CN111398584A - Protein chip for detecting HIV1/2 antibody and preparation method thereof - Google Patents
Protein chip for detecting HIV1/2 antibody and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of immunochemistry, and particularly relates to a protein chip for detecting HIV1/2 antibody and a preparation method thereof. The protein chip comprises a replaceable aldehyde group modified detection substrate, wherein the surface of the detection substrate comprises a detection area formed by detection points, and HIV recombinant antigens are fixed on the detection points. The protein chip for detecting the HIV1/2 antibody has the characteristics of less sample consumption, time saving, labor saving and high accuracy when used for detecting the HIV antibody.
Description
Technical Field
The invention belongs to the field of immunochemistry, and particularly relates to a protein chip for detecting HIV1/2 antibody and a preparation method thereof.
Background
Human Immunodeficiency Virus (HIV), i.e., the Virus of AIDS, a retrovirus that is a lentivirus that infects cells of the human immune system (L entivirus).
Protein chip (proteincchip) is a high-grade new technology integrating microelectronics, micromachine, chemical physical technology and computer technology into one body, and is considered as a high-efficiency tool in life science research, and is characterized by that various proteins are orderly fixed on a carrier to be a chip for detection, then the protein or other components marked with specific fluorescent antibody are used to make action with the chip, and the component which can not be complementarily combined with protein on the chip can be washed away by rinsing, then the fluorescent intensity of every point on the chip can be measured by using fluorescent scanner or laser confocal scanning technology, and the interaction relationship between protein and protein can be analyzed by means of fluorescent intensity so as to attain the goal of measuring various protein functions.
The protein chip technology has the advantages of high flux, miniaturization, rapid parallel analysis, small sample amount, high sensitivity, high specificity and the like, and is widely applied to the fields of clinical tumor, infectious diseases, autoimmune disease detection, drug research and development and the like. High throughput protein microarray methods can classify HIV monoclonal antibodies and variant antigens.
Polyclonal immune responses in plasma samples of HIV-infected subjects show different binding patterns, and antibodies to humoral responses to HIV can be found with precise diversity and specificity in response to blotted different antigenic proteins. Therefore, at present, the researchers use protein chip technology to coat recombinant HIV antigen to diagnose AIDS, and the research of the method has high specificity (99.97%) and sensitivity (100%), and improves the diagnosis efficiency and accuracy. In addition, HIV chips can be used to monitor the course of disease and antibody responses during therapy.
According to the national AIDS detection technical Specification (revised 2015), the laboratory detection of HIV mainly comprises HIV antibody detection, HIV nucleic acid qualitative and quantitative detection and CD4+T lymphocyte count, HIV drug resistance detection, and the like. HIV1/2 antibody (human immunity)The most common method for clinical HIV antibody screening is immunoenzyme-linked immunosorbent assay (E L ISA), HIV E L ISA reagent has been developed to the 4 th generation so far, the mainstream reagent and method for HIV detection in international and domestic at present is 3 rd generation reagent-antibody in double-antigen sandwich assay specimen, the sensitivity and specificity of detection are obviously improved compared with 1 st and 2 nd generation reagents, the reactivity of O group specimen is improved, the window period is shortened to 22 days, but compared with more advanced analytical techniques such as chemiluminescence and time-resolved fluorescence immunoassay, the method has lower sensitivity, false positive can occur between coated micropores for detection due to carrying pollution, the 4 th generation reagent increases the detection of P24 antigen compared with the 3 rd generation reagent, the detection positive rate is increased, but research shows that the specificity of the 4 th generation reagent is lower than that of the 3 rd generation reagent, because the former coated component contains HIV P2 antibody, and 539% of the antibody in blood of people with unknown reasons to 1% -2% of the antigen.
The only confirmation method for specifying HIV infection in China national AIDS detection technical Specification is an immunoblotting experiment (WB) which is the most specific and most sensitive method for confirming HIV infection and is a gold standard for antibody detection, HIV-1 and HIV-2 are found, HIV-1 is divided into M groups, 10 subtypes (A-J), O groups and N groups are totally included, HIV-2 also has 7 subtypes (A-G), HIV-1 is popular in China, more than 8 subtypes are detected, B subtypes are almost found in each region of China, C subtypes are mainly found in southwest and northwest regions of China, E subtypes are mostly found in southeast coastal regions and southwest border regions, a few HIV-2 infectors are found and confirmed in partial regions, the judgment standard of HIV western blotting experiments (WB) in China is that HIV-1 antibody positive, at least 2 env bands (gp41 and gp 35120), or gp1 and env 2 infectors are found in southwest regions, and are strictly confirmed in China, and the detection cost of HIV-2 is increased along with the gp 632, and the detection of HIV-2 infection is increased, and the detection of HIV-2 is increased by the detection of a high detection cost of HIV-2 and the detection of a high false blotting experiment (the detection of a high detection).
Disclosure of Invention
The invention is provided and completed in order to overcome the problems of high false positive rate of HIV third generation reagent and the limitation of immunoblotting experiment detection.
The invention aims to provide a protein chip for detecting HIV1/2 antibody.
Another object of the present invention is to provide a protein chip assay kit for detecting HIV1/2 antibody.
Another objective of the invention is to provide a preparation method of a protein chip for detecting HIV1/2 antibody.
The embodiment of the invention provides a protein chip for detecting HIV1/2 antibody of human immunodeficiency virus, which comprises a substrate, wherein HIV specific recombinant antigens gp36, gp41, gp120 and gp160 detection points are fixed on the surface of the substrate.
Preferably, the protein chip for detecting HIV1/2 antibody according to the present invention, wherein the substrate is aldehyde-modified glass.
Preferably, the protein chip for detecting HIV1/2 antibody of human immunodeficiency virus according to the invention, wherein, WB clinical verification detects HIV specific recombination antigens gp36, gp41, gp120, gp160 molecular weight bands are displayed in the form of chip point;
preferably, the protein chip for detecting HIV1/2 antibody of human immunodeficiency virus according to the present invention, wherein if the protein chip can perform synchronous detection against HIV specific recombinant antigens gp36, gp41, gp120, gp160 antibodies (IgG antibody and IgM antibody) of AIDS patients by replacing different labeled fluorescent agents;
preferably, the protein chip for detecting HIV1/2 antibody of human immunodeficiency virus according to the invention, wherein HIV specific recombinant antigens gp36, gp41, gp120, gp160 diluent are diluted with 2 × SCC (sodium citrate) buffer solution, which is beneficial to preservation of antigen activity.
Preferably, the protein chip for detecting HIV1/2 antibody of human immunodeficiency virus according to the invention, wherein the HIV specific recombinant antigen gp36 has a coating concentration of 3.125ug/m L, gp41 has a coating concentration of 12.5ug/m L, gp120 has a coating concentration of 200ug/m L, and gp160 has a coating concentration of 12.5ug/m L.
Preferably, the protein chip for detecting HIV1/2 antibody according to the present invention, wherein the control region has a negative control formed by fixing 10% Bovine Serum Albumin (BSA).
Preferably, the protein chip for detecting HIV1/2 antibody according to the present invention comprises a plurality of detection regions and a control region, wherein any detection region comprises at least one group of HIV-specific recombinant antigens gp36, gp41, gp120 and gp160 detection regions, and the concentration of the same antigen in the same detection region is the same.
Preferably, the protein chip for detecting HIV1/2 antibody according to the present invention, wherein the detection chip comprises ten detection regions, each detection region comprises 10 spots, the spots immobilized with HIV recombinant antigen have a diameter of 50 μm and a spot spacing of 100 μm, and the spots form an array of 2.5mm × 2.5.5 mm.
The embodiment of the invention provides a protein chip detection kit for detecting HIV1/2 antibody, wherein the detection kit comprises a protein chip and a detection reagent, the protein chip comprises a substrate, and HIV specific recombinant antigens gp36, gp41, gp120 and gp160 detection points are fixed on the surface of the substrate.
Preferably, the protein chip detection kit for detecting the HIV1/2 antibody is characterized in that the detection reagent is an enzyme-labeled antibody, more preferably a horseradish peroxidase-labeled antibody.
The embodiment of the invention provides a preparation method of a protein chip for detecting HIV1/2 antibody, which comprises the following steps:
the HIV recombinant antigen is fixed on the substrate of the protein chip, and the recombinant antigen comprises HIV antigens gp36, gp41, gp120 and gp 160.
Preferably, the method for preparing the protein chip for detecting the HIV1/2 antibody according to the present invention comprises the step of treating aldehyde groups on the surface of the substrate of the protein chip.
Compared with the prior art, the invention has the beneficial effects that:
1) the invention realizes that different HIV type antigens are simultaneously arrayed on one substrate, IgG and/or IgM antibodies of multiple human samples can be simultaneously detected on one glass slide, the required sample amount is small (15 mu L), a reaction result with multiple indexes can be obtained by only one reaction, the detection speed and efficiency are improved, and more indexes can be provided for clinic.
2) The protein chip of the invention needs less antigen, 1u L can sample at least 5 chips, the antigen quantity needed by detecting the blood sample of 1 patient is far lower than that of the E L ISA method, the invention is simple and rapid, and the detection cost is reduced.
3) The protein chip of the invention has high detection specificity, stable detection result and high reliability, and is beneficial to clinical diagnosis and treatment. The method avoids false positive caused by misjudgment of strips and improper operation in the existing method and solves the problem that the detection result of 4-20% of sample WB for primary detection of HIV positive is 'antibody uncertain' in the existing detection method.
4) The manufacturing process is simple, the operation is safe, the automation can be realized, and the practicability is high.
In conclusion, the protein chip for detecting the HIV1/2 antibody has the characteristics of less sample dosage, time saving, labor saving and high accuracy when used for detecting the HIV antibody.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a block antigen layout of a glass chip prepared according to example 1 of the present invention, in which 10 persons per chip;
FIG. 2 is a diagram of scanning entity of the aldehyde chip protein IgG coating detection line quality control experiment chip in the preparation process according to example 1 of the present invention;
FIG. 3 is a chemiluminescence scan of a coating antigen concentration gradient and a plasma dilution gradient during preparation in accordance with example 1 of the invention;
FIG. 4 is a chemiluminescence scan of a concentration gradient experiment of avidin HRP-labeled secondary antibody.
FIG. 5 shows fluorescence intensity scans of plasma results from HIV protein chip assays for patients, including 8 HIV patients and 1 normal control and 1 blank control.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The technical solutions provided by the embodiments of the present application are described in detail below with reference to the accompanying drawings.
The invention provides a protein chip for detecting HIV1/2 antibody, which comprises a substrate, wherein the surface of the substrate is replaceable detection paper, the surface of the detection paper comprises one or more detection areas and a control area, the detection areas comprise one or more detection points, HIV recombinant antigens are fixed on the detection points, the HIV recombinant antigens comprise HIV specific recombinant antigens gp36, gp41, gp120 and gp160, and each detection point corresponds to a plasma or serum sample.
According to the technical scheme of the invention, the substrate of the protein chip can be a glass sheet or a silicon sheet, and the aldehyde group of the active functional group is introduced to the surface of the substrate, so that the protein can be effectively and firmly fixed.
The surface of the substrate is adhered with gummed paper to physically separate the chip into ten independent detection areas which can be used alternatively.
The method can utilize a high-speed full-automatic sample applicator to sample a 1-10n L recombinant antigen solution and a negative control solution on a substrate, wherein the sample application conditions are that the temperature is 25 ℃ and the drying environment is adopted, the sample application form can be improved under the sample application conditions, so that the distribution of an antibody on a glass slide is more uniform, proteins are connected to the substrate treated by aldehyde groups through chemical bonds to form Schiff alkali to be fixed, the Schiff alkali is kept at 4 ℃ for overnight, and then the vacuum drying is carried out for 1-4 hours.
One of HIV specific recombinant antigens gp36, gp41, gp120 or gp160 is fixed at any detection point, and the detection region at least comprises one gp36, gp41, gp120 and gp160 detection point.
The size of the coating of the antigen and the control dots can be changed according to the size of the dots and the change of the dot spacing, and the array arrangement and distribution quantity can be changed according to the number of samples to be detected, for example, the glass chip block antigen arrangement diagram shown in FIG. 1 is provided with a plurality of detection areas on a substrate, each detection area comprises 4 detection dots arranged in 4 rows, the control area comprises 4 control dots arranged in 1 row, the detection points and the control dots are arranged in 5 parallel rows, the detection areas are separated by detachable adhesive separation adhesive tape, a specific frame structure is adopted before spotting, the chip lattice is optimized, so that the samples can be detected in batches, the operation is simple, the sample consumption is small, the samples are not cross-polluted, the diameter of the antigen and the control dots is 50 μm, when the dot spacing is 100 μm, the size of the antigen array is × 2.5.5 mm, and 10 detection areas can be simultaneously arranged on one glass substrate, and 10 samples can be detected on each chip.
The method for detecting the AIDS antibody by using the protein chip comprises the following steps:
(1) sealing the nonspecific sites on the surface of the substrate by using a sealing solution containing 10% fetal calf serum, and incubating for 1-1.5 hours at 37 ℃; the 10% fetal bovine serum blocking solution is obtained by diluting fetal bovine serum with PBST.
(2) Washing with PBST solution for 3-4 times, each time for 10 s, washing with PBS, centrifuging, and removing excessive blocking solution;
(3) dropping a patient plasma or serum sample 15u L on the chip, and placing the chip in a wet box to incubate for 30 minutes at 37 ℃ so as to ensure that the antigen and the antibody fully react;
(4) washing with PBST for 3-4 times, each time for 10 seconds, washing with PBS, centrifuging, and removing redundant samples;
(5) dropping HRP-labeled Anti-Human secondary antibody (HRP-Anti-Human-IgG) diluted by PBS and mixed solution with the concentration of 0.4ug/ml, and incubating for 30 minutes at 37 ℃ in a dark place;
(6) washing with PBST for 3 times, each time for 10 s, washing with PBS, and centrifuging;
(7) scanning and collecting signals by using a chemiluminescence scanner, and analyzing a detection result according to scanning display.
Obviously, additional detection reagents are necessary for the detection of AIDS antibodies using the above protein chip. The detection reagent may be an enzyme-labeled antibody commonly used in chemiluminescent reaction methods, such as a horseradish peroxidase-labeled antibody.
The concentration of the enzyme-labeled antibody is related to the detection effect, and the concentration of the enzyme-labeled antibody is preferably more than or equal to 0.4ug/m L in the embodiment of the invention.
The equipment and reagents used in the following examples were:
1. the main instruments and equipment comprise a sample applicator, a German NanoPluter NP 2.1/2GeSim ultramicro sample applicator, a chemiluminescence scanner, a Bio-rad Berle ChemiDoc MP chemiluminescence imaging system, a centrifuge, and a Thermoscientific Sorvall L egend Micro 17&21 series centrifuge.
2. Main reagents and sources thereof: aldehyde based chips were purchased from baiao corporation, shanghai; human IgG was purchased from abcam, england; the HRP-labeled donkey anti-human IgG antibody is purchased from Shanghai Biotech engineering Co., Ltd; immobilon Western HRP substrate purchased from Merckmillipore corporation; PBS powder, Tween20, and fetal Bovine Serum (BSA) powder were purchased from Sigma; PBST buffer: commercial PBS powder was dissolved in deionized water to form PBS buffer at a concentration of 0.01M, pH ═ 7.4; and adding Tween-20, and mixing uniformly to obtain a PBST solution, wherein the volume concentration of Tween20 in the PBST solution is 0.1%.
The source of the HIV-1/2 recombinant antigen is shown in Table 1.
Example 1
Quality control experiment
1. Aldehyde chip protein IgG coating detection line quality control experiment
1) Human IgG solution and PBS blank control solution which are diluted by gradient and 200 mu g/m L, 100 mu g/m L, 50 mu g/m L0, 25 mu g/m L, 12.5 mu g/m L, 6.25 mu g/m L, 3.13 mu g/m L, 1.56 mu g/m L, 0.78 mu g/m L, 0.39 mu g/m L and 0.19 mu g/m L are respectively spotted on a detection area on a substrate, 3 duplicate spots are spotted on each concentration of IgG, and the substrate is incubated for 12 hours at 4 ℃;
2) the chip is sealed by 10% fetal bovine serum albumin for 100 min;
3) washing the chip with PBST for 2min 3 times;
4) the concentration gradient of HRP-labeled secondary antibody 15 mu L labeled secondary antibody with different known concentration gradients added to the detection zones 1-9 on the chip is 1.6 mu g/m L, 0.8 mu g/m L0, 0.4 mu g/m L, 0.2 mu g/m L, 0.1 mu g/m L, 0.05 mu g/m L, 0.025 mu g/m L, 0.0125 mu g/m L and 0.006 mu g/m L, the blank control PBS 15 mu L is added to the detection zone 10, and the incubation is carried out for 30min at 37 ℃;
5) PBST is washed for 3 times, each time for 2 min;
6) 15 μ L of HRP luminescent substrate was added to each detection zone and incubated for 2min at room temperature.
Scanning is carried out by using a chemiluminescence imaging system, namely, a quality control experiment of the aldehyde group modified chip protein IgG coating detection line is carried out, the result is shown in figure 2, the intensity of a generated luminescence signal is changed along with the change of the concentration of human IgG in a certain range, the signal intensity is obviously different from that of a blank control, and meanwhile, the signal intensity is changed along with the reduction of the concentration of an HRP-labeled secondary antibody. Therefore, the aldehyde group modified chip can be well combined with protein biomolecules.
HIV-1/2 recombinant antigen coating concentration and plasma dilution experiment
The optimal envelope concentration and plasma dilution of the HIV-1/2 recombinant antigen are determined by indirect E L ISA chessboard titration, and 10 parts of HIV-I positive serum are confirmed by WB to contain an antibody corresponding to the expressed HIV-I recombinant antigen and 10 parts of HIV-I negative serum.
1) Diluting 4 HIV recombinant antigens with gradient of 50 μ g/m L, 12.5 μ g/m L, 3.125 μ g/m L, 0.78 μ g/m L, 0.195 μ g/m L and 0 μ g/m L6 by using 0.1% PBST-BSA coating solution, respectively spotting the 4 HIV recombinant antigens with different concentrations in a detection area on a substrate, performing 6 spotting on each antigen, and incubating at 4 ℃ for 12 h;
2) the chip is sealed by 10% fetal bovine serum albumin for 100 min;
3) washing the chip with PBST for 2min 3 times;
4) adding known positive plasma 15 μ L with different concentrations into detection zones 1-9 on the chip, adding blank control PBS 15 μ L into detection zone 10, and incubating at 37 deg.C for 30 min;
5) PBST is washed for 3 times, each time for 2 min;
6) add 15 μ L avidin HRP-labeled secondary antibody to each detection zone, incubate for 30min at 37 ℃;
7) after PBST cleaning, removing non-specific binding, adding HRP luminescent substrate mixed solution, incubating at room temperature for 2min, and scanning with a chemiluminescence scanner.
The results of the gradient concentration experiments for different plasma concentrations corresponding to different HIV recombinant antigens were obtained by scanning the chip with a chemiluminescence scanner, and are shown in FIG. 3In the detection area of the chip 1-9, the corresponding plasma concentration is as follows: stock solution, 1: 10,1: 102，1：103，1：104The 5 th and 10 th detection zones are blank controls, from which the optimal coating concentration and plasma dilution concentration for each antigen can be obtained as shown in Table 2.
TABLE 2 coating concentrations of various antigens and plasma dilutions
Second, preparation of protein chip
In each detection square, HIV recombinant antigens gp36, gp41, gp120 and gp160 and a negative control solution are sequentially spotted on the chip according to a pattern arrangement mode by using a high-speed full-automatic sample spotting instrument to form detection spots and control spots, and the detection spots and the control spots are subjected to vacuum drying for 2 hours after standing overnight at the temperature of 4 ℃; further, after vacuum-pumped drying, the mixture was pumped and sealed with a water vapor-resistant plastic pouch and stored at 4 ℃.
Third, chip detection HRP-antibody concentration gradient experiment
The protein chip prepared by the method is rewarmed for 30min at room temperature, 10% fetal bovine serum albumin is sealed for 100min, PBST is cleaned for 3 times, each time is 2min, centrifugal drying is carried out, positive plasma with the experimental concentration is added into a detection zone of 1-10, incubated for 30min at 37 ℃, PBST is cleaned for 3 times, each time is 2min, centrifugal drying is carried out, HRP-labeled anti-human IgG secondary antibody solutions with the gradient dilution of 1.6 mu g/m L, 0.8 mu g/m L, 0.4 mu g/m L, 0.2 mu g/m L, 0.1 mu g/m L, 0.05 mu g/m L, 0.025 mu g/m L, 0.0125 mu g/m L and 0.06 mu g/m L are spotted on the chip at the detection zone of 1-9, BSA with the 10% sample is taken as a blank control and is placed in a wet box, and after 30min at 37 ℃, cleaned by PBST buffer solution and centrifuged.
The chip is scanned by using a chemiluminescence scanner, namely, an experiment for determining the concentration gradient of the HRP-labeled detection antibody is carried out, the result is shown in figure 4, under the condition that the concentration conditions of antigen and plasma are not changed, when the incubation concentration of the HRP-labeled secondary antibody is more than 0.4 mu g/m L, the intensity of the generated signal is obviously different from the intensity of a fluorescence signal generated by a negative control, so that the optimal concentration of the goat anti-human IgG antibody for incubating the HRP-labeled goat is more than 0.4 mu g/ml, and the intensity of the generated fluorescence signal is also changed along with the change of the concentration of the human HRP-labeled secondary antibody in a certain range and is obviously different from the intensity of the fluorescence signal generated by a blank control.
Example 2 sample detection assay
Serum samples:
50 known HIV positive plasma samples from the specimen bank of the affiliated Youtanan Hospital, university of capital medical science;
5 parts of plasma from a known normal healthy person;
5 blanks (blank 1 × PBS).
The operation flow of the protein chip is as follows:
(1) the protein chip prepared in the above example 1 was left at room temperature for 30 min;
(2) sealing the nonspecific sites on the surface of the substrate by using a sealing solution containing 10% fetal calf serum, and incubating for 1-1.5 hours at 37 ℃;
(3) washing with PBST solution for 3-4 times, each time for 10 s, washing with PBS, and centrifuging;
(4) dropping patient plasma or serum sample on the chip, and incubating in a wet box at 37 deg.C for 30min to make antigen and antibody fully react;
(5) washing with PBST for 3-4 times, each time for 10 seconds, washing with PBS, centrifuging, and removing redundant samples;
(6) dropping HRP-antihuman IgG diluted by PBS and mixed solution with the concentration of 0.4 mu g/m L, and incubating for 30 minutes at 37 ℃ in the dark;
(7) washing with PBST for 3 times, each time for 10 s, washing with PBS, and centrifuging;
(8) dropping HRP luminescent substrate mixed solution, and incubating for 2min at room temperature;
(9) the signals were scanned and collected by a chemiluminescence scanner, and the results were shown in FIG. 5 according to the scanning, and the results of the analysis and detection are shown in Table 3 below.
Table 350 cases of clinical specimen test results
The detection result of the chip is as follows:
AFP (100%) was detected in 50 out of 50 known HIV positive patient plasma, with all internal negative controls negative. And (3) proving that: the chip and the method have detection sensitivity of 50/50-100 percent and specificity of 100 percent, and have reliable clinical application value. The above data show that the chip and the method of the invention have good stability, accuracy and reliability.
The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.
Claims (10)
1. A protein chip for detecting HIV1/2 antibody is characterized by comprising a substrate, wherein HIV specific recombinant antigens gp36, gp41, gp120 and gp160 detection points are fixed on the surface of the substrate.
2. The protein chip for detecting HIV1/2 according to claim 1, wherein the substrate is an aldehyde-modified glass plate.
3. The protein chip for detecting HIV1/2 antibody according to claim 1, wherein the concentration of HIV specific recombinant antigen gp36 is 3.125 μ g/m L, the concentration of HIV specific recombinant antigen gp41 is 12.5 μ g/m L specific recombinant antigen gp120 is 200 μ g/m L specific recombinant antigen gp160 is 12.5 μ g/m L.
4. The protein chip for detecting HIV1/2 according to claim 1, wherein the surface of the substrate comprises a plurality of detection regions and control regions, any detection region comprises at least one group of HIV-specific recombinant antigens gp36, gp41, gp120 and gp160, and the concentration of the same antigen in the same detection region is the same.
5. The protein chip for detecting HIV1/2 antibody according to claim 1, wherein the spots immobilized with HIV recombinant antigen have a diameter of 50 μm and a spot spacing of 100 μm, and the spots form an array of 2.5mm × 2.5.5 mm.
6. A protein chip detection kit for detecting HIV1/2 antibody is characterized in that the detection kit comprises a protein chip and a detection reagent,
the protein chip comprises a substrate, wherein HIV specific recombinant antigens gp36, gp41, gp120 and gp160 detection points are fixed on the surface of the substrate.
7. The protein chip test kit for testing HIV1/2 antibody according to claim 6, wherein the test reagent is enzyme-labeled antibody.
8. The protein chip test kit for detecting HIV1/2 antibody according to claim 7, wherein the concentration of the enzyme-labeled antibody is 0.4 μ g/m L or more.
9. The protein chip detection kit for detecting HIV1/2 antibody according to claim 7, wherein the detection reagent is horseradish peroxidase-labeled antibody.
10. A method for preparing a protein chip for detecting HIV1/2 antibody, which comprises the following steps:
the HIV recombinant antigen is fixed on the substrate of the protein chip, and the recombinant antigen comprises HIV antigens gp36, gp41, gp120 and gp 160.
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| PCT/CN2021/081827 WO2021185357A1 (en) | 2020-03-20 | 2021-03-19 | Protein chip for detecting human immunodeficiency virus hiv1/2 antibody and preparation method therefor |
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| WO2021185357A1 (en) * | 2020-03-20 | 2021-09-23 | 北京良芯生物科技发展有限公司 | Protein chip for detecting human immunodeficiency virus hiv1/2 antibody and preparation method therefor |
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