CN114236127A - Hepatitis C virus antigen-antibody joint detection kit and detection method thereof - Google Patents
Hepatitis C virus antigen-antibody joint detection kit and detection method thereof Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N2469/10—Detection of antigens from microorganism in sample from host
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- G01N2469/00—Immunoassays for the detection of microorganisms
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Abstract
The invention discloses a hepatitis C virus antigen-antibody joint inspection kit and a detection method thereof, wherein the kit comprises a micro-fluidic chip, a sample processing solution, a sample diluent and a reference substance, each reaction unit of the micro-fluidic chip comprises an A/B detection area, whether a first compound formed by a donor-hepatitis C virus antibody-receptor exists in the A detection area or not is detected, whether a second compound formed by the donor-hepatitis C virus antigen-receptor exists in the B detection area or not is detected, and a light-excited chemiluminescence reaction reagent is also arranged in each reaction unit of the micro-fluidic chip. The invention organically combines the light-activated chemiluminescence technology with the microfluidic chip technology, is a multi-index joint detection system, avoids the defects of narrow linear range, complex operation and the like of an enzyme-linked immunoassay kit, and overcomes the defects of low sensitivity, poor stability, low flux and the like.
Description
Technical Field
The invention relates to a hepatitis C virus antigen-antibody joint inspection kit and a detection method thereof, belonging to the field of molecular immunology.
Background
Hepatitis C is a global infectious disease caused by infection with Hepatitis C Virus (HCV). It is estimated that 1.7 hundred million hepatitis C virus infected patients exist all over the world at present, the hepatitis C infection rate in China is about 30%, and at least 4000-6000 million hepatitis C patients exist at present. 80-85% of infected patients develop chronic hepatitis C, 20% of infected patients can develop hepatic fibrosis, and 4-5% of patients with hepatic fibrosis finally develop hepatocellular carcinoma, which is very serious in harm. At present, no specific therapeutic drug aiming at hepatitis C virus exists, and the development of hepatitis C vaccine is difficult due to the rapid variation of HCV, and the development is difficult to break through in a short period. Therefore, the early diagnosis of HCV is of great significance for screening HCV infection sources, guiding clinical treatment and prognosis judgment.
The detection of hepatitis C virus has been developed from the previous reagent only detecting virus antibody to the current reagent for detecting antigen and antibody combination. This avoids the risk of missed detection in patients with a window period who have only viral antigens and no corresponding antibodies or who have insufficient corresponding antibody titers to be detected. The hepatitis C antigen-antibody joint detection kit can be used for screening the blood source of HCV and detecting hepatitis C antigen and hepatitis C antibody at the same time, so that the blood use and blood transfusion safety can be improved, the working efficiency is improved, and the cost is saved; in addition, the kit and the detection method thereof can distinguish whether the hepatitis C virus antigen-antibody joint inspection kit and the preparation method thereof are HCV current infection or existing infection, can be used for the early detection and treatment evaluation of HCV, and provide an important hepatitis C virus antigen-antibody joint inspection kit and the detection evaluation index of the preparation method thereof for guiding the clinic.
Related methods for combined detection of hepatitis C antigen and antibody have been introduced in many patent documents, including: a resolution immunofluorescence assay (such as CN102928595A), an enzyme immunoassay (such as CN103018455A), a direct labeling chemiluminescence assay (such as CN103018455A), an indirect labeling chemiluminescence assay (such as CN104237520B), a homogeneous immunoassay (such as CN111272997A), and the like. Among them, the currently used ELISA and chemiluminescence immunoassay methods are heterogeneous immunoassays. Heterogeneous immunoassays require the separation and removal of unreacted labeled antibody distributed in the liquid phase by repeated washing of the solid phase (microparticles) before detection of a signal. Multiple washing inevitably brings washing errors to immunoassay, and affects the precision of the analysis method, thereby affecting the accuracy and the analysis sensitivity of the analysis method.
The microfluidic technology is a newly developed technology in recent years, mainly uses analytical chemistry, controls fluid in a micron-sized structure, performs reaction detection, and has the advantages of small volume, large specific surface area, short reaction time, less reagent and sample consumption, capability of simultaneously detecting multiple samples and multiple indexes and the like. Has important practical value for the discovery of clinical disease markers and early diagnosis of diseases. Conventional immunoassays require relatively long assay times, cumbersome liquid handling procedures, relatively low throughput (only one sample or a few samples of a single item can be tested at a time), and relatively high amounts of biologically active reagents. The microfluidic nano chip can effectively overcome the defects and has extremely strong advantages. The detection which usually needs a sample amount of a few milliliters needs only a sample amount of a few microliters after the microfluidic chip technology is adopted, so that the consumption of samples and reagents is greatly saved, the performance index is superior to that of the common detection method, and the method has wide application prospect and important application value.
At present, analysis by referring to a microfluidic technology is a trend, but no mature microfluidic detection product exists in the aspect of hepatitis C detection, and the design of a microfluidic detection kit is particularly important for meeting the needs of the broad market.
Disclosure of Invention
In view of the above problems in the prior art, the present invention aims to obtain a hepatitis c virus antigen-antibody joint inspection kit and a detection method thereof.
In order to realize one of the above purposes, the technical scheme of the hepatitis C virus antigen-antibody joint inspection kit adopted by the invention is as follows:
the kit comprises a microfluidic chip, sample treatment liquid, sample diluent and a reference substance; each reaction unit of the microfluidic chip comprises an A/B detection area, the A detection area detects whether a first complex formed by a donor-hepatitis C virus antibody-receptor exists, and the B detection area detects whether a second complex formed by a donor-hepatitis C virus antigen-receptor exists.
Preferably, the micro-fluidic chip comprises a freeze-dried product of the photo-activated chemiluminescent reaction reagent. The light-activated chemiluminescence reaction reagent comprises a photosensitive particle-labeled hepatitis C virus capture antibody (antigen) and a luminous particle-labeled hepatitis C virus detection antibody (antigen).
Preferably, the photosensitive particles have a diameter of 180-220nm and are soluble in an aqueous medium, and a sensitizer comprising photoactivation can generate a singlet state in an excited state. The photosensitive compound is excited by light with a special wavelength of 680nm, more than 6 ten thousand singlet oxygen molecules can be generated per second, signals are amplified, but the half-life of singlet oxygen is only 4us, the singlet oxygen is quickly attenuated in an aqueous medium and can be transmitted to a distance of 200nm furthest, if a receptor approaches a donor through a certain mode, such as antibody immunoadsorption, avidin-streptomycin recognition and the like, a fluorescent group in the receptor can quickly absorb singlet oxygen, and a 450-620nm optical signal is generated in an above conversion mode and is detected by a detector. The photosensitive microparticles are linked to a capture antibody (antigen) by avidin-streptomycin.
Preferably, the luminescent fine particles, which comprise an olefin compound and a metal chelate compound, are in a non-particulate form and are soluble in an aqueous medium; the receptor is filled with luminescent compound and lanthanide polymer particles, wherein the lanthanide element in the A detection area is Eu3+, the lanthanide element in the B detection area is Tb3+, and the diameter is 180-220 nm.
Preferably, the capture antigen contained in the A detection area is Core, NS3 and NS4 chimeric antigen 1, and the detection antigen is chimeric antigen 2; the capture antibody contained in the B detection region is an N-terminal epitope aiming at the HCV core antigen, and the detection antibody is a C-terminal epitope aiming at the HCV core antigen.
Preferably, the kit further comprises a hepatitis c virus antigen antibody control, the mixed human serum is used as a negative control, the HCV antibody is added to the negative control to be used as a positive control 1, and the HCV antigen is added to the negative control to be used as a positive control 2.
Preferably, the sample treatment agent is: 12% urea, 3% n-butanol, 0.5% Tween-20, 0.7% CTAB and 10% sodium chloride.
Preferably, the sample diluent is PBS buffer containing BSA (0.5g/L), PC-300 (0.1%).
The invention also discloses a detection method using the kit, which comprises the following steps:
a) taking out the microfluidic chip in the kit and balancing to room temperature;
b) mixing a serum sample to be detected and a sample diluent into 100 mul of liquid to be detected 1 according to the volume of 1:10, and mixing the serum sample to be detected and a sample treatment liquid into 100 mul of liquid to be detected 2 according to the proportion of 1: 1;
c) respectively adding liquid 1 and liquid 2 to be detected into A, B detection areas;
d) incubating at 37 ℃ for 15 min;
e) the reaction wells were irradiated with laser light, and the light signal value of each reaction well was measured.
Preferably, the preparation of the light-activated chemiluminescent reagent is prepared by the following steps:
1) photosensitive microparticle-labeled capture antibody (antigen): adding the capture antibody (antigen) into a sodium carbonate buffer solution, adding activated biotin, reacting for 12-18 hours at 37 ℃, and dialyzing to prepare the capture antibody (antigen) marked by the biotin; coupling the photosensitive particles combined with streptavidin with a capture antibody (antigen), and storing the coupled photosensitive particles in a 0.01M PBS (containing 3% BSA) with pH7.4;
2) luminescent-microparticle-labeled detection antibody (antigen): adding a receptor into a sodium carbonate buffer solution, adding a detection antibody (antigen), rapidly and uniformly mixing, carrying out light-shielding reaction at 37 ℃, adding a carbonate buffer solution containing 75mg/ml Gly, removing liquid to obtain a conjugate of the detection antibody (antigen) and the receptor, and carrying out redissolution and storage by using 0.01M PBS (containing 3% B photosensitive microspheres) with pH7.4;
3) modification of nano-cellulose: preparing the antibodies of 1) and 2) into a reaction reagent according to a proper proportion, and adding nano-cellulose accounting for 0.1% of the volume of the reaction reagent for modification for later use; and respectively preparing the HCV antigen antibody light-induced chemiluminescence reaction reagent.
Preferably, the packaging of the microfluidic chip is prepared by the following steps: adding 100ul of the prepared 2 photo-activated chemiluminescence reaction reagents into a detection area of a microfluidic chip respectively, specifically adding a cHCV antibody and an HCV antigen photo-activated chemiluminescence reaction reagent into the detection area A and the detection area B respectively correspondingly, pre-freezing for 2 hours at-70 ℃, and then freeze-drying for 20 +/-2 hours in a freeze-dryer. And buckling the upper cover of the chip, and carrying out ultrasonic welding to form a closed microfluidic chip for later use.
Compared with the prior art, the invention organically combines the light-activated chemiluminescence technology and the microfluidic chip technology, is a multi-index joint detection system, not only avoids the defects of narrow linear range, complex operation and the like of an enzyme-linked immunoassay kit, but also overcomes the defects of low sensitivity, poor stability, low flux and the like. The invention adopts the rapid, accurate, sensitive and high-flux micro-flow control light-excited chemiluminescence homogeneous immunoassay reagent to detect the hepatitis C virus antigen antibody in the blood plasma, the whole detection process has high flux, small sample amount and no separation and washing process, thereby not only saving the detection time, but also not generating extra waste liquid of the hepatitis C virus antigen antibody joint detection kit and the preparation method thereof, avoiding the washing error and having high precision and sensitivity.
Drawings
FIG. 1 is a schematic diagram of the detection of HCV antigens according to the present invention.
Detailed Description
The present invention provides a hepatitis C virus antigen-antibody joint inspection kit and a detection method thereof, which are described in detail and fully below with reference to examples. The following examples are illustrative only and are not to be construed as limiting the invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.
The kit adopts homogeneous immunization, and is matched with a light-activated chemiluminescence detector by using a light-activated chemiluminescence analysis method to determine the content of hepatitis C virus antigen antibodies in a human body sample. As shown in fig. 1, the technical principle of the reaction is: the sample, photosensitive particle marked hepatitis C virus capture antibody (antigen) and luminous particle marked hepatitis C virus detection antibody (antigen) are mixed under homogeneous phase conditions. At this time, the hepatitis C virus capture antibody (antigen) and the luminescent microspheres coupled with the hepatitis C virus detection antibody (antigen) rapidly and effectively identify the target molecules of the detection sample to form an immune sandwich compound. Forming a photosensitive microsphere-HCV capture antibody (antigen) -antigen (antibody) -HCV detection antibody (antigen) -luminous microsphere immune complex.
Under the irradiation of laser (with the wavelength of 680nm), the photosensitizer on the photosensitive microsphere converts oxygen in the surrounding environment into more active singlet oxygen. The singlet oxygen diffuses into the luminescent microsphere to react with the chemiluminescent reagent on the luminescent microsphere, so that the fluorescent group on the luminescent microsphere is further activated to emit fluorescence, and the wavelength is 615nm (detection area A) or 543nm (detection area B). The half-life of singlet oxygen is 4 mus and the diffusion distance in solution is around 200 nm. If the biomolecules do not have interaction, the singlet oxygen cannot diffuse to the luminescent microspheres, and no fluorescence signal is generated. Within the measuring range, the luminous intensity is in direct proportion to the concentration of the hepatitis C virus antigen antibody in the sample, and the concentration of the hepatitis C virus antigen antibody in the sample to be measured can be quantitatively measured by using improved four-parameter Logistic equation fitting.
Preparing a capture antibody (antigen) marked by photosensitive particles:
(1) preparing a component photosensitive microsphere-streptavidin conjugate:
a. photosensitive microsphere (donor) suspension treatment: centrifuging 10mg of photosensitive microspheres in a high-speed refrigerated centrifuge, removing supernatant, adding 0.2ml of MES buffer solution, and performing ultrasound on an ultrasonic cell disruption instrument until the particles are resuspended;
b. preparing a streptavidin solution: weighing 1mg of streptavidin, and adding 0.2ml of MES buffer solution for dissolving;
c. mixing: mixing the processed photosensitive microsphere (donor) suspension and streptavidin solution in an equal volume ratio, and quickly mixing uniformly;
d. reaction: adding 10ul of NaBH3CN solution prepared by MES buffer solution at a concentration of 40mg/ml, rapidly mixing, and performing rotary reaction at 37 ℃ for 48 hours;
e. and (3) sealing: adding 40ul MES buffer solution to prepare 150mg/ml Gly solution and 25ul 40mg/ml NaBH3CN solution, mixing uniformly, and carrying out rotary reaction at 37 ℃ for 2 hours; then 0.3ml of 200mg/ml BSA solution (MES buffer) was added, and the reaction was carried out for 16 hours at 37 ℃ with rotation;
f. cleaning: centrifuging, discarding the supernatant, adding fresh MES buffer solution, performing ultrasonic resuspension, centrifuging again, and cleaning for 3 times;
g. luminescence reagent buffer solution preparation: 5% (v/v) HEPES, 1% (wt/v) trehalose, 0.5% (wt/v) TrionX-100, (wt/v) 0.1% dextran, pH 7.5. + -. 0.10;
h. diluting and suspending: finally, the suspension was diluted with luminescence reagent buffer at a donor concentration of 200. mu.g/mL.
(2) Preparation of component biotin-labeled capture antibody (antigen):
a. antibody (antigen) treatment: dialyzing the HCV capture antibody (antigen) in 0.1M NaHCO3 solution for 4 h/time, changing the solution for 1 time, measuring the antibody concentration and adjusting to 0.5 mg/ml;
b. marking: collecting 200ul of treated HCV capture antibody (antigen), adding 8ul of 5mg/ml biotin solution, rapidly mixing, standing at 2-8 deg.C for 12-16 hr;
c. and (3) dialysis: dialyzing the reacted biotin-labeled antibody in a biotin-labeled dialysis buffer solution (pH 8.00), dialyzing at 2-8 ℃, and changing the dialysate at least 1 time for at least 4-5 hours each time;
d. diluting and suspending: dialyzed biotinylated antibody was pipetted into a clean centrifuge tube and adjusted to 20ug/ml with 0.01M PBS pH7.4 (containing 3% BSA).
(3) Preparation of photosensitive microparticle-labeled capture antibody (antigen): the photosensitive microsphere-streptavidin conjugate and the biotin-labeled capture antibody (antigen) were mixed in equal volumes and incubated at 37 ℃ for 10 min.
Preparation of (II) component luminous microsphere-detection antibody (antigen) conjugate
a. Antibody (antigen) treatment: dialyzing 0.3mg HCV detection antibody (antigen) in 0.1M NaHCO3 solution for 4 h/time, changing solution for 1 time, measuring antibody concentration and adjusting to 1 mg/ml;
b. receptor treatment: adding 4mg of receptor into a centrifuge tube, adding 0.05M NaHCO3 buffer solution, centrifuging at 7000rpm for 10min, discarding the supernatant, adding 800ul of 0.05M NaHCO3 buffer solution into the centrifuge tube, ultrasonically cleaning, centrifuging again, and discarding the supernatant;
c. mixing and reacting: adding 400ul of 0.05M CB buffer solution with the pH value of 9.6 to resuspend the receptor to ensure that the concentration of the receptor is 10mg/ml, adding 0.2mg of dialyzed HCV detection antibody, uniformly mixing, placing the centrifugal tube at 37 ℃, vertically rotating the mixer, keeping away from light at 25-40 rpm, and uniformly mixing overnight; cooling the centrifugal tube at the temperature of 2-8 ℃ for 10min, taking 8ul of 8mg/ml NaBH4 solution, immediately adding the solution into the centrifugal tube, uniformly mixing, and reacting for 2 hours on a room-temperature mixer;
d. and (3) sealing: adding 70ul of 75mg/ml Gly carbonate buffer solution into a centrifugal tube, uniformly mixing, and reacting for 1 hour at 25-40 rpm on a vertical rotary mixer;
e. cleaning: centrifuging at 7500rpm for 5min, discarding the supernatant, adding 0.1M PBST buffer solution with pH of 7.4, performing ultrasonic cleaning, and repeating twice;
f. diluting and suspending: diluted with 0.01M PBS (containing 3% BSA) pH7.4 to an acceptor concentration of 200. mu.g/mL.
Example 2 test method of kit
(1) Taking out the microfluidic chip in the kit and balancing to room temperature;
(2) mixing 10 mul of serum sample to be detected with 90 mul of sample diluent to obtain 100 mul of solution to be detected 1, and mixing 50 mul of serum sample to be detected with 50 mul of sample treatment solution to obtain 100 mul of solution to be detected 2;
(3) respectively adding liquid 1 and liquid 2 to be detected into A, B detection areas;
(4) incubating at 37 ℃ for 15 min;
(5) the reaction wells were irradiated with laser light, and the light signal value of each reaction well was measured.
Example 3 clinical Performance of the kit
After clinical samples are verified, 135 samples are detected by adopting 3.00pg/ml of plasma HCV as a cut-off value, and the comparative data of the kit and a hepatitis C virus antigen-antibody joint detection kit (enzyme linked immunosorbent assay) of Shandong Leibo Biotechnology limited company are shown in the following table:
clinical detection performance table of plasma HCV kit
Negative coincidence rate: 68/72 ═ 94.44%;
positive compliance rate: 63/63-100%;
the hepatitis C virus antigen antibody light-induced chemiluminescence homogeneous immunoassay kit has good clinical applicability and advancement.
Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.
Claims (10)
1. The kit is characterized by comprising a microfluidic chip, sample processing liquid, sample diluent and a reference substance, wherein each reaction unit of the microfluidic chip comprises an A/B detection area, the A detection area is used for detecting whether a first compound formed by a donor-hepatitis C virus antibody-receptor exists or not, the B detection area is used for detecting whether a second compound formed by the donor-hepatitis C virus antigen-receptor exists or not, and each reaction unit of the microfluidic chip is internally provided with a light-excited chemiluminescence reaction reagent.
2. The hepatitis C virus antigen-antibody joint inspection kit according to claim 1, wherein the light-activated chemiluminescent reaction reagent is a lyophilized product comprising a photosensitive particle-labeled hepatitis C virus capture antibody and a luminescent particle-labeled hepatitis C virus detection antibody.
3. The hepatitis C virus antigen-antibody joint inspection kit of claim 2, wherein the photosensitive particles comprise a light-activated sensitizer with a diameter of 180-220nm, and the photosensitive particles are connected with the capture antibody through avidin-streptomycin.
4. The hepatitis c virus antigen-antibody joint inspection kit of claim 2, wherein the luminescent microparticles comprise an olefinic compound and a metal chelate; the receptor is filled with luminescent compound and lanthanide polymer particles, wherein the lanthanide element in the A detection area is Eu3+, the lanthanide element in the B detection area is Tb3+, and the diameter is 180-220 nm.
5. The hepatitis C virus antigen-antibody joint test kit of claim 4, wherein the capture antigen contained in the detection region A is Core, NS3, NS4 chimeric antigen 1, and the detection antigen is chimeric antigen 2; the capture antibody contained in the B detection region is an N-terminal epitope aiming at the HCV core antigen, and the detection antibody is a C-terminal epitope aiming at the HCV core antigen.
6. The hepatitis c virus antigen-antibody joint inspection kit according to claim 1, wherein the control is a negative control of mixed human serum, the HCV antibody is added to the negative control as a positive control 1, and the HCV antigen is added to the negative control as a positive control 2.
7. The hepatitis c virus antigen-antibody joint inspection kit of claim 1, wherein the sample treatment agent is: 12% urea, 3% n-butanol, 0.5% Tween-20, 0.7% CTAB and 10% sodium chloride.
8. The hepatitis C virus antigen-antibody joint inspection kit of claim 1, wherein the sample diluent is PBS buffer containing BSA 0.5g/L and 0.1% PC-300.
9. A detection method using the hepatitis C virus antigen-antibody joint detection kit according to any one of claims 1 to 8, characterized in that the detection method comprises the following steps:
a) taking out the microfluidic chip in the kit and balancing to room temperature, wherein a light-excited chemiluminescence reaction reagent is also arranged in each reaction unit of the microfluidic chip;
b) mixing a serum sample to be detected and a sample diluent into 100 mul of liquid to be detected 1 according to the volume of 1:10, and mixing the serum sample to be detected and a sample treatment liquid into 100 mul of liquid to be detected 2 according to the proportion of 1: 1;
c) adding the solutions to be detected 1 and 2 in the step b) into A, B two detection areas respectively;
d) incubating at 37 ℃ for 15 min;
e) the reaction wells were irradiated with laser light, and the light signal value of each reaction well was measured.
10. The method for detecting the combined detection of the hepatitis C virus antigen and the antibody according to claim 9, wherein the preparation of the light-activated chemiluminescent reagent comprises the following steps:
i. photosensitive microparticle-labeled capture antibody: adding the capture antibody into a sodium carbonate buffer solution, adding activated biotin, reacting for 12-18 hours at 37 ℃, and dialyzing to prepare a capture antibody marked by the biotin; coupling the photosensitive particles combined with the streptavidin with a capture antibody, and re-dissolving and storing the coupled photosensitive particles and the capture antibody by using PBS (phosphate buffer solution) containing 3% BSA and 0.01M pH7.4;
luminescent microparticle-labeled detection antibody: adding an acceptor into a sodium carbonate buffer solution, adding a detection antibody, quickly and uniformly mixing, carrying out light-shielding reaction at 37 ℃, adding a carbonate buffer solution containing 75mg/ml Gly, removing liquid to obtain a conjugate of the detection antibody and the acceptor, and carrying out redissolving and storage by using 0.01M PBS (phosphate buffer solution) containing 3% B photosensitive microspheres and pH7.4;
nanocellulose modification: preparing the antibodies in the steps i and ii into a reaction reagent according to a proper proportion, and adding nano-cellulose accounting for 0.1% of the volume of the reaction reagent for modification for later use; and respectively preparing the HCV antigen antibody light-induced chemiluminescence reaction reagent.
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| CN116429754A (en) * | 2023-02-13 | 2023-07-14 | 科美博阳诊断技术(上海)有限公司 | HCV detection kit and using method thereof |
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