Example 2: C1s antibody FITC labeling experiment

S1: Use a balance to weigh FITC and dissolve it in DMSO to make a solution with a concentration of 10 mg/mL;

S2: Use an ultrafiltration tube to replace the buffer system of C1s antibody with a 10 mM carbonate buffer system: add the antibodies to the ultrafiltration tubes respectively, and add 10 mM carbonate buffer, 5000*g for 30 min, 4 degrees for 30 min.

S3: Repeat step S2 twice;

S4: Aspirate the ultrafiltered antibody, put it into an EP tube, add 5UL of FITC solution, and place it in a constant temperature mixer for reaction for 3 hours (25 degrees, 500 rpm).

S5: Ultrafiltration of the reacted antibodies: Add the antibodies to the ultrafiltration tubes respectively, and add 2 mL of 0.2M pH=7.4 PBS (EDTA) buffer. Centrifuge at 5000×g for 30 min at 4 degrees.

S6: Repeat step S5 twice;

S7: Take out the ultrafiltered antibody, rinse the ultrafiltration tube with 200uL 0.2M PH=7.4 PBS (EDTA) buffer, and add the rinse solution to the antibody.

Embodiment 3:

Platelet C1s-FITC flow cytometry assay

1. C1s-FITC flow cytometry of normal platelet count (100-300×10 ⁹ ) samples:

1. Specimen collection: Use EDTA-K ₂ anticoagulation vacuum blood collection tube to draw 2 mL of venous blood.

2. Staining and fixing cells:

① For negative control, take 50uL PBS (ph=7.2) and add it to the labeled A1 test tube, then add 1ul whole blood and 5ul of the corresponding isotype control as the negative control of fluorescent antibody. Vortex and incubate in the dark for 15min, centrifuge (1500rpm, 5min), aspirate the supernatant (avoid aspirating red blood cells), add 450uL PBS and mix thoroughly before testing.

② For sample testing, take 50uL PBS and add it to the labeled B1 test tube, then add 1uL whole blood, add 5uL of CD61-PE and C1s-FITC fluorescent antibodies respectively, vortex and incubate for 15min, centrifuge (1500rpm, 5min), aspirate the supernatant (avoid aspirating red blood cells), add 450uL PBS and mix thoroughly before testing.

3. Test and analyze the results on the machine. Use FACSComp to adjust the voltage and compensation of the instrument's forward angle (FSC), side angle (SSC), fluorescence 1 (FL1), and fluorescence 2 (FL2). Use SSC as the ordinate and CD61-PE as the abscissa to obtain data (see Figure 2-A). Select the platelet groups and set the gates on Figure A (see Figure 2-A). Use CELLQuest software to obtain 5,000 platelets for analysis, use IgG1-PE as an isotype control (see Figure 2-B), and calculate the proportion of C1s-positive platelets (see Figure 2-C).

(II) C1s-FITC flow cytometry experiment for reduced platelet count (<100×10 ⁹ )

1. Specimen collection: Use EDTA-K ₂ anticoagulation vacuum blood collection tube to draw 2 mL of venous blood.

2. Specimen processing: Centrifuge at 3000rpm for 5min. After centrifugation, gently take out and place on the test tube rack.

3. Staining and fixing cells:

① For negative control, take 40uL PBS and add it to the labeled A1 test tube, then add 10uL upper plasma, and add 5uL of the corresponding isotype control as a fluorescent antibody negative control, vortex and incubate in the dark for 15min, centrifuge (1500rpm, 5min), aspirate the supernatant (avoid aspirating red blood cells), add 450uL PBS and mix thoroughly before testing.

② For sample testing, take 40uL PBS and add it to the labeled A1 test tube, then add 10uL of upper plasma, and add 5uL of CD61-PE and C1s-FITC fluorescent antibodies respectively. After vortexing, incubate in the dark for 15min, centrifuge (1500rpm, 5min), aspirate the supernatant (avoid aspirating red blood cells), add 450uL PBS and mix thoroughly for testing.

3. Test and analyze the results on the machine. Use FACSComp to adjust the voltage and compensation of the instrument's forward angle (FSC), side angle (SSC), fluorescence 1 (FL1), and fluorescence 2 (FL2). Use SSC as the ordinate and CD61-PE as the abscissa to obtain data (see Figure 2-A). Select the platelet groups and set the gates on Figure A (see Figure 2-A). Use CELLQuest software to obtain 5,000 platelets for analysis, use IgG1-PE as an isotype control (see Figure 2-B), and calculate the proportion of C1s-positive platelets (see Figures 2-D and E).

Please refer to Figure 2. The results show that the proportion of C1s-positive platelets in ITP patients is significantly higher than that in the normal population and patients with other types of thrombocytopenia (see Figure 2-D, E, F).

In summary, the specific application of C1s antibody in the diagnosis of ITP through specific examples in this application provides a reliable experimental basis and provides ideas for further research and development of diagnostic methods for ITP in the future.

Claims (8)

1. Application of C1s antibody in the preparation of ITP diagnostic products, characterized in that: the sequence of the C1s antibody is as shown in SEQ ID NO: 1-10, wherein SEQ ID NO: 1-3 is the heavy chain gene sequence of the C1s antibody, SEQ ID NO: 4-5 is the heavy chain amino acid sequence of the C1s antibody, SEQ ID NO: 6-8 is the light chain gene sequence of the C1s antibody, and SEQ ID NO: 9-10 is the light chain amino acid sequence of the C1s antibody. 2. The use of C1s antibody in the preparation of ITP diagnostic products according to claim 1, characterized in that: the diagnostic product comprises a kit and a detection reagent, and the kit and the detection reagent use C1s antibody as a specific detection reagent for the target C1s to be detected. 3. The use of the C1s antibody according to claim 1 in the preparation of an ITP diagnostic product, characterized in that the use comprises a non-diagnostic validation method, wherein the non-diagnostic validation method comprises the following contents: S1: Antibody performance verification experiment; S2: C1s antibody FITC labeling experiment; S3: Platelet C1s-FITC flow cytometry experiment. 4. The use of the C1s antibody according to claim 3 in the preparation of an ITP diagnostic product, characterized in that the antibody performance verification experiment includes an ELISA binding experiment and an SDS-PAGE test. 5. The use of the C1s antibody according to claim 3 in the preparation of an ITP diagnostic product, characterized in that the platelet C1s-FITC flow cytometry experiment includes two groups of experiments, the two groups of experiments are respectively a normal human platelet C1s-FITC flow cytometry experiment and a thrombocytopenia C1s-FITC flow cytometry experiment. 6. The use of the C1s antibody according to claim 5 in the preparation of an ITP diagnostic product, wherein the platelet C1s-FITC flow cytometry detection comprises the following steps: A1: Specimen collection and processing; A2: staining and fixing cells; Specifically including negative control and sample testing: Negative control: add 50ul PBS at pH 7.2 to the labeled A1 test tube, then add 1ul whole blood, and add 5ul of the corresponding isotype control as the negative control of the fluorescent antibody, vortex and incubate in the dark for 15min, centrifuge at 1500rpm for 5min, aspirate the supernatant, add 450ul PBS and mix well before testing; Sample detection: Take 50ul PBS and add it to the marked B1 test tube, then add 1ul whole blood, add 5ul CD61-PE and C1s-FITC fluorescent antibodies respectively, incubate in the dark for 15min after vortexing, centrifuge at 1500rpm for 5min, aspirate the supernatant, add 450ul PBS and mix well before testing; A3: Test on the computer and analyze the results. 7. A kit for diagnosing ITP, characterized in that: the kit uses C1s on platelets as a detection target, and the kit also includes the C1s antibody according to claim 1. 8. A kit for ITP diagnosis according to claim 7, characterized in that the kit further comprises other medically acceptable reagents.