WO2017008179A1 - Reagent for high throughput combined detection of hepatitis c virus antigen and antibody - Google Patents

Abstract

Disclosed is a reagent for high throughput combined detection of a hepatitis C virus antigen and antibody. Using the flow cytometry principle, different concentrations of FITC fluorescein-labeled microspheres correspond to coated antibodies and antigens; after addition of a detection sample, the microspheres, the detection sample and a PE-labeled paired antibody and antigen form a sandwich structure; and under the action of 480nm exciting light, the effects of simultaneous detection of a hepatitis C virus antigen and antibody can be achieved in a high throughput way according to different emitted light intensities of fluoresceins at different concentrations. By means of the utilization of the fluorescent coloration effect, the reaction sensitivity is enhanced, and a hepatitis C virus antibody and core antigen can be detected simultaneously by the means of utilization of FITC-labeled microspheres for simultaneous detection of the antigen and antibody.

Description

High-throughput reagent for detecting hepatitis C virus antigen antibody Technical field

The invention belongs to the technical field of hepatitis C detection, and particularly relates to a reagent for high-throughput combined detection of hepatitis C virus antigen-antibody.

Background technique

Hepatitis C is a global infectious disease caused by hepatitis C virus (HCV) infection. It is estimated that there are currently 170 million people infected with hepatitis C virus in the world. The hepatitis C infection rate in China is about 30%, and there are at least 40-40 million hepatitis C patients. Among them, 80-85% of infected people will develop chronic hepatitis C, and 20% of them can develop liver fibrosis. Finally, 4 to 5% of patients with liver fibrosis develop hepatocellular carcinoma, and the damage is very serious.

At present, there is no specific treatment for hepatitis C virus. The development of hepatitis C vaccine is also difficult due to the rapid mutation of HCV. It is difficult to make breakthroughs in the short term. Therefore, early diagnosis of HCV is of great significance for screening HCV infection sources, guiding clinical treatment and prognosis.

The existing clinical detection methods of HCV mainly include PCR detection, hepatitis C virus antibody detection and hepatitis C virus core antigen detection kit. Among the three detection methods, the PCR detection method has high sensitivity and accurate results, but the method operation is cumbersome. The required personnel have strong technical ability and are not easy to promote in various hospitals; the hepatitis C virus antibody detection reagent is the most common hepatitis C detection method in clinical practice, but there is a “window period” in the detection, that is, infection in hepatitis C. In the early stage, there was no immune antibody in the patient's body, and the test results could not be obtained at an early stage, resulting in a missed diagnosis. The hepatitis C virus core antigen detection reagent has gradually been accepted by hospitals in recent years, and the market share has also increased. However, for the follow-up detection of hepatitis C patients, there is no good tracking effect.

Summary of the invention

In order to solve the problems of the above various detection methods, the present invention provides a reagent for high-throughput detection of hepatitis C virus antibodies and antigens. The reagent utilizes the principle of flow cytometry to coat the antibody and the antigen in different concentrations of FITC-fluorescein-labeled microspheres, and after sandwiching the test sample, the sandwiched sandwich structure with the PE-labeled paired antibody and antigen constitutes a sandwich structure at 480 nm. Under the action, different concentrations of fluorescein emit light with different intensity, which can achieve high-throughput simultaneous detection of hepatitis C antigen and antibody. The principle of fluorescence detection is adopted to improve the sensitivity of detection, and the antigen and antibody can be qualitatively detected at the same time, and it is possible to determine whether the detection sample has hepatitis C virus infection, and has high clinical application value.

The present invention is achieved by the following measures:

A chemiluminescence method combined with detection of hepatitis C virus antibody-antigen reagent, the components of which are as follows:

Component 1 (R1) FITC Fluorescent Microsphere Reagent:

Prepared with deionized water and adjusted to pH 7.6 with HCl.

The FITC high-brightness microsphere preparation process is: dissolving fluorescein isothiocyanate (FITC) into dimethyl sulfoxide to prepare a 1 mg/mL concentrated dye solution, and the surface is simultaneously aminated and carboxylated polystyrene. The microspheres were mixed at a ratio of 1:1, stored in the dark for 2 hours, washed with absolute ethanol, centrifuged at 15000 r/min for 5 min, the liquid was removed, and the centrifuged solid was dispersed into 100 mL of HEPES buffer (0.1 M, pH=7.4). ), placed at 2-8 ° C, and stored.

The preparation process of FITC low-light microspheres is: dissolving fluorescein isothiocyanate (FITC) into dimethyl sulfoxide to prepare a concentrated dye solution of 1 mg/mL, and the surface is simultaneously aminated and carboxylated polystyrene micro The balls were mixed at a ratio of 1:3, stored in the dark for 2 hours, washed with absolute ethanol, centrifuged at 5000 r/min for 5 min, the liquid was removed, and the centrifuged solid was dispersed into 100 mL of HEPES buffer (0.1 M, pH = 7.4). Place it at 2-8 ° C and store.

Preparation of FITC highlight microspheres coated with hepatitis C core antigen antibody: 1 mL of the above FITC highlight microspheres was added, and 1 mL of carbonate buffer containing 0.005 g of N-hydroxysuccinimide and 0.005 g of carbodiimide was added. (0.1M, pH=8.0), room temperature, protected from light, placed for 2h, centrifuged at 5000r/min for 5min, added 100μL of hepatitis C core antigen coated antibody, added 1mL phosphate buffer (0.2M, pH=7.4), shake at room temperature Incubate for 4 h, add 1 mL of solution containing 01% BSA and 0.5% Tween 20, incubate for 12 h at 4 °C, wash and centrifuge three times with 1 mL of phosphate buffer (0.2 M, pH=7.4), and add 00 mL of HEPES buffer (0.1 M, pH = 7.4), placed at 2-8 ° C, and stored.

Preparation of FITC low-light microspheres coated with hepatitis C virus protein: 1 mL of the above FITC low-light microspheres was added, and 1 mL of a carbonate buffer containing 0.005 g of N-hydroxysuccinimide and 0.005 g of carbodiimide was added ( 0.1M, pH=8.0), room temperature, protected from light, placed for 2h, centrifuged at 5000r/min for 5min, added 100μL of hepatitis C virus mixed protein (NS3, NS4 and NS5), the ratio of various concentrations was 1:1:1, added 1mL phosphate buffer (0.2M, pH=7.4), incubate for 4h at room temperature, add 1mL solution containing 01% BSA and 0.5% Tween 20, incubate for 12h at 4°C, and use 1mL phosphate buffer (0.2M, pH=7.4) Wash and centrifuge three times, add 100 mL of HEPES buffer (0.1 M, pH=7.4), place at 2-8 ° C, and store.

Component 2 (R2) PE Labeling Reagent:

Prepared with deionized water and adjusted to pH 7.6 with HCl.

The PE labeling antibody or protein preparation process is: (1) Preparation of phycoerthrin PE: 600 μL of 15.5 mg/mL iminothiolane hydrochloride is added to 1.2 mL of 3.6 mg/mL. In PE (phycoerythrin), mix with 1.2 mL of phosphate buffer (0.2 M, pH 6.8), place in a dialysis bag and place in 50 mmol/L pH 6.8 phosphate buffer for dialysis, overnight at 4 ° C. The cells were dialyzed against 50 mmol/L pH 7.5 phosphate buffer for 6 h. (2) PE-labeled antibody or protein: Take 30 μL of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide (SPDP) 1.1 mg/mL in ethanol, and add 700 μL of 4.2 mg/mL antibody or The protein was phosphate buffered (50 mmol/L, pH 7.5) and allowed to react at room temperature for 2.5 h. 400 μL of the above thiolated phycoerythrin was further added, and reacted at room temperature for 12 hours. 100 μl of 50 mmol/L sodium iodoacetate was added to block the residual sulfhydryl group, and dialyzed against 50 mmol/L pH 7.5 phosphate buffer at 4 ° C overnight, and 0.01% Na ₃ N was added. ₃ packs, placed at 2-8 ° C, and stored.

Cleaning fluid

All components were dissolved in deionized water to prepare a solution.

Positive control

Negative control

BSA 4%

Dissolved with calf serum.

The kit of the present invention is used as follows:

(1) Prepare a flat-bottomed test tube according to the needs of the experiment, and make a number;

(2) Add 15 μl of positive control, negative control or sample to the test tube;

(3) using a continuous pipette to add 30 μl of R2 (PE labeling reagent) to the test tube;

(4) placed on the shaker and gently shake until thoroughly mixed, and incubated at 37 ° C for 5 minutes;

(5) Add 30 μl of R1 (FITC fluorescent microsphere reagent) to the test tube with a continuous pipette, and ensure that the fluorescent microsphere reagent is thoroughly mixed during the loading;

(6) placed on a shaker and gently shake until thoroughly mixed, and incubated at 37 ° C for 5 minutes;

(7) Centrifuge the tube for 5 min using a centrifuge at 5000 r/min, let stand for 2 minutes, then pour the supernatant into the arc;

(8) using a continuous pipette in a test tube to add 300 μl of the cleaning solution, placed on a shaker at 1500 ~ 2000 rpm for 20 seconds, until thoroughly mixed, repeat the (7) operation once;

(9) repeating the operation of (8) twice;

(10) 1000 μl of the washing solution was added to each tube, and the luminescence intensity FL2-H was measured by flow cytometry.

Threshold determination: Negative control mean FL2-H value × 2.1 (if the negative control average FL2-H value ≤ 5, calculated as 5; OD > 5, according to the actual measurement negative control average FL2-H value × 2.1). The FL2-H value of the test specimen is less than the critical value and is HCV negative. The FL2-H value in the test specimen is equal to or greater than the critical value and is HCV positive.

The reaction principle diagram of the kit of the present invention is shown in FIG.

According to the technical scheme of the present invention, a combined detection kit for simultaneously detecting a hepatitis C virus antigen-antibody can be obtained, which is coated by binding antibodies and antigens at different concentrations of FITC-fluorescein-labeled microspheres. After detecting the sample, the PE-labeled paired antibody and antigen form a sandwich structure. Under the action of 480 nm excitation light, different concentrations of fluorescein emit different light intensity, which can achieve high-throughput simultaneous detection of hepatitis C antigen and antibody. The sensitivity of the reaction is enhanced by the fluorescent color development effect, and the hepatitis C virus antibody and the core antigen can be simultaneously detected by using FITC-labeled microspheres to simultaneously detect antigens and antibodies. The high-throughput detection kit can simultaneously monitor the hepatitis C core antigen and hepatitis C antibody on the flow cytometer, and more intuitively identify the infection period of hepatitis C, and has high application value for clinical hepatitis C detection.

The result of detecting the positive result of the kit of the present invention is shown in FIG. 2 .

The innovation of the kit for detecting hepatitis C virus antigen-antibody of the present invention is:

(1) Using high-concentration FITC-labeled microspheres to coat anti-HCV core antigen antibodies, while using low-concentration FITC-labeled microspheres to coat other fragments of hepatitis C antigen (NS3, NS4, NS5), the immune reaction can be used in the sample. The antibodies to hepatitis C core antigen and various fragment antigens can be adsorbed to the surface of the microspheres by an immune reaction, thereby avoiding missed detection.

(2) The PE label in the kit is a PE enzyme label for the corresponding antibody and antigen paired with the coated antibody and the antigen, and can be paired with the coated antibody and the antigen to ensure the specificity of the kit detection and accurate detection. Hepatitis C virus.

(3) The kit of the invention adopts the principle of flow cytometry, and uses the fluorescence color rendering technology to calculate the result, thereby effectively ensuring the sensitivity of the kit, and can be detected even in a trace state, and the effect of early detection is achieved.

(4) The component of the kit of the invention is a liquid component, which is free from the influence of the solid phase carrier by the enzyme-linked immunosorbent assay, is not restricted by the test sample, and the detection result is more intuitive. In some studies, the combined detection of hepatitis C core antigen-hepatitis C antibody by enzyme-linked immunosorbent assay has resulted in waste of slats due to the number of samples, and it is not possible to visually express the content of hepatitis C core antigen or antibody. The kit of the present invention uses different FITC contents. The labeled microspheres can distinguish different detection items, and can achieve the purpose of simultaneously detecting hepatitis C core antigen and hepatitis C antibody, and have good clinical application value for detection and treatment tracking of hepatitis C.

DRAWINGS

Figure 1 is a schematic diagram of the reaction principle;

Figure 2 is a graph showing positive results;

Figure 3 is a graph showing the results of different infection days in Example 1, wherein a, the test results of 1-6 days of infection (both core antigen and antibody are negative); b, 7-38 days of infection (core antigen positive, Antibody negative); c, test results after 39 days of infection (both core antigen and antibody are positive).

detailed description

For a better understanding of the invention, it will be further described below in conjunction with the specific embodiments.

Example 1

The composition and concentration of the detection reagent are

Component 1 (R1) FITC Fluorescent Microsphere Reagent:

Prepared with deionized water and adjusted to pH 7.6 with HCl.

Component 2 (R2) PE Labeling Reagent:

Prepared with deionized water and adjusted to pH 7.6 with HCl.

Cleaning fluid:

All components were dissolved in deionized water to prepare a solution.

Positive control:

Dissolved with calf serum.

Negative control:

BSA 4%

Dissolved with calf serum.

The kit of the present invention is used as follows:

(1) Prepare a flat-bottomed test tube according to the needs of the experiment, and make a number;

(2) Add 15 μl of positive control, negative control or sample to the test tube;

(3) using a continuous pipette to add 30 μl of R2 (PE labeling reagent) to the test tube;

(4) placed on the shaker and gently shake until thoroughly mixed, and incubated at 37 ° C for 5 minutes;

(5) Add 30 μl of R1 (FITC fluorescent microsphere reagent) to the test tube with a continuous pipette, and ensure that the fluorescent microsphere reagent is thoroughly mixed during the loading;

(6) placed on a shaker and gently shake until thoroughly mixed, and incubated at 37 ° C for 5 minutes;

(7) Centrifuge the tube for 5 min using a centrifuge at 5000 r/min, let stand for 2 minutes, then pour the supernatant into the arc;

(8) using a continuous pipette in a test tube to add 300 μl of the cleaning solution, placed on a shaker at 1500 ~ 2000 rpm for 20 seconds, until thoroughly mixed, repeat the (7) operation once;

(9) repeating the operation of (8) twice;

(10) 1000 μl of the washing solution was added to each tube, and the luminescence intensity FL2-H was measured by flow cytometry.

Example 2

The composition and concentration of the detection reagent are

Component 1 (R1) FITC Fluorescent Microsphere Reagent:

Prepared with deionized water and adjusted to pH 7.6 with HCl.

Component 2 (R2) PE Labeling Reagent:

Prepared with deionized water and adjusted to pH 7.6 with HCl.

The washing solution, positive control, negative control and detection method were as in Example 1.

Example 3

The composition and concentration of the detection reagent are

Component 1 (R1) FITC Fluorescent Microsphere Reagent:

Prepared with deionized water and adjusted to pH 7.6 with HCl.

Component 2 (R2) PE Labeling Reagent:

Prepared with deionized water and adjusted to pH 7.6 with HCl.

The washing solution, positive control, negative control and detection method were as in Example 1.

Effect test 1:

Accuracy analysis of kits for detection of hepatitis C virus antigen-antibody by flow cytometry, using ortho's kit as a gold standard, for 40 clinical samples, for comparison of results, if the test results are different, send The third-party inspection agency uses the hepatitis C virus PCR detection method for verification. The test results are shown in Table 1:

Serial number Example 1 Example 2 Third party verification Serial number Example 1 Example 2 Third party verification 1 - - twenty one - - 2 + + twenty two + + 3 - - twenty three - - 4 - - twenty four + - + 5 + + 25 + + 6 + + 26 + + 7 - - 27 - - 8 + - + 28 - - 9 + + 29 - - 10 + + 30 + + 11 + + 31 - - 12 + + 32 + + 13 - - 33 + + 14 - - 34 - - 15 - - 35 + + 16 - - 36 + + 17 - - 37 + + 18 + + 38 - - 19 + + 39 + + 20 - - 40 - -

After comparison test, the results of the samples No. 8 and No. 24 were abnormal, and were verified by a third party. Example 1 The results of the detection of the present invention are more accurate, and the two samples which are abnormal are mainly autoimmune antibodies, and the concentration of the hepatitis C virus core antigen is relatively low, resulting in missed detection. According to the detection result, the detection result of the kit of the present invention is more accurate, and the occurrence of the miss detection is prevented.

Effect Test 2:

The sensitivity of the kit was verified by dilution of the positive sample. The specific implementation is as follows:

A hepatitis C virus-positive sample was subjected to gradient dilution using a normal negative sample, and each of Example 1, Example 2, Example 3, and the orthovirus hepatitis C virus core antigen detection kit (ELISA) was used. The positive samples of the dilution gradient were tested, and the test results are shown in Table 2.

Dilution ratio 1 1/2 1/4 1/8 1/16 1/32 Example 1 test results + + + + + + Example 2 test results + + + + + + Example 3 test results + + + + + + US ortho reagent test results + + + + - -

According to the results of Table 2, the sensitivity of the ortho company is 1/8, and the sensitivity of the embodiment 1, the embodiment 2, and the embodiment 3 can reach 1/32. This result indicates that the kit of the present invention has higher sensitivity. Even if the concentration of hepatitis C virus in the sample is very low, it can be well detected, and the effect of early detection and early prevention can be achieved.

Effect Test 3:

For a case of HCV patient infection cycle serial serum samples, use Example 1, Roche's hepatitis C virus nucleic acid quantitative detection kit (PCR-fluorescence), ortho's hepatitis C virus core antigen detection kit (enzyme-linked immunosorbent assay) The method was tested with the hepatitis C virus antibody diagnostic kit (enzyme-linked immunosorbent assay), and the PCR-fluorescence assay was positive for the first day. The results of the method tests for HCV patients after infection were as shown in Table 3:

The results of Example 1 for different infection days are shown in Figure 3.

From the results of Table 3, it is shown that Example 1 of the present invention can detect hepatitis C infection earlier than the hepatitis C virus core antigen detection kit of Ortho Division (enzyme-linked immunosorbent assay), while utilizing high-brightness microspheres and low-light micro-micro The ball distinguishes different detection items, and can directly obtain the positive status of hepatitis C core antigen and hepatitis C antibody, which has guiding significance for different infection cycles.

The traditional hepatitis C virus detection kit is detected by enzyme-linked immunosorbent assay, which has poor mobility, low sensitivity and unacceptable accuracy. The present invention utilizes the principle of flow cytometry by passing microspheres labeled with FITC at different concentrations. Corresponding coated antibody and antigen, after adding the test sample, and sandwiching the PE-labeled paired antibody and antigen to form a sandwich structure (as shown in Fig. 1), under the excitation light of 480 nm, different concentrations of fluorescein emit light intensity is different, can be high The flux achieves the simultaneous detection of hepatitis C antigens and antibodies. The reagent kit is a liquid reagent, which is free from the limitation of the slats of the 96-well plate and can be more maneuverable. The sensitivity of PE fluorescence is much higher than that of the enzyme, which enhances the sensitivity of the reaction, and uses micro-labels with different FITC content. The ball can distinguish different detection items, and can achieve the purpose of simultaneously detecting hepatitis C core antigen and hepatitis C antibody. The detection of hepatitis C is more accurate and will not be missed, and the effect of early detection is achieved, which has high application value for the prevention of hepatitis C.

Claims (1)

The invention relates to a high-throughput combined detection reagent for hepatitis C virus antigen antibody, which comprises the components 1, the component 2, the cleaning solution, the positive control and the negative control, and the specific composition thereof is as follows: 1) Component 1 is a FITC fluorescent microsphere reagent comprising components at the following concentrations:

The above reagent was prepared with deionized water, and the pH of HCl was adjusted to 7.6; The preparation process of FITC high-brightening microspheres is as follows: dissolving fluorescein isothiocyanate into dimethyl sulfoxide to prepare a concentrated dye solution of 1 mg/mL, and polystyrene microspheres with simultaneous surface amination and carboxylation Mix in a ratio of 1:1, store in the dark for 2h, wash with absolute ethanol, centrifuge at 15000r/min for 5min, remove the liquid, and disperse the centrifuged solid into 100mL HEPES buffer pH=7.4, 2-8°C Place, save; The preparation process of FITC low-light microspheres is as follows: dissolving fluorescein isothiocyanate into dimethyl sulfoxide to prepare a concentrated dye solution of 1 mg/mL, and the polystyrene microspheres with the surface simultaneously aminated and carboxylated according to 1 The ratio of 3 was mixed, stored in the dark for 2 hours, washed with absolute ethanol, centrifuged at 5000 r/min for 5 min, the liquid was removed, and the centrifuged solid was dispersed into 100 mL of HEPES buffer at pH=7.4, placed at 2-8 °C. ,save; Preparation of FITC high-brightening microspheres coated with hepatitis C core antigen antibody: 1 mL of the above FITC highlight microspheres was added, and 1 mL of carbon having pH=8.0 containing 0.005 g of N-hydroxysuccinimide and 0.005 g of carbodiimide was added. Acidate buffer, room temperature, protected from light, place for 2h, centrifuge at 5000r/min for 5min, add 100μL of hepatitis C core antigen coated antibody, add 1mL phosphate buffer with pH=7.4, incubate for 4h at room temperature, add 0.1% BSA and 1 mL of 0.5% Tween 20 solution, incubate for 12 h at 4 °C, and centrifuge three times with 1 mL of pH=7.4 phosphate buffer, add 100 mL of HEPES buffer at pH=7.4, place at 2-8 °C, and store; Preparation procedure of FITC low-light microspheres coated with hepatitis C virus protein: 1 mL of the above FITC low-light microspheres was added, and 1 mL of pH=8.0 carbonate containing 0.005 g of N-hydroxysuccinimide and 0.005 g of carbodiimide was added. Buffer, room temperature, protected from light, placed for 2h, centrifuged at 5000r/min for 5min, added 100μL of Hepatitis C virus mixed protein NS3, NS4 and NS5, the ratio of various concentrations was 1:1:1, added 1mL pH=7.4 phosphate buffer Incubate for 4 h at room temperature, add 1 mL of solution containing 0.1% BSA and 0.5% Tween 20, incubate for 12 h at 4 °C, wash and centrifuge three times with 1 mL of pH=7.4 phosphate buffer, and add 100 mL of HEPES with pH=7.4. Place in buffer at 2-8 ° C and store; 2) The component 2 is prepared from the following concentrations of reagents:

Component 2 was prepared with deionized water, and HCl was adjusted to pH 7.6; The preparation process of the PE-labeled antibody or protein is: (1) preparation of thiolated phycoerythrin: 600 μL of 15.5 mg/mL sterol imidate is added to 1.2 mL of 3.6 mg/mL phycoerythrin PE, and 1.2 mL, pH 6.8 phosphate buffer, mixed, placed in a dialysis bag, placed in 50 mmol / L pH 6.8 phosphate buffer for dialysis, overnight at 4 ° C, then replaced with pH 7.5, 50 mmol / L phosphate buffer Dialysis for 6 h; (2) PE-labeled antibody or protein: Take 30 μL of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide SPDP 1.1 mg/mL in ethanol, and add 700 μL of 4.2 mg/mL antibody. Or protein pH 7.5 phosphate buffer, reacted at room temperature for 2.5h; add the above thiolated phycoerythrin 400μL, react at room temperature for 12h, add 100μl of 50mmol / L sodium iodoacetate to block the residual sulfhydryl group, with 50mmol / L Dialysis overnight at pH 7.5 phosphate buffer at 4 ° C, adding 0.01% Na ₃ N _{3 in 5} parts, placed at 2-8 ° C, and stored; 3) The cleaning solution comprises components of the following concentrations:

All components are dissolved in deionized water to prepare a solution state; 4) The positive control contains components at the following concentrations:

The solvent is calf serum; 5) The negative control is a 4% BSA solution in which calf serum is dissolved.

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