CN104697830B - For acidic treatment agent, sample preprocessing method, kit and the detection method of HIV detections - Google Patents
For acidic treatment agent, sample preprocessing method, kit and the detection method of HIV detections Download PDFInfo
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Abstract
The invention discloses a kind of acidic treatment agent, sample preprocessing method, kit and detection methods for HIV detections, belong to in-vitro diagnosis detection technique field.The acidic treatment agent is mainly mixed by the inorganic acid solution of the organic acid soln of a concentration of 0.1 1.0mol/L and a concentration of 0.01 0.1mol/L or the organic acid soln for a concentration of 0.1 1.2mol/L, and the pH value of the acidic treatment agent is 2.5 4.5.Using above-mentioned acidic treatment agent, p24 antigens and p24 antibody in separating sample can be very good.Using the HIV detection kits and sample preprocessing method and detection method of above-mentioned acidic treatment agent, there is high sensitivity.
Description
Technical field
The present invention relates to in-vitro diagnosis detection technique fields, and HIV (human immunodeficiencies are used for more particularly to one kind
Poison) detection acidic treatment agent, sample preprocessing method, kit and detection method.
Background technology
After inhibition of HIV (human immunodeficiency virus) intrusion human body, surface glycoprotein gp120 and cell surface receptor egg
White CD4 is combined with high-affinity, is adsorbed onto on host cell；Gp120 interacts again with host cell surface accessory receptor, makes
Virus is closer with host cell membrane；Gp41 generates a series of conformation changes, the fusion peptide fragment Insertion Into Host Cell of N-terminal
Film causes peplos to be merged with the final of cell membrane, and viral RNA enters cell.After HIV infection, it can monitor at first
The p24 antigens of viral RNA followed by virus, are finally antibody.According to the spy of HIV gene structures, immunology and epidemiology
Sign, HIV are divided into two major class of HIV-1 and HIV-2.The appeal of HIV-1 is stronger, is the main disease of world today HIV prevalences
Strain.
The method of detection HIV has more than 100 kinds at present, can be divided into two major class of antibody test and viral diagnosis on the whole.
Viral diagnosis includes cell culture (virus purification), the detection of p24 antigens and viral nucleic acid detection.Early stage is main to the diagnosis of HIV
It is the antibody that AntiHIV1 RT activity is detected by serological test, diagnoses HIV infection indirectly.Antibody is typically 3~8 weeks energy after infection
Enough it is detected.HIV infection person more than 70% can just detect antibody after infecting 6 months, in gay community, this
A number is more than 80%；In addition, newborn generates ANTI-HIV DRUGS generally after birth 1 year, the ANTI-HIV DRUGS from parent is easy
Cause newborn's false positive；Simple detection antibody method increases the danger of HIV " window phase " propagation.Antigen can be in individual
It is detected after infection prior to 2~18d of seroconversion.Therefore, the seroconversion phase by detect p24 antigens have it is very big excellent
Gesture, can be as a kind of method of early stage auxiliary diagnosis HIV infection.
But the detection of conventional use of p24 antigens there are sensitivity it is relatively low the problem of.Such as independent p24 antigen detecting agents
Detection level be about 10pg/mL, the p24 antigen detections level of joint-detection is even more that independent detection level is not achieved.But both
Make when virus actively replicates, the amount of antigen in serum also rarely reaches this level.It is also, conventional to use chemiluminescence
When immunization detects HIV, often there is false negative and false positive, cause the inaccuracy of HIV diagnostic results, occur failing to pinpoint a disease in diagnosis or miss
It examines, consequence is extremely serious.
Invention content
Based on this, the defects of it is an object of the invention to overcome the prior art, provide at a kind of acidity for HIV detections
Agent is managed, sample is pre-processed using the acidic treatment agent, the detection sensitivity of HIV can be improved.
To achieve the above object, the present invention takes following technical scheme：
A kind of acidic treatment agent for HIV detections, mainly by the organic acid soln of a concentration of 0.1-1.0mol/L and dense
Spend the organic acid soln mixed for the inorganic acid solution of 0.01-0.1mol/L or for a concentration of 0.1-1.2mol/L, the acid
Property inorganic agent pH value be 2.5-4.5.
The present inventor after numerous studies experiment investigation by having found, the low original of p24 antigen detection sensitivities in routine techniques
Cause is that the p24 antigens in HIV infection person's serum are generally all combined into compound with anti-p24 antibody, and detection method is in itself
Antigen antibody complex cannot be detected, needs complex dissociation can be detected.But complex dissociation when
It waits, just p24 antigens and anti-p24 antibody dissociations can be made by needing to handle by acid solution, in order to which all p24 antigens are released
Come, need to add in the sour agent processing of excess or high-temperature heating sample, and such as only add in hydrochloric acid inorganic acid, due to by addition salt
The amount of acid is limited, and the hydrochloric acid of addition by Sample Dilution, can only be made to be enough to discharge all p24 antigens, and could meet detection will
It asks.But sample just greatly reduces detection sensitivity after being diluted.And it is heated at high temperature sample to discharge p24 antigens, and may
Harmful effect is generated to biological sample.
Also, can not achieve the purpose that avoid diluted sample simply by improving content of hydrochloric acid, be on the one hand due to
When concentration of hydrochloric acid is excessively high, volatility is also corresponding larger, has higher requirements to instrument, thereby increases and it is possible to can influence antigen or antibody
Activity has some impact on detection accuracy；For another aspect, when concentration of hydrochloric acid is excessively high, too strong acidity can be to sample
Originally harmful effect is caused, so as to reduce the accuracy of detection.
And the acidic treatment agent of the present invention, it is that the mixed acid of high molar concentration organic acid and low molar concentration inorganic acid is molten
Liquid makes organic acid and inorganic acid match or be used alone high molar concentration organic acid soln；Using the biological of organic acid,
And the property contacted with some organic matters is easier, organic acid is made lenitively to discharge p24 antigens, a small amount of nothing can also be added in
Hydrogen ion necessary to machine acid provides, to supplement acidity.The two cooperates or is used alone the organic acid soln of high molar concentration
Can the p24 antigens in separating sample and anti-p24 antibody well, without by Sample Dilution, so that it may discharge completely therein
P24 antigens, reach testing requirements.So as to improve detection sensitivity.
The molar ratio of the organic acid and unitary inorganic acid is 1-20 in one of the embodiments,:1；It is or described organic
The molar ratio of acid and dibasic inorganic acid is 0.5-10:1；Or the molar ratio of the organic acid and ternary inorganic acid is 0.3-6.6:1.
The unitary inorganic acid, which refers to a molecule such as hydrochloric acid, can ionize out a hydrionic inorganic acid, and the dibasic inorganic acid refers to sulfuric acid
Two hydrionic inorganic acids can be ionized out Deng a molecule, the ternary inorganic acid, which refers to a molecule such as phosphoric acid, can ionize out three
A hydrionic inorganic acid.Organic acid and inorganic acid according to aforementioned proportion are coordinated, there is better p24 antigens releasing effect.
The molar ratio of the organic acid and unitary inorganic acid is 7-13 in one of the embodiments,:1；It is or described organic
The molar ratio of acid and dibasic inorganic acid is 3.5-6.5:1；Or the molar ratio of the organic acid and ternary inorganic acid is 2.3-4.3:
1。
The organic acid is acetic acid, benzoic acid, ethanedioic acid, succinic acid, malic acid, lemon in one of the embodiments,
At least one of acid, salicylic acid.
The inorganic acid is sulfuric acid, in hydrochloric acid, nitric acid, hydrofluoric acid, sulfurous acid, nitrous acid in one of the embodiments,
At least one.
The acidic treatment agent in one of the embodiments, further includes buffer solution, the buffer solution is formate buffer,
Acetate buffer, citrate buffer, Succinate Buffer, citrate buffer, sulfate buffer, nitrate
At least one of buffer solution, borate buffer solution, phosphate buffer or Tris buffer solutions.Using buffer solution, can reduce
Determinand in reagent components and sample is by the ability of the impact of acid.
The invention also discloses a kind of preprocess methods of HIV samples to be tested, will be to be measured using above-mentioned acidic treatment agent
Sample is with the acidic treatment agent according to 0.5-5:1 volume ratio mixing, obtains solution to be measured.
After sample to be tested is mixed with the acidic treatment agent in one of the embodiments, also by solution to be measured in 20-
It incubates 1-10 minutes at being incubated 1-30 minutes, preferably 20-40 DEG C at 60 DEG C, is incubated 3-7 minutes more preferably at 30-40 DEG C.It is logical
The mode incubated is crossed, more preferably p24 antigens and anti-p24 antibody can be detached faster, obtain free p24 antigens.
The invention also discloses a kind of HIV detection kits, including following components：
1) acidic treatment agent：Above-mentioned acidic treatment agent；
2) magnetic microsphere system：Magnetic microsphere including HIV recombinant antigens 1 connected directly or indirectly and/or directly
Connect or be indirectly connected with the magnetic microsphere of ANTI-HIV DRUGS A；
3) objects system is marked：HIV recombinant antigens 2 including corresponding label tracer connected directly or indirectly and/
Or the ANTI-HIV DRUGS B of label tracer connected directly or indirectly；
The site that the HIV recombinant antigens 1 and HIV recombinant antigens 2 are combined with test antibodies is different, the AntiHIV1 RT activity
The site that antibody A and ANTI-HIV DRUGS B are combined with determined antigen is different.
Above-mentioned " being directly connected to " be directly in conjunction with connection, above-mentioned " being indirectly connected with " be by biotin and strepto- it is affine
Side of the bridging object that element or fluorescein isothiocynate and anti-fluorescein isothiocynate antibody etc. can be combined with each other to combine indirectly
Formula connects.Wherein antibody A and antibody B both can be monoclonal antibody, or polyclonal antibody does not influence its detection effect
Fruit.
In above-mentioned detection kit, both it can detect HIV antibody using HIV recombinant antigens system or be resisted using AntiHIV1 RT activity
System unites to detect HIV antigens, can also come detect HIV simultaneously using HIV recombinant antigens system and ANTI-HIV DRUGS system simultaneously
Antibody and HIV antigens (such as HIV p24 antigens), can improve detection sensitivity.
Also, in above-mentioned magnetic microsphere system, in the HIV recombinant antigens 2 of the linkage flag tracer, HIV recombinations are anti-
The preferred 1-20 μ g/mL of working concentration, the preferred 0.1-5.0% of working concentration of magnetic microsphere of original 1；The connection ANTI-HIV DRUGS A
Magnetic microsphere in, the preferred 1-20 μ g/mL of working concentration of ANTI-HIV DRUGS A, the preferred 0.1- of working concentration of magnetic microsphere
5.0%.In above-mentioned label objects system, in the HIV recombinant antigens 2 of the linkage flag tracer, the work of HIV recombinant antigens 2
The preferred 2-20 μ g/mL of concentration mark the preferred 0.1-1mg/L of working concentration of tracer；The AntiHIV1 RT activity of the linkage flag tracer
In antibody B, the preferred 2-20 μ g/mL of working concentration of ANTI-HIV DRUGS B mark the preferred 0.1-1mg/L of working concentration of tracer.
It is suitable for the invention magnetic microsphere and is also referred to as magnetic bead or magnetic ball, can be magnetic microsphere commonly used in the art.
Preferably, the magnetic ball that the present invention uses, is by nano level Fe2O3Or Fe3O4Magnetic particle and high-molecular organic material carry out
It is compound, the micron-sized solid phase microballoon with superparamagnetism and huge amount protein adsorption capacity is formed, has and makees in externally-applied magnetic field
It can be magnetized rapidly under, the attribute that remanent magnetism is zero after magnetic field is withdrawn.Wherein, the type of the high-molecular organic material does not have
Especially limitation, can be selected as needed.
Magnetic microsphere used in the present invention should be able to meet a diameter of 0.1-5 μm, and magnetic microsphere can also be changed by surface
Property and with various active functional group, including but not limited to-OH ,-COOH ,-NH2。
The magnetic microsphere is Fe in one of the embodiments,2O3Or Fe3O4Magnetic nano-particle and organic polymer
The complex of material, and with 0.1-5 μm of grain size, also, the magnetic microsphere is optionally by surface modification and with one
Kind or various active functional group.
The detection kit further includes following component in one of the embodiments,：
4) immune interference object bonding agent：Including bonding agent and auxiliary element；The bonding agent is newborn bovine serum, sheep blood
Clearly, at least one of horse serum, bovine serum albumin(BSA), goat anti-human igg/IgM, rabbit anti-human igg/IgM, mouse anti-human igg/IgM；
The auxiliary element is at least one in glycerine, ethylene glycol, polyethylene glycol, sucrose, sodium chloride, edetate (EDTA)
Kind.
For the present inventor through a large number of experiments the study found that in conventional H IV detections, specificity is low with sensitivity another
It is a the reason is that having a large amount of chaff interferents in the sample, such as：Blood plasma, haemocyanin (Rheumatoid factors, binding protein), the thermophilic opposite sex are anti-
The interference of body, Human Anti animal Antibody (HAAA) and drug and its caused metabolin etc., especially latter two antibody is most.
On the basis of the studies above, in detection kit of the invention, immune interference object bonding agent is also added into, it is therein
Bonding agent as protide can be with nonspecific combination such as the heterophil antibody in sample and the wet factor of class point, and assists
Ingredient can eliminate the influence of tissue metabolism's object, so as to avoid interference of the chaff interferent to detection process, improve detection specificity and
Sensitivity.
A concentration of 0.1-20g/L of the bonding agent in one of the embodiments, the auxiliary element it is a concentration of
0.2-10g/L。
It is further included in the chaff interferent bonding agent in one of the embodiments,：Surfactant I, surfactant II,
Ascorbic acid oxidase and bilirubin oxidase, the surfactant I is zwitterionic surfactant, such as lecithin, ammonia
Base acid type, betaine type etc.；The surfactant II be nonionic surfactant, such as fatty glyceride, aliphatic acid mountain
Pears are smooth, polysorbate etc.；The preferred 0.01-0.1mol/L of concentration of the surfactant I, the concentration of the surfactant II
It is preferred that 0.01-0.1mol/L, the preferred 1-20KU/L of concentration of the ascorbic acid oxidase, the concentration of the bilirubin oxidase
It is preferred that 1-20KU/L.
Wherein：Addition surfactant is conducive to the dispersion effect of chaff interferent in sample, and ascorbic acid oxidase and courage
Red pigment oxidizing ferment can reduce the influence of ascorbic acid and bilirubin to result in sample, each ingredient in immune interference object bonding agent
It cooperates, preferable Anti-Jamming can be reached.
The HIV recombinant antigens are HIV 1+2 type recombinant antigens in one of the embodiments, and the ANTI-HIV DRUGS is
AntiHIV1 RT activity p24 antibody.The HIV 1+2 types recombinant antigen corresponds to gp36 for corresponding 2 type of gp41, gp120 and HIV of 1 types of HIV
Recombinant protein.
Above-mentioned label tracer includes following several：1st, the label that can directly shine that chemiluminescence immune assay uses
Object, such as luminol and its derivative, different luminol or derivatives thereof, acridinium ester；2nd, chemiluminescence enzyme immunoassay uses
The marker for coordinating corresponding luminous substrate that can shine, such as alkaline phosphatase or peroxidase.
The label tracer is luminous marker in one of the embodiments, is selected from：Adamantane, luminol and its
Derivative, different luminol and its derivative, acridinium ester.It is and upper it is preferred that N- (4- aminobutyls)-N- ethyls different luminol (ABEI)
The oxidative system for stating luminous marker cooperation includes H2O2Microperoxisome, H2O2Catalase, H2O2Lactoperoxidase
Enzyme, H2O2Deuterohemin, H2O2Hemin, hypochlorite-CoCl2, persulfate, potassium peroxide, sodium metaperiodate,
H2O2-K3Fe(CN)6, xanthine-hypoxanthine oxidase, at least one of potassium tert-butoxide.
Above-mentioned luminous marker refers to participate in energy transfer in luminescence-producing reaction and finally be discharged in the form of emitting photon
The compound of energy, the catalysis and the oxidation of oxidant which can be through catalyst, the intermediate of one excitation state of formation,
When this excitation state intermediate returns to stable ground state, while launch photon (hM).
Or the label tracer is chemiluminescent catalysts, is selected from：Alkaline phosphatase, peroxidase.In use, match
It closes corresponding chemiluminescent substrate and can shine and be qualitatively detected, the chemiluminescent substrate includes NaOH and H2O2, further include gold
At least one of firm alkane, luminol and its derivative, different luminol or derivatives thereof, preferably N- (4- aminobutyls)-N- second
Base different luminol (ABEI).
In one of the embodiments, in 2) the magnetic microsphere system, the magnetic for being indirectly connected with HIV recombinant antigens 1
Property microballoon by being coated with the magnetic microsphere of anti-fluorescein isothiocynate antibody and marking the HIV recombinant antigens 1 of fluorescein isothiocynate
Composition；Or the magnetic microsphere for being indirectly connected with HIV recombinant antigens 1 is given birth to by the magnetic microsphere and label of coating Streptavidin
The HIV recombinant antigens 1 of object element form；Or the magnetic microsphere for being indirectly connected with HIV recombinant antigens 1 is resisted by being coated with anti-label protein
The magnetic microsphere of body and the HIV recombinant antigens 1 with label protein form；
The magnetic microsphere for being indirectly connected with ANTI-HIV DRUGS A (i.e. AntiHIV1 RT activity p24 antibody) is by being coated with anti-isosulfocyanic acid fluorescence
The magnetic microsphere of plain antibody and the ANTI-HIV DRUGS A compositions for marking fluorescein isothiocynate；Or described it is indirectly connected with ANTI-HIV DRUGS
The magnetic microsphere of A is made of the magnetic microsphere of coating Streptavidin and the ANTI-HIV DRUGS A of label biotin；It is or described indirect
The magnetic microsphere of ANTI-HIV DRUGS A is connected by being coated with the magnetic microsphere of anti-label protein antibody and there is the AntiHIV1 RT activity of label protein
Antibody A forms.
Above-mentioned anti-fluorescein isothiocynate (FITC) antibody both can be monoclonal antibody, or polyclonal antibody.
Also, in above-mentioned magnetic microsphere system, in the magnetic microsphere of the anti-fluorescein isothiocynate antibody of coating, resist different
The preferred 1-20 μ g/mL of working concentration of thiocyanic acid anti-fluorescein antibody, the working concentration preferred 0.1-2mg/mL of magnetic microsphere, it is described
In the HIV recombinant antigens 1 (or ANTI-HIV DRUGS A) for marking fluorescein isothiocynate, the working concentration of fluorescein isothiocynate is preferred
The preferred 0.1-10 μ g/mL of working concentration of 5-500ng/mL, HIV recombinant antigen 1 (or ANTI-HIV DRUGS A)；The coating strepto- parent
In the magnetic microsphere of element, the preferred 1-20 μ g/mL of working concentration of Streptavidin, the preferred 0.1- of working concentration of magnetic microsphere
2mg/mL, the preferred 10-500 μ g/mL of working concentration of the HIV recombinant antigens 1 (or ANTI-HIV DRUGS A) of the label biotin.
It is described in one of the embodiments, 3) to mark in objects system, the HIV recombinations for being indirectly connected with label tracer
Antigen 2 is made of the HIV recombinant antigens 2 of label biotin and the label tracer of labelled streptavidin；Or it is described between in succession
The HIV recombinant antigens 2 of label tracer are connect by the HIV recombinant antigens 2 of label fluorescein isothiocynate and the anti-isothiocyanic acid of label
The label tracer composition of anti-fluorescein antibody；Or the HIV recombinant antigens 2 for being indirectly connected with label tracer are by with label egg
White HIV recombinant antigens 2 and the label tracer composition of the anti-label protein antibody of label；
The ANTI-HIV DRUGS B for being indirectly connected with label tracer is by the ANTI-HIV DRUGS B of label biotin and label strepto-
The label tracer composition of Avidin；Or the ANTI-HIV DRUGS B for being indirectly connected with label tracer is by label isosulfocyanic acid fluorescence
The ANTI-HIV DRUGS B of element and the label tracer composition of the anti-fluorescein isothiocynate antibody of label；Or it described is indirectly connected with marking
The ANTI-HIV DRUGS B of tracer is by the ANTI-HIV DRUGS B with label protein and the label tracer of the anti-label protein antibody of label
Composition.
The above-mentioned preferred Flag label proteins of label protein, Flag label proteins are the hydrophilic polypeptides for encoding 8 amino acid
(DYKDDDDK), the Kozak sequences while in carrier built cause the table in eukaryotic expression system of the fusion protein with Flag
Up to more efficient
Said program enriches magnetic microsphere system used in detection and label objects system, can be flexible according to different demands
Selection.Such as HIV recombinant antigens 1 (or ANTI-HIV DRUGS A) directly can be connected into magnetic microsphere in magnetic microsphere system,
Can HIV recombinant antigens 1 (or ANTI-HIV DRUGS A) and magnetic microsphere be connected by above-mentioned indirect bridging mode.It is also possible to
Label tracer is directly connected to HIV recombinant antigens 2 (or ANTI-HIV DRUGS B) in objects system is marked, it can also be by above-mentioned
Indirect bridging mode linkage flag tracer and HIV recombinant antigens 2 (or ANTI-HIV DRUGS B).Also, above-mentioned magnetic microsphere system
It influences each other or limits with being had no in label objects system using which kind of connection mode (be directly connected to or connection of putting up a bridge indirectly), both may be used
It, also can magnetic simultaneously using the mode that is directly connected to or simultaneously using indirect connections in magnetic microsphere system and label objects system
Property the selection of microballoon system be directly connected to, label objects system is marked using being indirectly connected with or the selection of magnetic microsphere system is indirectly connected with
Note objects system, which uses, to be directly connected to.
It should be understood that the detection kit further includes in one of the embodiments,：
5) calibration object solution：(HIV inactivated is positive for negative calibration object (HIV negative serums product) and positive calibration object
Serum product) solution.
By the proportionate relationship of sample and calibration object solution relative light intensity (RLU), measurement result is calculated.
Contain bovine serum albumin(BSA) (BSA) and anti-corrosion in the detection kit each component in one of the embodiments,
Agent, a concentration of 0.01-0.5g/ml of BSA, preservative is potassium sorbate, sodium benzoate, Sodium azide, sodium nitrite, Proclin
At least one of series.
The invention also discloses a kind of HIV detection methods, using above-mentioned detection kit, include the following steps：
1) it pre-processes：Sample preprocessing is carried out according to above-mentioned preprocess method；
2) it reacts：Magnetic microsphere system and label objects system are added in above-mentioned solution to be measured, it is compound to form electrochemiluminescent immunoassay
Object；
3) it detects：Above-mentioned electrochemiluminescent immunoassay complex precipitate, removal supernatant after cleaning, are added in the bottom that shines by externally-applied magnetic field
Object detects the relative light intensity sent out, and the content of test antibodies and/or determined antigen is calculated.
In one of the embodiments, in the step 2) reaction, it is additionally added above-mentioned immune interference object bonding agent.Make to exempt from
Epidemic disease chaff interferent bonding agent is combined with the chaff interferent in sample, so as to avoid interference of the chaff interferent to detection process, improves detection
Sensitivity.
The invention also discloses a kind of application of above-mentioned HIV detection kits on chemiluminescent analyzer.This is tried
Agent box is applied to chemiluminescent analyzer, has the advantages of working specification, nothing is artificially introduced error and full-automation.
Compared with prior art, the invention has the advantages that：
The present invention a kind of acidic treatment agent, organic acid and inorganic acid are matched, using organic acid be easier and some
The property of organic matter contact, makes organic acid lenitively detach p24 antigens and anti-p24 antibody, and inorganic acid offer can also be used must
The hydrogen ion needed, to supplement acidity.The two cooperates or is used alone organic acid, can be well in separating sample
P24 antigens and anti-p24 antibody, when being detected, without by Sample Dilution or high-temperature heating, so that it may discharge completely therein
P24 antigens reach testing requirements, improve detection sensitivity.
A kind of preprocess method of sample to be tested of the present invention, using above-mentioned acidic treatment agent, in sample preprocessing,
It need not be by Sample Dilution or high-temperature heating, it is possible to which p24 antigens and anti-p24 antibody in good separating sample discharge completely
P24 antigens therein, reach testing requirements.Also, this method is in sample preprocessing, also by solution to be measured at 20-60 DEG C
It incubates 1-30 minutes.By way of incubation, more preferably p24 antigens from anti-p24 antibody can be discharged faster, dissociated
P24 antigens not only improve detection efficiency, but also will not generate harmful effect to sample due to high temperature.
A kind of HIV detection kits of the present invention, in addition to above-mentioned acidic treatment agent is employed, are also added into immune interference object
Bonding agent by avoiding interference of the chaff interferent to detection process, further improves detection sensitivity.Make the inspection of p24 antigens
Go out level and reach 0.625U/mL (L6), reached the requirement of HIV antigen-antibody combined detection kits, be even up to China
Pharmaceutical biological product calibrating institute about the independent detection reagent of HIV p24 antigens limit of identification require.HIV p24 are reached
Antigen detection assays sensitivity is less than 1pg/mL, Functional Sensitivity is less than the purpose of 5pg/mL.And theoretically, detect P24 antigens
Window phase generally in 4-14d or so, reduce 2/3 compared with the HIV antibody detection window phase, press on towards the window level of HIV-RNA, and examine
Survey cost but reduces much compared with p24 antigens and detection of nucleic acids.
A kind of HIV detection methods of the present invention, wherein sample use above-mentioned preprocess method, by organic acid and inorganic acid
Organic acid is matched or be used alone as acidic treatment agent, detection sensitivity can be improved.Particularly, also coordinate immune interference
The use of object bonding agent by avoiding interference of the chaff interferent to detection process, further improves detection sensitivity.
Specific embodiment
The present invention is described further with reference to embodiments, but does not cause any restrictions to the present invention.
In following embodiment：
HIV 1+2 types 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 ABEI are marked, purchased from meridian life science.
HIV 1+2 type recombinant antigens with label protein, purchased from meridian life science.
AntiHIV1 RT activity p24 polyclonal antibody B, purchased from meridian life science.
Goat-anti FITC polyclonal antibodies, purchased from hundred great bio tech ltd of Beijing.
Magnetic microsphere, source：It is produced for Shenzhen NPD projects biomedicine limited company.
FITC：Purchased from Sigma.
ABEI：It is produced for Shenzhen NPD projects biomedicine limited company.
Biotin, Streptavidin：It is purchased from Roche.
Negative calibration object and positive calibration object：It is purchased from meridian life science.
Embodiment 1
A kind of HIV detection kits, including following components：
1) acidic treatment agent：
The hydrochloric acid solution of the acetic acid solution of a concentration of 0.5mol/L and a concentration of 0.05mol/L, according to wherein acetic acid and salt
The molar ratio of acid is 10：1 mixing；And by volume 1:1 adds in the acetate buffer of 0.05mol/L, obtains acidic treatment agent,
The pH value of the acidic treatment agent is 3.5.
2) magnetic microsphere system：The magnetic microsphere solution of HIV 1+2 types recombinant antigen 1 is coated with, wherein：HIV 1+2 type weights
The working concentration of group antigen 1 is 10 μ g/mL, and the working concentration of magnetic microsphere is 0.5mg/mL.
The magnetic microsphere solution of AntiHIV1 RT activity p24 monoclonal antibodies A is coated with, wherein：AntiHIV1 RT activity p24 monoclonal antibodies A's is dense
It spends for 10 μ g/mL, the working concentration of magnetic microsphere is 0.5mg/mL.
3) objects system is marked：Mark the antibody-solutions of the anti-label protein of ABEI, working concentration 0.2mg/L.
HIV 1+2 types recombinant antigen 2 with label protein, working concentration are 1 μ g/mL；
Mark AntiHIV1 RT activity p24 the polyclonal antibody B, working concentration 0.5mg/L of ABEI.
The label protein is Flag label proteins.
4) immune interference object bonding agent, including：
Bonding agent：Concentration expressed in percentage by volume is the newborn bovine serum of 0.1-20%, 0.1-20g/L bovine serum albumin(BSA)s, 0.01-
5g/L goat anti-human iggs/IgM, 0.01-5g/L rabbit anti-human igg/IgM；
Auxiliary element：0.01-5g/L ethylene glycol, 0.01-5g/L EDTA-2K, 0.01-0.2mol/L Macrogol 6000s；
Buffering component：0.01-5g/L Tris, 0.01-0.2mol/L PBS buffers；
Surfactant：Lecithin (the source of 0.01-0.1mol/L：Upper marine base row chemical industry), 0.01-0.1mol/L
Fatty glyceride (source：Upper marine base row chemical industry)；
Ascorbic acid oxidase：1-20KU/L；
Bilirubin oxidase：1-20KU/L.
5) calibration object solution：Negative calibration object (HIV negative serums product) and positive calibration object (HIV positive serum products,
Inactivate) solution.
Above-mentioned each component is containing bovine serum albumin(BSA) (BSA) and preservative, a concentration of 0.05g/ml of BSA, preservative master
It is NaN to want ingredient3, a concentration of 0.02g/ml.
In the preparation method of the HIV detection kits of the present embodiment, in addition to following reagent, remaining is conventionally made
It is standby.
The magnetic microsphere of above-mentioned coating HIV 1+2 types recombinant antigen 1 and the magnetism of coating AntiHIV1 RT activity p24 monoclonal antibodies A
Microballoon is prepared by the following method：Using 1- cyclohexyl -2- morpholine ethyl carbodiimide tosilate (CMC) etc. as taking
Bridge substance connection antigen (or antibody) and the magnetic microsphere for having adsorbed carboxylic group after processing, 30-40 DEG C incubates 1-2 hours,
It can be used using the washing removal impurity of 3-5 magnetic ball suspension.
The antibody AntiHIV1 RT activity p24 polyclonal antibodies of ABEI (or label) of the anti-label protein of above-mentioned label ABEI by with
It is prepared by lower section method：ABEI is connected using n-hydroxysuccinimide (NHS) etc. as bridging substance with after antibody (or antigen), passing through
It crosses G-25 gel chromatographies and crosses column and obtain purified.
The method for carrying out HIV antigens and antibody combined detection using the detection kit of the present embodiment, includes the following steps：
1) it pre-processes：50 μ L samples, 50 μ L feminine genders calibration objects and 50 μ L positive calibration objects are added separately in reaction cup,
And 50 μ L acidic treatment agent are added in, mixing incubates 5 minutes at 37 DEG C.
2) first step is reacted：The magnetic microsphere solution of 20 μ L coating HIV 1+2 types recombinant antigen 1 is added in, 20 μ L coatings are anti-
The magnetic microsphere solution of HIV p24 monoclonal antibodies A, 50 μ L carry 2 solution (its of HIV 1+2 types recombinant antigen of label protein
Include the immune interference object bonding agent that mass percentage is 50%), mixing.20min is incubated at 37 DEG C, is placed under magnetic environment
Cleaning 3 times.
3) second step reacts：The antibody-solutions of the anti-label protein of 100 μ L labels ABEI are added in, 100 μ L carry label egg
The AntiHIV1 RT activity p24 polyclonal antibody B solutions of white 2 solution of HIV 1+2 types recombinant antigen and 100 μ L labels ABEI, formation include
Magnetic microsphere, HIV 1+2 types recombinant antigen 1, test antibodies, 2 electrochemiluminescent immunoassay compound with ABEI of HIV 1+2 types recombinant antigen are answered
It closes object and is formed and include magnetic microsphere, AntiHIV1 RT activity p24 monoclonal antibody A, HIV p24 antigens, AntiHIV1 RT activity p24 monoclonal antibodies B
The electrochemiluminescent immunoassay compound compound with ABEI.
4) it detects：Externally-applied magnetic field delays above-mentioned electrochemiluminescent immunoassay complex precipitate, removal supernatant with automatic filling system
Fliud flushing is cleaned, and is added in chemiluminescence excimer, is detected the relative light intensity sent out, bent by the revised work of calibration object
The HIV antibody concentration of sample and the content of HIV p24 antigens can be calculated in line.
Embodiment 2
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) acidic treatment agent：Hydrochloric acid mainly by the acetic acid solution of a concentration of 0.1mol/L and a concentration of 0.01mol/L is molten
Liquid, the molar ratio according to wherein acetic acid and hydrochloric acid are 20：1 mixes, and the pH value of the acidic treatment agent is 3.5.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 3
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) acidic treatment agent：Hydrochloric acid mainly by the acetic acid solution of a concentration of 0.5mol/L and a concentration of 0.05mol/L is molten
Liquid, the molar ratio according to wherein acetic acid and hydrochloric acid are 1：1 mixes, and the pH value of the acidic treatment agent is 3.5.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 4
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) acidic treatment agent：Hydrochloric acid mainly by the acetic acid solution of a concentration of 0.5mol/L and a concentration of 0.05mol/L is molten
Liquid, the molar ratio according to wherein acetic acid and hydrochloric acid are 13：1 mixes, and the pH value of the acidic treatment agent is 3.5.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 5
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) acidic treatment agent：Hydrochloric acid mainly by the acetic acid solution of a concentration of 0.5mol/L and a concentration of 0.05mol/L is molten
Liquid, the molar ratio according to wherein acetic acid and hydrochloric acid are 7：1 mixes, and the pH value of the acidic treatment agent is 2.5.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 6
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) acidic treatment agent：It is mainly formed by the acetic acid solutions of a concentration of 0.1mol/L, the pH of the acidic treatment agent
Be worth is 3.5.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 7
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) acidic treatment agent：It is mainly formed by the acetic acid solutions of a concentration of 1.2mol/L, the pH of the acidic treatment agent
Be worth is 4.5.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 8
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) in acidic treatment agent, acetic acid is replaced with into salicylic acid.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 9
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) in acidic treatment agent, acetic acid is replaced with into salicylic acid, hydrochloric acid is replaced with into sulfurous acid.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 10
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：The kit
Component in there is no immune interference object bonding agent.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：It is right using this
The detection kit of ratio is detected, and without adding in immune interference object bonding agent step in sample-adding reacts.
Embodiment 11
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
1) in immune interference object bonding agent, auxiliary element is not added.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 12
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
2) magnetic microsphere system：The magnetic microsphere solution of Streptavidin (SA) is coated with, wherein：The work of Streptavidin
A concentration of 10 μ g/mL, the working concentration of magnetic microsphere is 1mg/mL.
1 solution of HIV 1+2 types recombinant antigen of biotin labeling, working concentration are 10 μ g/mL；
The AntiHIV1 RT activity p24 monoclonal antibody solution As of biotin labeling, working concentration are 10 μ g/mL.
3) objects system is marked：Mark 2 solution of HIV 1+2 types recombinant antigen of ABEI, working concentration 0.5mg/L.
Mark the AntiHIV1 RT activity p24 polyclonal antibodies of ABEI, working concentration 0.5mg/L.
The preparation method with reference to embodiment 1 of the HIV detection kits of the present embodiment obtains.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 13
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：
2) magnetic microsphere system：The magnetic microsphere solution of goat-anti FITC polyclonal antibodies is coated with, wherein：More grams of goat-anti FITC
The working concentration of grand antibody is 10 μ g/mL, and the working concentration of magnetic microsphere is 1mg/mL.
1 solution of HIV 1+2 types recombinant antigen of FITC labels, working concentration are 10 μ g/mL；
The AntiHIV1 RT activity p24 monoclonal antibody solution As of FITC labels, working concentration are 10 μ g/mL.
3) objects system is marked：Mark the antibody-solutions of the anti-label protein of ABEI, working concentration 0.2mg/L.
2 solution of HIV 1+2 types recombinant antigen with label protein, working concentration are 1 μ g/mL.
HIV p24 polyclonal antibodies with label protein, working concentration 0.5mg/L.
The preparation method with reference to embodiment 1 of the HIV detection kits of the present embodiment obtains.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：Using this reality
The detection kit for applying example is detected.
Embodiment 14
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 12, the difference lies in：The reagent
There is no immune interference object bonding agent in the component of box.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 12, the difference lies in：Using this
The detection kit of comparative example is detected, and without adding in immune interference object bonding agent step in sample-adding reacts.
Embodiment 15
A kind of method that detection kit using embodiment 1 carries out HIV detections, with the detection method base in embodiment 1
This is identical, the difference lies in：
1) it pre-processes：50 μ L samples and 100 μ L acidic treatment agent are added in reaction cup simultaneously mixing, 5 are incubated at 37 DEG C
Minute.
Embodiment 16
A kind of method that detection kit using embodiment 1 carries out HIV detections, with the detection method base in embodiment 1
This is identical, the difference lies in：
1) it pre-processes：50 μ L samples and 10 μ L acidic treatment agent are added in reaction cup simultaneously mixing, 5 points are incubated at 37 DEG C
Clock.
Comparative example 1
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：The kit
Component in there is no acidic treatment agent and immune interference object bonding agent.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：It is right using this
The detection kit of ratio is detected, and is done without 1) pre-treatment step and that addition is not immune in reaction is loaded
Disturb object bonding agent step.
Comparative example 2
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 12, the difference lies in：The reagent
There is no acidic treatment agent and immune interference object bonding agent in the component of box.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 12, the difference lies in：Using this
The detection kit of comparative example is detected, and immune without adding in without 1) pre-treatment step and in sample-adding reacts
Chaff interferent bonding agent step.
Comparative example 3
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 1, the difference lies in：The kit
Component in there is no acidic treatment agent.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 1, the difference lies in：It is right using this
The detection kit of ratio is detected, and without 1) pre-treatment step.
Comparative example 4
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 12, the difference lies in：The reagent
There is no acidic treatment agent in the component of box.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 12, the difference lies in：Using this
The detection kit of comparative example is detected, and without 1) pre-treatment step.
Comparative example 5
A kind of HIV detection kits, it is essentially identical with the detection kit of embodiment 10, the difference lies in：
1) acidic treatment agent：The acetic acid solution of a concentration of 0.01mol/L, the pH value of the acidic treatment agent is 2.5.
A kind of method of HIV detections, it is essentially identical with the detection method in embodiment 10, the difference lies in：Using this
The detection kit of embodiment is detected.
Experimental example
Using the detection kit in above-described embodiment, comparative example and detection method in chemiluminescent analyzer (manufacturer：
Shenzhen New Industries Biomedical Engineering Co., Ltd., instrument model：Maglumi 2000) on carry out determination experiment, survey
Sample originally includes：It purchases from the HIV antibody of Nat'l Pharmaceutical ＆ Biological Products Control Institute and the National reference of HIV p24 antigens.Knot
Fruit is as follows：
1 sensitivity technique result of table
In upper table, sensitivity for analysis adds twice of standard deviation, as M+2SD for the mean value of test result.
It can be seen that the following in from the above：
1st, it can be found by the Comparative result of embodiment 1-16 and comparative example 1-4：In 1-16 of the embodiment of the present invention, because adding
Acidic treatment agent, sensitivity for analysis are improved, higher than comparative example 1-4.
2nd, it can be sent out by the Comparative result and embodiment 12 of embodiment 1 and embodiment 10 and the Comparative result of embodiment 13
It is existing：After immune interference bonding agent is added in, the sensitivity for analysis of embodiment 1 and embodiment 12 is greatly improved, respectively
1.302pg/ml and 3.945pg/ml are reached, this is highly beneficial for the early clinical diagnosis of HIV p24, is also beneficial to shorten
Window phase.
3rd, it can be found by embodiment 1-7 and the Comparative result of comparative example 5：By inorganic acid and organic acid in accordance with the appropriate ratio
Coordinate or extremely important to the concentration selection of organic acid, such as only cause acidity is too strong may make antigen-antibody with inorganic acid
Inactivation.And the concentration of inorganic acid is too low (comparative example 5), due to acid deficiency, also affects the separation of p24 in the sample.
In short, suitable acidity is extremely important for reaction, such as organic acid and inorganic acid are matched with specific ratio
It closes, there is preferable detection sensitivity.
4th, it can be found by the Comparative result of embodiment 8-9：Acetic acid and hydrochloric acid are not unique choosing of organic acid and inorganic acid
It selects, other acid can also substitute.
5th, it can be found by the Comparative result of embodiment 1-13：We can see that using indirect connections to finally detecting
As a result best connection scheme can be selected to be tested according to their needs without influence substantially.
Functional Sensitivity is to detect the concentration that has of sample to repeat to correspond to when CV is 20% in the daytime, sensitive for function below
Spend experimental result：
2 embodiment 1 of table, the Functional Sensitivity testing result of embodiment 10
3 embodiment 12 of table, the Functional Sensitivity testing result of embodiment 14
4 comparative example 1 of table, the Functional Sensitivity testing result of comparative example 2
5 comparative example 3 of table, the Functional Sensitivity testing result of comparative example 4
In from the above as can be seen that in embodiment 1 and 12, because adding in immune interference bonding agent and p24 Ag-Abs
The acidic treatment agent of compound, Functional Sensitivity are apparently higher than corresponding comparative example.The function of embodiment 1 and 12 is sensitive simultaneously
Degree has respectively reached 2.19pg/mL and 2.33pg/mL, this is highly beneficial for the early clinical diagnosis of HIV p24, is also beneficial to
Shorten window phase.
10 embodiment of table and comparative example are for HIV p24 antigenic agents national standard measurement results
The testing requirements of HIV p24 antigenic agents national standards are：Negative National reference (N1-N20) result meets
Rate reaches 20/20；Positive National reference (P1-P10) result coincidence rate reaches 10/10；To sensitivity reference material, HIV antibody/
The limit of identification of P24 antigenic agents must not be higher than 10U/mL, and the limit of identification of HIV P24 antigenic agents must not be higher than
2.5U/mL.In the result of embodiment 1 and 12, negative National reference coincidence rate reaches 20/20；Positive National reference knot
Fruit coincidence rate reaches 10/10；In fact particularly embodiment 1 reaches 0.625U/mL (L6) levels, i.e., to the detection of sensitivity reference material
The requirement that 1 kit measurement result of example complies fully with HIV p24 antigenic agents national standards is applied, has reached HIV antigen-antibodies
The requirement of combined detection kit has been even up to individually being examined about HIV p24 antigens for Nat'l Pharmaceutical ＆ Biological Products Control Institute
The limit of identification requirement of test agent.
13 embodiment of table and comparative example HIV antibody reagent national standard measurement result
The testing requirements of HIV antibody reagent national standard are：Negative National reference (N1-N20) result coincidence rate reaches
To 20/20；Positive National reference (P1-P20) result coincidence rate reaches 20/20；Three parts of minimum detection limit samples are at least a
For the 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 minimum detection limit samples have at least 3 parts for the positive.I.e. embodiment 1 and 12 kit of embodiment are surveyed
Determine the requirement that result complies fully with HIV antibody reagent national standard.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (12)
1. a kind of HIV detection kits, which is characterized in that including following components：
1）Acidic treatment agent：Mainly by the organic acid soln of a concentration of 0.1-1.0 mol/L and a concentration of 0.01-0.1 mol/L
Inorganic acid solution mix, the pH value of the acidic treatment agent is 2.5-4.5；Wherein, the inorganic acid is unitary inorganic acid,
The molar ratio of the organic acid and unitary inorganic acid is 1-20:1；Or the inorganic acid be dibasic inorganic acid, the organic acid and
The molar ratio of dibasic inorganic acid is 0.5-10:1；Or the inorganic acid is ternary inorganic acid, the organic acid and ternary inorganic acid
Molar ratio be 0.3-6.6:1；
2）Magnetic microsphere system：It magnetic microsphere including HIV recombinant antigens 1 connected directly or indirectly and/or is directly connected to
Or it is indirectly connected with the magnetic microsphere of ANTI-HIV DRUGS A；
3）Mark objects system：Including the HIV recombinant antigens 2 of label tracer connected directly or indirectly accordingly and/or directly
Meet or be indirectly connected in succession the ANTI-HIV DRUGS B of label tracer；
The site that the HIV recombinant antigens 1 and HIV recombinant antigens 2 are combined with test antibodies is different, the ANTI-HIV DRUGS
The site that A and ANTI-HIV DRUGS B are combined with determined antigen is different.
2. HIV detection kits according to claim 1, which is characterized in that the organic acid and unitary inorganic acid rub
You are than being 7-13:1；Or the molar ratio of the organic acid and dibasic inorganic acid is 3.5-6.5:1；Or the organic acid and ternary without
The molar ratio of machine acid is 2.3-4.3:1.
3. HIV detection kits according to claim 1, which is characterized in that the organic acid is acetic acid, benzoic acid, second
At least one of diacid, succinic acid, malic acid, citric acid, salicylic acid.
4. HIV detection kits according to claim 1, which is characterized in that further include following component：
4）Immune interference object bonding agent：Including bonding agent and auxiliary element；The bonding agent is newborn bovine serum, sheep blood serum, horse
At least one of serum, bovine serum albumin(BSA), goat anti-human igg/IgM, rabbit anti-human igg/IgM, mouse anti-human igg/IgM；It is described
Auxiliary element is at least one of glycerine, ethylene glycol, polyethylene glycol, sucrose, sodium chloride, edetate.
5. HIV detection kits according to claim 4, which is characterized in that a concentration of 0.1-20 g/ of the bonding agent
L, a concentration of 0.2-10 g/L of the auxiliary element.
6. HIV detection kits according to claim 1, which is characterized in that the HIV recombinant antigens are HIV 1+2 types
Recombinant antigen, the ANTI-HIV DRUGS are AntiHIV1 RT activity p24 antibody.
7. HIV detection kits according to claim 1, which is characterized in that the label tracer is luminous marker,
It is selected from：Adamantane, luminol and its derivative, different luminol and its derivative, acridinium ester；
Or the label tracer is chemiluminescent catalysts, is selected from：Alkaline phosphatase, peroxidase.
8. HIV detection kits according to claim 1, which is characterized in that described 2）In magnetic microsphere system, between described
The magnetic microsphere of HIV recombinant antigens 1 is connect in succession by being coated with the magnetic microsphere of anti-fluorescein isothiocynate antibody and the different sulphur cyanogen of label
The HIV recombinant antigens 1 of sour fluorescein form；Or the magnetic microsphere for being indirectly connected with HIV recombinant antigens 1 is affine by coating strepto-
The magnetic microsphere of element and the HIV recombinant antigens 1 of label biotin form；Or the magnetism for being indirectly connected with HIV recombinant antigens 1
Microballoon is formed by being coated with the magnetic microsphere of anti-label protein antibody and the HIV recombinant antigens 1 with label protein；
The magnetic microsphere for being indirectly connected with ANTI-HIV DRUGS A is by being coated with the magnetic microsphere of anti-fluorescein isothiocynate antibody and marking
Remember the ANTI-HIV DRUGS A compositions of fluorescein isothiocynate；Or the magnetic microsphere for being indirectly connected with ANTI-HIV DRUGS A is by coating strepto-
The magnetic microsphere of Avidin and the ANTI-HIV DRUGS A compositions for marking biotin；Or the magnetism for being indirectly connected with ANTI-HIV DRUGS A
Microballoon is formed by being coated with the magnetic microsphere of anti-label protein antibody and the ANTI-HIV DRUGS A with label protein.
9. HIV detection kits according to claim 1, which is characterized in that described 3）It marks in objects system, it is described indirect
The HIV recombinant antigens 2 of linkage flag tracer are by the HIV recombinant antigens 2 of label biotin and the label of labelled streptavidin
Tracer forms；Or the HIV recombinant antigens 2 for being indirectly connected with label tracer are by the HIV weights of label fluorescein isothiocynate
The label tracer composition of group antigen 2 and the anti-fluorescein isothiocynate antibody of label；Or described it is indirectly connected with label tracer
HIV recombinant antigens 2 by the HIV recombinant antigens 2 with label protein and the anti-label protein antibody of label label tracer group
Into；
The ANTI-HIV DRUGS B for being indirectly connected with label tracer is affine by the ANTI-HIV DRUGS B and label strepto- of label biotin
The label tracer composition of element；Or the ANTI-HIV DRUGS B for being indirectly connected with label tracer is by label fluorescein isothiocynate
The label tracer composition of ANTI-HIV DRUGS B and the anti-fluorescein isothiocynate antibody of label；Or it is described be indirectly connected with label tracer
The ANTI-HIV DRUGS B of object by the ANTI-HIV DRUGS B with label protein and the anti-label protein antibody of label label tracer group
Into.
10. application of the claim 1-9 any one of them HIV detection kits on chemiluminescent analyzer.
11. a kind of preprocess method of HIV samples to be tested, which is characterized in that using claim 1-9 any one of them HIV
Detection kit, by sample to be tested and the acidic treatment agent according to 0.5-5:1 volume ratio mixing, obtains solution to be measured.
12. the preprocess method of HIV samples to be tested according to claim 11, which is characterized in that by sample to be tested and institute
After stating acidic treatment agent mixing, also solution to be measured is incubated 1-30 minutes at 20-60 DEG C.
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| CN106290864A (en) * | 2016-08-12 | 2017-01-04 | 江苏泽成生物技术有限公司 | A kind of human immunodeficiency virus antigen and TPPA test kit and preparation method |
| CN108680735A (en) * | 2018-05-21 | 2018-10-19 | 苏州佑君环境科技有限公司 | A kind of acridinium ester labeled antibody dilution and preparation method thereof |
| CN112362864B (en) * | 2021-01-12 | 2021-04-30 | 广州科方生物技术股份有限公司 | Treating agent for reducing background luminescence value of immunodiagnostic reagent and preparation method thereof |
| CN113295864A (en) * | 2021-04-25 | 2021-08-24 | 北京指真生物科技有限公司 | Kit and detection method for quantitative combined detection of HIV (human immunodeficiency Virus) antigen and antibody |
| CN114544978A (en) * | 2022-03-04 | 2022-05-27 | 美康生物科技股份有限公司 | IGF-1 detection kit, preparation method and detection method thereof |
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| US8216855B2 (en) * | 2006-02-13 | 2012-07-10 | Agency For Science, Technology And Research | Method of processing a biological and/or chemical sample |
| CN101281196A (en) * | 2008-05-16 | 2008-10-08 | 北京工业大学 | HIV-1 p24 antigen acridine ester chemiluminescence immune analyse detecting method |
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| CN103869073A (en) * | 2014-04-01 | 2014-06-18 | 广州市丰华生物工程有限公司 | Double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody and HIV-1p24 antigen |
| CN104090101A (en) * | 2014-07-21 | 2014-10-08 | 威海威高生物科技有限公司 | Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof |
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