CN104697830A - Acid treating agent for HIV detection, sample pretreatment method, kit and detection method - Google Patents
Acid treating agent for HIV detection, sample pretreatment method, kit and detection method Download PDFInfo
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Abstract
The invention discloses an acid treating agent for HIV detection, a sample pretreatment method, a kit and a detection method, and belongs to the technical field of in vitro diagnosis and detection. The acid treating agent is mainly formed by mixing an organic acid solution with concentration of 0.1-1.0mol/L and an inorganic acid solution with concentration of 0.01-0.1mol/L, or an organic acid solution which is with concentration of 0.1-1.2mol/L, wherein the pH value of the acid treating agent is 2.5-4.5. The acid treating agent can well separate out p24 antigen and p24 antibody in a sample. The invention also discloses the HIV detection kit adopting the acid treating agent, the sample pretreatment method and the detection method, and the HIV detection kit, the sample pretreatment method and the detection method have the advantage of high sensitivity.
Description
Technical field
The present invention relates to in-vitro diagnosis detection technique field, particularly relate to a kind of detect for HIV (human immunodeficiency virus) acidic treatment agent, sample preprocessing method, kit and detection method.
Background technology
After HIV virus (human immunodeficiency virus) invades human body, its surface glycoprotein gp120 is combined with high-affinity with cell surface receptor protein CD4, is adsorbed onto on host cell; Gp120 interacts with host cell surface accessory receptor again, make virus and host cell membrane closer to; Gp41 produces a series of conformation change, the fusogenic peptide fragment Insertion Into Host Cell film that its N holds, and cause the final fusion of peplos and cell membrane, viral RNA enters cell.After HIV, can monitor at first viral RNA, be then virus p24 antigen, be finally antibody.According to HIV gene structure, immunology and EPDML feature, HIV is divided into the large class of HIV-1 and HIV-2 two.The appeal of HIV-1 is comparatively strong, is the main diseases strain that world today HIV is popular.
The method of current detection HIV has kind more than 100, can be divided into antibody test and the large class of Viral diagnosis two on the whole.Viral diagnosis comprises cell chulture (virus purification), p24 antigen detects and viral nucleic acid detects.In early days the diagnosis of HIV is mainly detected to the antibody of AntiHIV1 RT activity by serological test, indirectly diagnose HIV.Antibody normally can be detected after infection for 3 ~ 8 weeks.HIV person more than 70% just can detect antibody in infection after 6 months, and in gay community, this numeral is more than 80%; In addition, neonate produces ANTI-HIV DRUGS generally in birth after 1 year, and the ANTI-HIV DRUGS from parent easily causes neonate's false positive; Simple detection antibody method adds the danger that HIV " window phase " propagates.Antigen can be detected prior to seroconversion 2 ~ 18d after individuality infects.Therefore, there is very large advantage in the seroconversion phase by detecting p24 antigen, can as a kind of method of early stage auxiliary diagnosis HIV.
But the conventional p24 antigen used detects exists the lower problem of sensitivity.The level that detects as independent p24 antigen detecting agent is approximately 10pg/mL, and the p24 antigen of joint-detection detects level and do not reach independent detection level especially.But both made when virus is enlivened and copied, the antigen amount in serum also seldom reaches this level.Further, when conventional use Chemiluminescence immunoassay detects HIV, often occur false negative and false positive, cause the inaccuracy of HIV diagnostic result, occur failing to pinpoint a disease in diagnosis or mistaken diagnosis, consequence is very serious.
Summary of the invention
Based on this, the object of the invention is to the defect overcoming prior art, a kind of acidic treatment agent detected for HIV is provided, adopts this acidic treatment agent to carry out pre-service to sample, the detection sensitivity of HIV can be improved.
For achieving the above object, the present invention takes following technical scheme:
A kind of acidic treatment agent detected for HIV, inorganic acid solution primarily of concentration to be the organic acid soln of 0.1-1.0mol/L and concentration be 0.01-0.1mol/L mixes, or for concentration be the organic acid soln of 0.1-1.2mol/L, the pH value of this acidic treatment agent is 2.5-4.5.
The present inventor is found after being investigated by a large amount of research experiment, the reason that in routine techniques, p24 antigen detection sensitivity is low, that p24 antigen in HIV person's serum is generally all combined into compound with anti-p24 antibody, and detection method itself can not detectable antigens antibody complex, needs complex dissociation to be detected.But, when complex dissociation, need just can make p24 antigen and anti-p24 antibody dissociation by acid solution process, in order to all p24 antigen is discharged, need to add excessive sour agent process or high-temperature heating sample, and as only added the mineral acids such as hydrochloric acid, because the amount being subject to adding hydrochloric acid limited, can only, by Sample Dilution, the hydrochloric acid added be made to be enough to, by all p24 antigen release, could testing requirement be met.But, after sample is diluted, just reduce detection sensitivity greatly.And high-temperature heating sample discharges p24 antigen, harmful effect may be produced to biological specimen again.
Further, can not reach simply by raising content of hydrochloric acid the object avoiding diluted sample, be due to when concentration of hydrochloric acid is too high on the one hand, its volatility is also corresponding larger, instrument is had higher requirements, and the activity of antigen or antibody may be affected, certain influence is caused to detection accuracy; On the other hand, when concentration of hydrochloric acid is too high, excessively strong acidity can cause harmful effect to sample, thus reduces the accuracy of detection.
And acidic treatment agent of the present invention, be the mixed acid solution of high volumetric molar concentration organic acid and low volumetric molar concentration mineral acid, organic acid and mineral acid are matched, or be used alone high volumetric molar concentration organic acid soln; Utilize organic acid biological, and the character more easily contacted with some organism, p24 antigen is discharged by organic acid lenitively, also can add the hydrogen ion that a small amount of mineral acid provides required, with supplementary acidity.The two cooperatively interacts or is used alone the organic acid soln of high volumetric molar concentration can p24 antigen well in separating sample and anti-p24 antibody, without the need to by Sample Dilution, just can discharge p24 antigen wherein completely, reach testing requirement.Thus can detection sensitivity be improved.
Wherein in an embodiment, the mol ratio of described organic acid and unitary inorganic acid is 1-20:1; Or the mol ratio of described organic acid and dibasic inorganic acid is 0.5-10:1; Or the mol ratio of described organic acid and ternary mineral acid is 0.3-6.6:1.Described unitary inorganic acid refers to that molecular energies such as hydrochloric acid ionize out a hydrionic mineral acid, described dibasic inorganic acid refers to that molecular energies such as sulfuric acid ionize out two hydrionic mineral acids, and described ternary mineral acid refers to that molecular energies such as phosphoric acid ionize out three hydrionic mineral acids.Organic acid and mineral acid are coordinated according to aforementioned proportion, there is better p24 antigen releasing effect.
Wherein in an embodiment, the mol ratio of described organic acid and unitary inorganic acid is 7-13:1; Or the mol ratio of described organic acid and dibasic inorganic acid is 3.5-6.5:1; Or the mol ratio of described organic acid and ternary mineral acid is 2.3-4.3:1.
Wherein in an embodiment, described organic acid is at least one in acetic acid, benzoic acid, ethane diacid, succinic acid, malic acid, citric acid, salicylic acid.
Wherein in an embodiment, described mineral acid is at least one in sulfuric acid, hydrochloric acid, nitric acid, hydrofluorite, sulphurous acid, nitrous acid.
Wherein in an embodiment, this acidic treatment agent also comprises damping fluid, and described damping fluid is at least one in formate buffer, acetate buffer, citrate buffer, Succinate Buffer, citrate buffer, sulfate buffer, nitrate damping fluid, borate buffer solution, phosphate buffer or Tris damping fluid.Use damping fluid, the ability of the determinand in reagent components and sample by the impact of acid can be reduced.
The invention also discloses a kind of preprocess method of HIV sample to be tested, adopt above-mentioned acidic treatment agent, sample to be tested is mixed according to the volume ratio of 0.5-5:1 with described acidic treatment agent, obtains solution to be measured.
Wherein in an embodiment, after sample to be tested is mixed with described acidic treatment agent, also by solution to be measured incubation 1-30 minute at 20-60 DEG C, incubation 1-10 minute at preferred 20-40 DEG C, more preferably incubation 3-7 minute at 30-40 DEG C.By the mode of incubation, better faster p24 antigen can be separated with anti-p24 antibody, obtain free p24 antigen.
The invention also discloses a kind of HIV detection kit, comprise following component:
1) acidic treatment agent: above-mentioned acidic treatment agent;
2) magnetic microsphere system: the magnetic microsphere comprising HIV recombinant antigen 1 connected directly or indirectly, and/or the magnetic microsphere of ANTI-HIV DRUGS A connected directly or indirectly;
3) label system: the HIV recombinant antigen 2 comprising corresponding mark tracer connected directly or indirectly, and/or the ANTI-HIV DRUGS B of mark tracer connected directly or indirectly;
The site that described HIV recombinant antigen 1 and HIV recombinant antigen 2 are combined with test antibodies is different, and the site that described ANTI-HIV DRUGS A and ANTI-HIV DRUGS B is combined with determined antigen is different.
Above-mentioned " directly connecting " is the connection directly combined, and above-mentioned " indirectly connecting " is by biotin and Streptavidin, or fluorescein isothiocynate is connected in the mode indirectly combined with the bridging thing that anti-fluorescein isothiocynate antibody etc. can be combined with each other.Wherein antibody A and antibody B both can be monoclonal antibody, can be also polyclonal antibody, all not affect its Detection results.
In above-mentioned detection kit, both HIV recombinant antigen system can have been used to detect HIV antibody, or use ANTI-HIV DRUGS system to detect HIV antigen, also HIV recombinant antigen system and ANTI-HIV DRUGS system can be used simultaneously simultaneously to detect HIV antibody and HIV antigen (as HIV p24 antigen), can detection sensitivity be improved.
Further, in above-mentioned magnetic microsphere system, in the HIV recombinant antigen 2 of described linkage flag tracer, the working concentration preferred 1-20 μ g/mL of HIV recombinant antigen 1, the preferred 0.1-5.0% of working concentration of magnetic microsphere; In the magnetic microsphere of described connection ANTI-HIV DRUGS A, the working concentration preferred 1-20 μ g/mL of ANTI-HIV DRUGS A, the preferred 0.1-5.0% of working concentration of magnetic microsphere.In above-mentioned label system, in the HIV recombinant antigen 2 of described linkage flag tracer, the working concentration preferred 2-20 μ g/mL of HIV recombinant antigen 2, the preferred 0.1-1mg/L of working concentration of mark tracer; In the ANTI-HIV DRUGS B of described linkage flag tracer, the working concentration preferred 2-20 μ g/mL of ANTI-HIV DRUGS B, the preferred 0.1-1mg/L of working concentration of mark tracer.
Being applicable to magnetic microsphere of the present invention also referred to as magnetic bead or magnetic ball, can be magnetic microsphere conventional in this area.Preferably, the magnetic ball that the present invention uses is by nano level Fe
2o
3or Fe
3o
4magnetic particle and high-molecular organic material carry out compound, and form the micron-sized solid phase microballoon with superparamagnetism and huge amount protein adsorption capacity, have and can be magnetized rapidly under additional magnetic fields, after withdrawing magnetic field, remanent magnetism is the attribute of zero.Wherein, the kind of described high-molecular organic material is not particularly limited, and can select as required.
It is 0.1-5 μm that magnetic microsphere used in the present invention should be able to meet diameter, and magnetic microsphere with various active functional group, can also include but not limited to-OH ,-COOH ,-NH by surface modification
2.
Wherein in an embodiment, described magnetic microsphere is Fe
2o
3or Fe
3o
4the complex of magnetic nano-particle and high-molecular organic material, and the particle diameter with 0.1-5 μm, and, described magnetic microsphere optionally by surface modification with one or more activity functional groups.
Wherein in an embodiment, this detection kit also comprises following component:
4) immune interference thing bonding agent: comprise bonding agent and auxiliary element; Described bonding agent is at least one in NBCS, sheep blood serum, horse serum, bovine serum albumin(BSA), goat anti-human igg/IgM, rabbit anti-human igg/IgM, mouse-anti human IgG/IgM; Described auxiliary element is at least one in glycerine, ethylene glycol, polyglycol, sucrose, sodium chloride, edetate (EDTA).
The present inventor be experimental studies have found that by a large amount of, in conventional H IV detects, specificity and the low Another reason of sensitivity have a large amount of chaff interference in the sample, as: blood plasma, haemocyanin (Rheumatoid factors, associated proteins), heterophil antibody, Human Anti animal Antibody (HAAA) and medicine and the metabolin etc. caused thereof, especially latter two antibody interferes with is maximum.
On above-mentioned Research foundation, in detection kit of the present invention, also add immune interference thing bonding agent, the bonding agent as protide wherein can divide nonspecific combinations such as the wet factor with the heterophil antibody in sample and class, and auxiliary element can eliminate the impact of tissue metabolism's thing, thus avoid interference the interference of thing to testing process, improve detection specificity and sensitivity.
Wherein in an embodiment, the concentration of described bonding agent is 0.1-20g/L, and the concentration of described auxiliary element is 0.2-10g/L.
Wherein in an embodiment, also comprise in described chaff interference bonding agent: surfactant I, surfactant II, ascorbic acid oxidase and bilirubin oxidase, described surfactant I is zwitterionic surfactant, as lecithin, amino acid pattern, and betaine type etc.; Described surfactant II is non-ionic surfactant, and as fatty glyceride, fatty acid sorb is smooth, polysorbate etc.; The preferred 0.01-0.1mol/L of concentration of described surfactant I, the preferred 0.01-0.1mol/L of concentration of described surfactant II, the preferred 1-20KU/L of concentration of described ascorbic acid oxidase, the preferred 1-20KU/L of concentration of described bilirubin oxidase.
Wherein: add the dispersion effect that surfactant is conducive to chaff interference in sample, and ascorbic acid oxidase and bilirubin oxidase can reduce ascorbic acid in sample and cholerythrin to the impact of result, in immune interference thing bonding agent, each composition cooperatively interacts, and can reach good Anti-Jamming.
Wherein in an embodiment, described HIV recombinant antigen is HIV 1+2 type recombinant antigen, and described ANTI-HIV DRUGS is AntiHIV1 RT activity p24 antibody.Described HIV 1+2 type recombinant antigen is the recombinant protein of the corresponding gp36 of the gp41 that HIV 1 type is corresponding, gp120 and HIV 2 type.
Above-mentioned mark tracer comprises following several: 1, chemiluminescence immune assay use can directly luminescence label, as luminol and derivant, different luminol or derivatives thereof, acridinium ester etc.; 2, the label that the corresponding luminous substrate of cooperation of chemiluminescence enzyme immunoassay use can be luminous, as alkaline phosphatase or peroxidase etc.
Wherein in an embodiment, described mark tracer is luminous marker, is selected from: diamantane, luminol and derivant thereof, different luminol and derivant thereof, acridinium ester.The preferred N-different luminol of (4-aminobutyl)-N-ethyl (ABEI), the oxidative system coordinated with above-mentioned luminous marker comprises H
2o
2-microperoxisome, H
2o
2-hydrogen peroxidase, H
2o
2-lactoperoxidase, H
2o
2-deuterohemin, H
2o
2-protohemin, hypochlorite-CoCl
2, persulfate, potassium peroxide, sodium metaperiodate, H
2o
2-K
3fe (CN)
6, xanthine-hypoxanthine oxidase, at least one in potassium tert-butoxide.
Above-mentioned luminous marker refers to and participate in energy trasfer and the final compound released energy with the form of launching photon in luminescence-producing reaction, this compound can through the oxidation of the catalysis of catalyzer and oxygenant, form the intermediate of an excited state, when this excited state intermediate gets back to stable ground state, launch photon (hM) simultaneously.
Or described mark tracer is chemiluminescent catalysts, is selected from: alkaline phosphatase, peroxidase.During use, coordinate corresponding chemical luminous substrate luminescence to be qualitatively detected, described chemical luminous substrate comprises NaOH and H
2o
2, also comprise at least one in diamantane, luminol and derivant thereof, different luminol or derivatives thereof, the preferred N-different luminol of (4-aminobutyl)-N-ethyl (ABEI).
Wherein in an embodiment, described 2), in magnetic microsphere system, the magnetic microsphere of described indirect connection HIV recombinant antigen 1 is by wrapping by the magnetic microsphere of anti-fluorescein isothiocynate antibody, and the HIV recombinant antigen 1 of mark fluorescein isothiocynate forms; Or the magnetic microsphere of described indirect connection HIV recombinant antigen 1 is by wrapping by the magnetic microsphere of Streptavidin, and the HIV recombinant antigen 1 of mark biotin forms; Or the magnetic microsphere of described indirect connection HIV recombinant antigen 1 is by wrapping by the magnetic microsphere of anti-label protein antibody, and the HIV recombinant antigen 1 with label protein forms;
The magnetic microsphere of described indirect connection ANTI-HIV DRUGS A (i.e. AntiHIV1 RT activity p24 antibody) is by wrapping by the magnetic microsphere of anti-fluorescein isothiocynate antibody, and the ANTI-HIV DRUGS A of mark fluorescein isothiocynate forms; Or the magnetic microsphere of described indirect connection ANTI-HIV DRUGS A is by wrapping by the magnetic microsphere of Streptavidin, and the ANTI-HIV DRUGS A of mark biotin forms; Or the magnetic microsphere of described indirect connection ANTI-HIV DRUGS A is by wrapping by the magnetic microsphere of anti-label protein antibody, and the ANTI-HIV DRUGS A with label protein forms.
Above-mentioned anti-fluorescein isothiocynate (FITC) antibody both can be monoclonal antibody, also can be polyclonal antibody.
And, in above-mentioned magnetic microsphere system, described bag is by the magnetic microsphere of anti-fluorescein isothiocynate antibody, the working concentration preferred 1-20 μ g/mL of anti-fluorescein isothiocynate antibody, the preferred 0.1-2mg/mL of working concentration of magnetic microsphere, in the HIV recombinant antigen 1 (or ANTI-HIV DRUGS A) of described mark fluorescein isothiocynate, the working concentration preferred 0.1-10 μ g/mL of working concentration preferred 5-500ng/mL, the HIV recombinant antigen 1 (or ANTI-HIV DRUGS A) of fluorescein isothiocynate; Described bag is by the magnetic microsphere of Streptavidin, the working concentration preferred 1-20 μ g/mL of Streptavidin, the preferred 0.1-2mg/mL of working concentration of magnetic microsphere, the working concentration preferred 10-500 μ g/mL of the HIV recombinant antigen 1 (or ANTI-HIV DRUGS A) of described mark biotin.
Wherein in an embodiment, described 3), in label system, the HIV recombinant antigen 2 of described indirect linkage flag tracer is by the HIV recombinant antigen 2 of mark biotin, and the mark tracer composition of labelled streptavidin; Or the HIV recombinant antigen 2 of described indirect linkage flag tracer is by the HIV recombinant antigen 2 of mark fluorescein isothiocynate, and the mark tracer composition of the anti-fluorescein isothiocynate antibody of mark; Or the HIV recombinant antigen 2 of described indirect linkage flag tracer is by the HIV recombinant antigen 2 with label protein, and the mark tracer composition of the anti-label protein antibody of mark;
The ANTI-HIV DRUGS B of described indirect linkage flag tracer is by the ANTI-HIV DRUGS B marking biotin, and the mark tracer composition of labelled streptavidin; Or the ANTI-HIV DRUGS B of described indirect linkage flag tracer is by the ANTI-HIV DRUGS B marking fluorescein isothiocynate, and the mark tracer composition of the anti-fluorescein isothiocynate antibody of mark; Or the ANTI-HIV DRUGS B of described indirect linkage flag tracer is by the ANTI-HIV DRUGS B with label protein, and the mark tracer composition of the anti-label protein antibody of mark.
The preferred Flag label protein of above-mentioned label protein, Flag label protein is coding 8 amino acid whose hydrophilic polypeptides (DYKDDDDK), and the Kozak sequence simultaneously built in carrier makes the fusion expression efficiency in eukaryotic expression system with Flag higher
Such scheme has enriched the magnetic microsphere system and label system that detect and use, can select flexibly according to different demand.Such as in magnetic microsphere system, directly HIV recombinant antigen 1 (or ANTI-HIV DRUGS A) magnetic microsphere can be connected, also HIV recombinant antigen 1 (or ANTI-HIV DRUGS A) and magnetic microsphere can be connected by above-mentioned indirect bridging mode.Equally, in label system, mark tracer can directly be connected HIV recombinant antigen 2 (or ANTI-HIV DRUGS B), also can by above-mentioned indirect bridging mode linkage flag tracer and HIV recombinant antigen 2 (or ANTI-HIV DRUGS B).And, above-mentioned magnetic microsphere system with adopt which kind of connected mode (being directly connected or connection of indirectly putting up a bridge) there is no in label system to influence each other or limit, both can in magnetic microsphere system and label system, adopt direct connected mode simultaneously or adopt indirect connections simultaneously, also magnetic microsphere system direct connection can be selected, label system adopts and indirectly connects, or magnetic microsphere system selects indirect connection, label system adopts and directly connects.
Understandable, wherein in an embodiment, this detection kit also comprises:
5) calibration object solution: negative calibration object (HIV negative serum goods) and positive calibration object (the HIV positive serum goods of deactivation) solution.
By the proportionate relationship of sample and calibration object solution relative light intensity (RLU), calculate measurement result.
Wherein in an embodiment, all containing bovine serum albumin(BSA) (BSA) and antiseptic in each component of this detection kit, the concentration of BSA is 0.01-0.5g/ml, and antiseptic is at least one in potassium sorbate, Sodium Benzoate, Sodium azide, sodium nitrite, Proclin series.
The invention also discloses a kind of HIV detection method, adopt above-mentioned detection kit, comprise the following steps:
1) pre-service: carry out sample preprocessing according to above-mentioned preprocess method;
2) react: in above-mentioned solution to be measured, add magnetic microsphere system and label system, form electrochemiluminescent immunoassay compound;
3) detect: externally-applied magnetic field, by above-mentioned electrochemiluminescent immunoassay complex precipitate, is removed supernatant, after cleaning, added luminous substrate, detects the relative light intensity sent, calculates the content of test antibodies and/or determined antigen.
Wherein in an embodiment, described step 2) in reaction, also add above-mentioned immune interference thing bonding agent.The chaff interference of immune interference thing bonding agent in sample is combined, thus avoids interference the interference of thing to testing process, improve detection sensitivity.
The invention also discloses a kind of above-mentioned application of HIV detection kit on chemiluminescent analyzer.This kit is applied to chemiluminescent analyzer, there is working specification, the unmanned advantage for introducing error and full-automation.
Compared with prior art, the present invention has following beneficial effect:
A kind of acidic treatment agent of the present invention, organic acid and mineral acid are matched, the character utilizing organic acid more easily to contact with some organism, makes organic acid be separated with anti-p24 antibody by p24 antigen lenitively, also the hydrogen ion that mineral acid provides required can be utilized, with supplementary acidity.The two cooperatively interacts or using organic acid singly, can p24 antigen well in separating sample and anti-p24 antibody, when detecting, without the need to by Sample Dilution or high-temperature heating, just can discharge p24 antigen wherein completely, reach testing requirement, improve detection sensitivity.
The preprocess method of a kind of sample to be tested of the present invention, adopts above-mentioned acidic treatment agent, when sample preprocessing, without the need to by Sample Dilution or high-temperature heating, just can well p24 antigen in separating sample and anti-p24 antibody, discharge p24 antigen wherein completely, reach testing requirement.Further, the method in sample preprocessing, also by solution to be measured incubation 1-30 minute at 20-60 DEG C.By the mode of incubation, better faster p24 antigen can be discharged from anti-p24 antibody, obtain free p24 antigen, both improve detection efficiency, harmful effect can not be produced due to high temperature to sample again.
A kind of HIV detection kit of the present invention, except have employed above-mentioned acidic treatment agent, also adding immune interference thing bonding agent, by avoiding interference the interference of thing to testing process, further improve detection sensitivity.The level that detects of p24 antigen is made to reach 0.625U/mL (L6), reach the requirement of HIV antigen-antibody combined detection kit, even reach the limit of identification requirement detecting separately reagent about HIV p24 antigen of Nat'l Pharmaceutical & Biological Products Control Institute.Reach the object that the sensitivity of HIV p24 antigen detection assays is less than 1pg/mL, Functional Sensitivity is less than 5pg/mL.And in theory, detect the window phase of P24 antigen generally at about 4-14d, comparatively the HIV antibody detection window phase reduces 2/3, presses on towards the window level of HIV-RNA, and testing cost comparatively p24 antigen and detection of nucleic acids reduce a lot.
A kind of HIV detection method of the present invention, wherein sample adopts above-mentioned preprocess method, organic acid and mineral acid are matched or using organic acid singly as acidic treatment agent, can detection sensitivity be improved.Particularly, also coordinating the use of immune interference thing bonding agent, by avoiding interference the interference of thing to testing process, further improve detection sensitivity.
Embodiment
Below in conjunction with embodiment, the present invention is described further, but do not cause any restriction to the present invention.
In following examples:
HIV 1+2 type recombinant antigen 1, purchased from meridian life science.
AntiHIV1 RT activity p24 monoclonal antibody A, purchased from meridian life science.
The HIV 1+2 type recombinant antibodies of the anti-label protein of mark ABEI, purchased from meridian life science.
With the HIV 1+2 type recombinant antigen of label protein, purchased from meridian life science.
AntiHIV1 RT activity p24 polyclonal antibody B, purchased from meridian life science.
Goat-anti FITC polyclonal antibody, purchased from great bio tech ltd, Beijing hundred.
Magnetic microsphere, source: biomedical incorporated company produces for Shenzhen NPD projects.
FITC: purchased from Sigma.
ABEI: biomedical incorporated company produces for Shenzhen NPD projects.
Biotin, Streptavidin: all purchased from Roche.
Negative calibration object and positive calibration object: all purchased from meridian life science.
Embodiment 1
A kind of HIV detection kit, comprises following component:
1) acidic treatment agent:
Concentration is the acetic acid solution of 0.5mol/L and concentration is the hydrochloric acid solution of 0.05mol/L, is 10:1 mixing according to the mol ratio of wherein acetic acid and hydrochloric acid; And 1:1 adds the acetate buffer of 0.05mol/L by volume, obtain acidic treatment agent, the pH value of this acidic treatment agent is 3.5.
2) magnetic microsphere system: wrap by the magnetic microsphere solution of HIV 1+2 type recombinant antigen 1, wherein: the working concentration of HIV1+2 type recombinant antigen 1 is 10 μ g/mL, and the working concentration of magnetic microsphere is 0.5mg/mL.
Wrap by the magnetic microsphere solution of AntiHIV1 RT activity p24 monoclonal antibody A, wherein: the concentration of AntiHIV1 RT activity p24 monoclonal antibody A is 10 μ g/mL, and the working concentration of magnetic microsphere is 0.5mg/mL.
3) label system: the antibody-solutions of the anti-label protein of mark ABEI, its working concentration is 0.2mg/L.
With the HIV 1+2 type recombinant antigen 2 of label protein, its working concentration is 1 μ g/mL;
The AntiHIV1 RT activity p24 polyclonal antibody B of mark ABEI, its working concentration is 0.5mg/L.
Described label protein is Flag label protein.
4) immune interference thing bonding agent, comprising:
Bonding agent: concentration expressed in percentage by volume is NBCS, 0.1-20g/L bovine serum albumin(BSA), 0.01-5g/L goat anti-human igg/IgM, 0.01-5g/L rabbit anti-human igg/IgM of 0.1-20%;
Auxiliary element: 0.01-5g/L ethylene glycol, 0.01-5g/L EDTA-2K, 0.01-0.2mol/L Macrogol 6000;
Buffering component: 0.01-5g/L Tris, 0.01-0.2mol/L PBS buffering agent;
The lecithin (source: upper marine base row chemical industry) of surfactant: 0.01-0.1mol/L, the fatty glyceride (source: upper marine base row chemical industry) of 0.01-0.1mol/L;
Ascorbic acid oxidase: 1-20KU/L;
Bilirubin oxidase: 1-20KU/L.
5) calibration object solution: negative calibration object (HIV negative serum goods) and positive calibration object (HIV positive serum goods, deactivation) solution.
Above-mentioned each component is all containing bovine serum albumin(BSA) (BSA) and antiseptic, and BSA concentration is 0.05g/ml, and antiseptic principal ingredient is NaN
3, concentration is 0.02g/ml.
In the preparation method of the HIV detection kit of the present embodiment, except following reagent, all the other are all conventionally prepared.
Above-mentioned bag is prepared by the magnetic microsphere of AntiHIV1 RT activity p24 monoclonal antibody A by the following method by the magnetic microsphere of HIV 1+2 type recombinant antigen 1 and bag: use 1-cyclohexyl-2-morpholine ethyl carbodiimide tosilate (CMC) etc. to be connected antigen (or antibody) and the magnetic microsphere having adsorbed carboxylic group after treatment as bridging material, 30-40 DEG C of incubation 1-2 hour, then can use through the washing removal impurity of 3-5 magnetic ball suspending liquid.
The antibody (or AntiHIV1 RT activity p24 polyclonal antibody of mark ABEI) of the anti-label protein of above-mentioned mark ABEI is prepared by the following method: after using N-hydroxy-succinamide (NHS) etc. to connect ABEI and antibody (or antigen) as bridging material, cross post obtain purified through G-25 gel chromatography.
Adopt the detection kit of the present embodiment to carry out the method for HIV antigen and antibody combined detection, comprise the following steps:
1) pre-service: 50 μ L samples, the negative calibration objects of 50 μ L and the positive calibration object of 50 μ L are joined in reaction cup respectively, and add 50 μ L acidic treatment agent, mixing, 37 DEG C of incubations 5 minutes.
2) first step reaction: add 20 μ L and wrap by the magnetic microsphere solution of HIV 1+2 type recombinant antigen 1,20 μ L wrap by the magnetic microsphere solution of AntiHIV1 RT activity p24 monoclonal antibody A, 50 μ L, with HIV 1+2 type recombinant antigen 2 solution (being the immune interference thing bonding agent of 50% comprising mass percentage) of label protein, mix.At 37 DEG C of incubation 20min, clean 3 times under being placed in magnetic environment.
3) second step reaction: add the antibody-solutions that 100 μ L mark the anti-label protein of ABEI, 100 μ L are with HIV 1+2 type recombinant antigen 2 solution of label protein, the AntiHIV1 RT activity p24 polyclonal antibody B solution of ABEI is marked with 100 μ L, form the electrochemiluminescent immunoassay compound comprising magnetic microsphere, HIV 1+2 type recombinant antigen 1, test antibodies, HIV 1+2 type recombinant antigen 2 and ABEI compound, and form the electrochemiluminescent immunoassay compound comprising magnetic microsphere, AntiHIV1 RT activity p24 monoclonal antibody A, HIV p24 antigen, AntiHIV1 RT activity p24 monoclonal antibody B and ABEI compound.
4) detect: externally-applied magnetic field is by above-mentioned electrochemiluminescent immunoassay complex precipitate, remove supernatant, and clean with automatic filling system buffer liquid, add chemiluminescence excimer, detect the relative light intensity sent, the HIV antibody concentration of sample and the content of HIV p24 antigen can be calculated by the revised working curve of calibration object.
Embodiment 2
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1) acidic treatment agent: be the acetic acid solution of 0.1mol/L and concentration primarily of concentration be the hydrochloric acid solution of 0.01mol/L, be that 20:1 mixes according to the mol ratio of wherein acetic acid and hydrochloric acid, the pH value of this acidic treatment agent is 3.5.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 3
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1) acidic treatment agent: be the acetic acid solution of 0.5mol/L and concentration primarily of concentration be the hydrochloric acid solution of 0.05mol/L, be that 1:1 mixes according to the mol ratio of wherein acetic acid and hydrochloric acid, the pH value of this acidic treatment agent is 3.5.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 4
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1) acidic treatment agent: be the acetic acid solution of 0.5mol/L and concentration primarily of concentration be the hydrochloric acid solution of 0.05mol/L, be that 13:1 mixes according to the mol ratio of wherein acetic acid and hydrochloric acid, the pH value of this acidic treatment agent is 3.5.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 5
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1) acidic treatment agent: be the acetic acid solution of 0.5mol/L and concentration primarily of concentration be the hydrochloric acid solution of 0.05mol/L, be that 7:1 mixes according to the mol ratio of wherein acetic acid and hydrochloric acid, the pH value of this acidic treatment agent is 2.5.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 6
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1) acidic treatment agent: the acetic acid solutions being 0.1mol/L primarily of concentration forms, the pH value of this acidic treatment agent is 3.5.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 7
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1) acidic treatment agent: the acetic acid solutions being 1.2mol/L primarily of concentration forms, the pH value of this acidic treatment agent is 4.5.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 8
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1), in acidic treatment agent, acetic acid is replaced with salicylic acid.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 9
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1), in acidic treatment agent, acetic acid is replaced with salicylic acid, hydrochloric acid is replaced with sulphurous acid.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 10
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is: do not have immune interference thing bonding agent in the component of this kit.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of this comparative example to detect, and does not add immune interference thing bonding agent step in application of sample reaction.
Embodiment 11
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
1), in immune interference thing bonding agent, auxiliary element is not added.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 12
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
2) magnetic microsphere system: wrap by the magnetic microsphere solution of Streptavidin (SA), wherein: the working concentration of Streptavidin is 10 μ g/mL, and the working concentration of magnetic microsphere is 1mg/mL.
Biotin labeled HIV 1+2 type recombinant antigen 1 solution, its working concentration is 10 μ g/mL;
Biotin labeled AntiHIV1 RT activity p24 monoclonal antibody solution A, its working concentration is 10 μ g/mL.
3) label system: HIV 1+2 type recombinant antigen 2 solution of mark ABEI, its working concentration is 0.5mg/L.
The AntiHIV1 RT activity p24 polyclonal antibody of mark ABEI, its working concentration is 0.5mg/L.
The preparation method of the reference embodiment 1 of the HIV detection kit of the present embodiment obtains.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 13
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is:
2) magnetic microsphere system: wrap by the magnetic microsphere solution of goat-anti FITC polyclonal antibody, wherein: the working concentration of goat-anti FITC polyclonal antibody is 10 μ g/mL, and the working concentration of magnetic microsphere is 1mg/mL.
HIV 1+2 type recombinant antigen 1 solution of FITC mark, its working concentration is 10 μ g/mL;
The AntiHIV1 RT activity p24 monoclonal antibody solution A of FITC mark, its working concentration is 10 μ g/mL.
3) label system: the antibody-solutions of the anti-label protein of mark ABEI, its working concentration is 0.2mg/L.
With HIV 1+2 type recombinant antigen 2 solution of label protein, its working concentration is 1 μ g/mL.
With the HIV p24 polyclonal antibody of label protein, its working concentration is 0.5mg/L.
The preparation method of the reference embodiment 1 of the HIV detection kit of the present embodiment obtains.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of the present embodiment to detect.
Embodiment 14
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 12, difference is: do not have immune interference thing bonding agent in the component of this kit.
The method that HIV detects, substantially identical with the detection method in embodiment 12, difference is: adopt the detection kit of this comparative example to detect, and does not add immune interference thing bonding agent step in application of sample reaction.
Embodiment 15
Adopt the detection kit of embodiment 1 to carry out a method for HIV detection, substantially identical with the detection method in embodiment 1, difference is:
1) pre-service: 50 μ L samples and 100 μ L acidic treatment agent to be joined in reaction cup and to mix, 37 DEG C of incubations 5 minutes.
Embodiment 16
Adopt the detection kit of embodiment 1 to carry out a method for HIV detection, substantially identical with the detection method in embodiment 1, difference is:
1) pre-service: 50 μ L samples and 10 μ L acidic treatment agent to be joined in reaction cup and to mix, 37 DEG C of incubations 5 minutes.
Comparative example 1
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is: do not have acidic treatment agent and immune interference thing bonding agent in the component of this kit.
A kind of method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of this comparative example to detect, and do not have 1) pre-treatment step, and immune interference thing bonding agent step is not added in application of sample reaction.
Comparative example 2
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 12, difference is: do not have acidic treatment agent and immune interference thing bonding agent in the component of this kit.
A kind of method that HIV detects, substantially identical with the detection method in embodiment 12, difference is: adopt the detection kit of this comparative example to detect, and do not have 1) pre-treatment step, and immune interference thing bonding agent step is not added in application of sample reaction.
Comparative example 3
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 1, difference is: do not have acidic treatment agent in the component of this kit.
The method that HIV detects, substantially identical with the detection method in embodiment 1, difference is: adopt the detection kit of this comparative example to detect, and do not have 1) pre-treatment step.
Comparative example 4
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 12, difference is: do not have acidic treatment agent in the component of this kit.
The method that HIV detects, substantially identical with the detection method in embodiment 12, difference is: adopt the detection kit of this comparative example to detect, and do not have 1) pre-treatment step.
Comparative example 5
A kind of HIV detection kit, substantially identical with the detection kit of embodiment 10, difference is:
1) acidic treatment agent: concentration is the acetic acid solution of 0.01mol/L, the pH value of this acidic treatment agent is 2.5.
The method that HIV detects, substantially identical with the detection method in embodiment 10, difference is: adopt the detection kit of the present embodiment to detect.
Experimental example
Detection kit in employing above-described embodiment, comparative example and detection method are in chemiluminescent analyzer (manufacturer: Shenzhen New Industries Biomedical Engineering Co., Ltd., INSTRUMENT MODEL: Maglumi 2000) on carry out determination experiment, test sample book comprises: the National reference purchasing HIV antibody from Nat'l Pharmaceutical & Biological Products Control Institute and HIV p24 antigen.Result is as follows:
Table 1 sensitivity technique result
In upper table, sensitivity for analysis is the standard deviation that the average of test result adds twice, is M+2SD.
Can find out from the above results following some:
1, can be found by the Comparative result of embodiment 1-16 and comparative example 1-4: in embodiment of the present invention 1-16, because adding acidic treatment agent, its sensitivity for analysis is improved, higher than comparative example 1-4.
2, by the Comparative result of embodiment 1 and embodiment 10, and the Comparative result of embodiment 12 and embodiment 13 can find: after adding immune interference bonding agent, the sensitivity for analysis of embodiment 1 and embodiment 12 is greatly improved, reach 1.302pg/ml and 3.945pg/ml respectively, this early clinical diagnosis for HIV p24 is highly beneficial, is also conducive to shortening window phase.
3, can being found by the Comparative result of embodiment 1-7 and comparative example 5: mineral acid and organic acid are coordinated in accordance with the appropriate ratio, or choose extremely important to organic acid concentration, as only used mineral acid, causing acid mistake may make antigen-antibody inactivation by force.And the concentration of mineral acid too low (comparative example 5), due to acid not enough, also have impact on p24 separation in the sample.
In a word, suitable acidity for reaction extremely important, as organic acid and mineral acid coordinated with specific ratio, there is good detection sensitivity.
4, can be found by the Comparative result of embodiment 8-9: acetic acid and hydrochloric acid are not the unique selection of organic acid and mineral acid, and other acid also can substitute.
5, can be found by the Comparative result of embodiment 1-13: we can find out adopt indirect connections on final detection result substantially without affect, best connection scheme can be selected to test according to self needing.
Functional Sensitivity repeats the corresponding concentration detecting sample and have when CV is 20% in the daytime, is below Functional Sensitivity experimental result:
Table 2 embodiment 1, the Functional Sensitivity testing result of embodiment 10
Table 3 embodiment 12, the Functional Sensitivity testing result of embodiment 14
Table 4 comparative example 1, the Functional Sensitivity testing result of comparative example 2
Table 5 comparative example 3, the Functional Sensitivity testing result of comparative example 4
As can be seen from the above results, in embodiment 1 and 12, because adding the acidic treatment agent of immune interference bonding agent and p24 antigen-antibody complex, its Functional Sensitivity is apparently higher than corresponding comparative example.The Functional Sensitivity of embodiment 1 and 12 reaches 2.19pg/mL and 2.33pg/mL respectively simultaneously, and this early clinical diagnosis for HIV p24 is highly beneficial, is also conducive to shortening window phase.
Table 10 embodiment and comparative example are for HIV p24 antigenic agents national standard measurement result
The testing requirement of HIV p24 antigenic agents national standard is: negative National reference (N1-N20) result coincidence rate reaches 20/20; Positive National reference (P1-P10) result coincidence rate reaches 10/10; To sensitivity reference material, the limit of identification of HIV antibody/P24 antigenic agents must not higher than 10U/mL, and the limit of identification of HIV P24 Ag reagent must not higher than 2.5U/mL.In the result of embodiment 1 and 12, negative National reference coincidence rate reaches 20/20; Positive National reference result coincidence rate reaches 10/10; Particularly embodiment 1, sensitivity reference material is detected and reaches 0.625U/mL (L6) level, namely embodiment 1 kit measurement result meets the requirement of HIV p24 antigenic agents national standard completely, reach the requirement of HIV antigen-antibody combined detection kit, even reach the limit of identification requirement detecting separately reagent about HIVp24 antigen of Nat'l Pharmaceutical & Biological Products Control Institute.
Table 13 embodiment and comparative example HIV antibody reagent national standard measurement result
The testing requirement of HIV antibody reagent national standard is: negative National reference (N1-N20) result coincidence rate reaches 20/20; Positive National reference (P1-P20) result coincidence rate reaches 20/20; Three parts of lowest detectable limit samples at least portion are positive.In the result of embodiment 1 and 12, negative National reference coincidence rate reaches 20/20; Positive National reference result coincidence rate reaches 20/20; 6 parts of lowest detectable limit samples have at least 3 parts for positive.Namely embodiment 1 and embodiment 12 kit measurement result meet the requirement of HIV antibody reagent national standard completely.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (17)
1. the acidic treatment agent detected for HIV, it is characterized in that, inorganic acid solution primarily of concentration to be the organic acid soln of 0.1-1.0mol/L and concentration be 0.01-0.1mol/L mixes, or for concentration be the organic acid soln of 0.1-1.2mol/L, the pH value of this acidic treatment agent is 2.5-4.5.
2. the acidic treatment agent detected for HIV according to claim 1, it is characterized in that, the mol ratio of described organic acid and unitary inorganic acid is 1-20:1; Or the mol ratio of described organic acid and dibasic inorganic acid is 0.5-10:1; Or the mol ratio of described organic acid and ternary mineral acid is 0.3-6.6:1.
3. the acidic treatment agent detected for HIV according to claim 1, it is characterized in that, the mol ratio of described organic acid and unitary inorganic acid is 7-13:1; Or the mol ratio of described organic acid and dibasic inorganic acid is 3.5-6.5:1; Or the mol ratio of described organic acid and ternary mineral acid is 2.3-4.3:1.
4. the acidic treatment agent detected for HIV according to claim 1, it is characterized in that, described organic acid is at least one in acetic acid, benzoic acid, ethane diacid, succinic acid, malic acid, citric acid, salicylic acid.
5. the acidic treatment agent detected for HIV according to claim 1, it is characterized in that, described mineral acid is at least one in sulfuric acid, hydrochloric acid, nitric acid, hydrofluorite, sulphurous acid, nitrous acid.
6. a preprocess method for HIV sample to be tested, is characterized in that, adopts the acidic treatment agent described in any one of claim 1-5, is mixed by sample to be tested, obtain solution to be measured with described acidic treatment agent according to the volume ratio of 0.5-5:1.
7. the preprocess method of HIV sample to be tested according to claim 6, is characterized in that, after being mixed with described acidic treatment agent by sample to be tested, also by solution to be measured incubation 1-30 minute at 20-60 DEG C.
8. a HIV detection kit, is characterized in that, comprises following component:
1) acidic treatment agent: the acidic treatment agent described in any one of claim 1-5;
2) magnetic microsphere system: the magnetic microsphere comprising HIV recombinant antigen 1 connected directly or indirectly, and/or the magnetic microsphere of ANTI-HIV DRUGS A connected directly or indirectly;
3) label system: the HIV recombinant antigen 2 comprising corresponding mark tracer connected directly or indirectly, and/or the ANTI-HIV DRUGS B of mark tracer connected directly or indirectly;
The site that described HIV recombinant antigen 1 and HIV recombinant antigen 2 are combined with test antibodies is different, and the site that described ANTI-HIV DRUGS A and ANTI-HIV DRUGS B is combined with determined antigen is different.
9. HIV detection kit according to claim 8, is characterized in that, also comprises following component:
4) immune interference thing bonding agent: comprise bonding agent and auxiliary element; Described bonding agent is at least one in NBCS, sheep blood serum, horse serum, bovine serum albumin(BSA), goat anti-human igg/IgM, rabbit anti-human igg/IgM, mouse-anti human IgG/IgM; Described auxiliary element is at least one in glycerine, ethylene glycol, polyglycol, sucrose, sodium chloride, edetate.
10. HIV detection kit according to claim 9, is characterized in that, the concentration of described bonding agent is 0.1-20g/L, and the concentration of described auxiliary element is 0.2-10g/L.
11. HIV detection kit according to claim 8, is characterized in that, described HIV recombinant antigen is HIV 1+2 type recombinant antigen, and described ANTI-HIV DRUGS is AntiHIV1 RT activity p24 antibody.
12. HIV detection kit according to claim 8, is characterized in that, described mark tracer is luminous marker, is selected from: diamantane, luminol and derivant thereof, different luminol and derivant thereof, acridinium ester;
Or described mark tracer is chemiluminescent catalysts, is selected from: alkaline phosphatase, peroxidase.
13. HIV detection kit according to claim 8, it is characterized in that, described 2) in magnetic microsphere system, the magnetic microsphere of described indirect connection HIV recombinant antigen 1 is by wrapping by the magnetic microsphere of anti-fluorescein isothiocynate antibody, and the HIV recombinant antigen 1 of mark fluorescein isothiocynate forms; Or the magnetic microsphere of described indirect connection HIV recombinant antigen 1 is by wrapping by the magnetic microsphere of Streptavidin, and the HIV recombinant antigen 1 of mark biotin forms; Or the magnetic microsphere of described indirect connection HIV recombinant antigen 1 is by wrapping by the magnetic microsphere of anti-label protein antibody, and the HIV recombinant antigen 1 with label protein forms;
The magnetic microsphere of described indirect connection ANTI-HIV DRUGS A is by wrapping by the magnetic microsphere of anti-fluorescein isothiocynate antibody, and the ANTI-HIV DRUGS A of mark fluorescein isothiocynate forms; Or the magnetic microsphere of described indirect connection ANTI-HIV DRUGS A is by wrapping by the magnetic microsphere of Streptavidin, and the ANTI-HIV DRUGS A of mark biotin forms; Or the magnetic microsphere of described indirect connection ANTI-HIV DRUGS A is by wrapping by the magnetic microsphere of anti-label protein antibody, and the ANTI-HIV DRUGS A with label protein forms.
14. HIV detection kit according to claim 8, it is characterized in that, described 3), in label system, the HIV recombinant antigen 2 of described indirect linkage flag tracer is by the HIV recombinant antigen 2 marking biotin, and the mark tracer composition of labelled streptavidin; Or the HIV recombinant antigen 2 of described indirect linkage flag tracer is by the HIV recombinant antigen 2 of mark fluorescein isothiocynate, and the mark tracer composition of the anti-fluorescein isothiocynate antibody of mark; Or the HIV recombinant antigen 2 of described indirect linkage flag tracer is by the HIV recombinant antigen 2 with label protein, and the mark tracer composition of the anti-label protein antibody of mark;
The ANTI-HIV DRUGS B of described indirect linkage flag tracer is by the ANTI-HIV DRUGS B marking biotin, and the mark tracer composition of labelled streptavidin; Or the ANTI-HIV DRUGS B of described indirect linkage flag tracer is by the ANTI-HIV DRUGS B marking fluorescein isothiocynate, and the mark tracer composition of the anti-fluorescein isothiocynate antibody of mark; Or the ANTI-HIV DRUGS B of described indirect linkage flag tracer is by the ANTI-HIV DRUGS B with label protein, and the mark tracer composition of the anti-label protein antibody of mark.
15. 1 kinds of HIV detection methods, is characterized in that, adopt the detection kit described in any one of claim 8-14, comprise the following steps:
1) pre-service: carry out sample preprocessing according to the preprocess method described in any one of claim 6-7;
2) react: in above-mentioned solution to be measured, add magnetic microsphere system and label system, form electrochemiluminescent immunoassay compound;
3) detect: externally-applied magnetic field, by above-mentioned electrochemiluminescent immunoassay complex precipitate, is removed supernatant, after cleaning, added luminous substrate, detects the relative light intensity sent, calculates the content of test antibodies and/or determined antigen.
16. HIV detection methods according to claim 15, is characterized in that, described step 2) reaction in, also add the immune interference thing bonding agent described in any one of claim 9-10.
The application of HIV detection kit on chemiluminescent analyzer described in 17. any one of claim 8-14.
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