CN106771218B - It is a kind of detect HIV-1 p24 antigen kit and its application - Google Patents
It is a kind of detect HIV-1 p24 antigen kit and its application Download PDFInfo
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Abstract
The invention discloses a kind of kits for detecting HIV-1 p24 antigen, including ELISA Plate, sample diluting liquid, enzyme conjugates, negative control object, positive control, substrate solution, developing solution, terminate liquid and cleaning solution；The ELISA Plate is coated with by mouse anti-HIV-1 p24 monoclonal coated antibody, and the mouse anti-HIV-1 p24 monoclonal coated antibody is 2-C6 antibody；Contain biotin labeling mouse anti-HIV-1 p24 monoclonal antibody in the sample diluting liquid, the biotin labeling mouse anti-HIV-1 p24 monoclonal antibody is biotin labeling 11-A4 antibody.Furthermore, invention additionally discloses the applications of the kit, it is experimentally confirmed, using present invention offer kit is not only easy to operate, testing result is accurate, clear, and HIV-1 p24 antigen is detected using the kit, antigen sensitive can be made to have reached 1.25IU/mL, be significantly better than the detection sensitivity of existing common HIV-1 p24 detection kit.
Description
Technical field
The present invention relates to field of biotechnology, specifically a kind of kit for measuring HIV-1 p24 antigen and its answer
With.
Background technique
AIDS, that is, acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV) infection.
Inhibition of HIV body is in ball-type, and 100~120nm of diameter, viral volume surrounding is lipoprotein envelope, wherein the sugared egg special embedded with virus
White gp120 and gp41；Gp41 is transmembrane protein, and gp120 is located at surface, constitutes the furcella of envelope membrane surface, and is passed through with gp41 non-
Covalent effect combines.Viral internal is the 20 symmetrical nucleocapsids of face body, and virus core contains viral RNA, reverse transcriptase and nucleocapsid
Albumen.Viral genome is two identical positive chain RNAs, and every chain is about 9.2-9.8kb.The highest capsid protein of specificity
P24 is encoded by gag gene.P24 is the essential core polypeptide of HIV-1, is nucleocapsid constitutive protein, constitutes the core clothing of virus
Shell.Gag gene can encode the polymeric precursors albumen of about 500 amino acid composition, form p17, p24 core egg through protease hydrolytic
It is white.The measurement of p24 core antigen is widely used in clinical early diagnosis, treating AIDS effect due to there is good clinical meaning
The instruction detection etc. of viral growth in evaluation, HIV infection cell culture.
It is well known that HIV spread is rapid, and it is very harmful, it there is no cure method so far.Therefore people at highest risk is tracked and is examined
It surveys, early detection infection, timely medication, can delay to fall ill and can be effectively controlled AIDS virus propagation.ANTI-HIV DRUGS detection is to examining
Disconnected, treatment, control HIV spread, play an important role.But HIV infection early stage, antibody not yet occur before (also referred to as
" window phase "), p24 antigen has existed, and has strong infectiousness at this time.Therefore, the method that detection HIV antibody is applied alone is diagnosed
HIV missing inspection certainly will be will cause.Especially for the screening of biological products blood plasma and blood donor, this missing inspection undoubtedly be will cause
HIV is wider to send out.P24 albumen is the core antigen of HIV, has preferable stability, is the specific proteins of HIV, can
With the indication diagnosed as HIV, detection to it can shorten " the window before the appearance of HIV infection initial stage HIV antibody
Phase " reduces blood transfusion and the defeated risk of infection with blood product.
Currently, the detection of HIV-1 p24 antigen uses serological diagnostic method, mainly there is double antibody sandwich ELISA, exempts from
Epidemic disease compound cracks detection method, hypersensitization EIA method, enzyme linked immunological fluorescence method etc..But it is deposited in practical applications in these methods
The problem of be otherwise detection sensibility is poor, testing result false positive or false negative it is higher or with preferable sensibility and
Detection limit, but cumbersome complexity, can not promote and apply at all.
Summary of the invention
An object of the present invention is to provide a kind of kit for detecting HIV-1 p24 antigen, to solve current HIV-1
The detection of p24 antigen has that detection sensibility is poor, detection result accuracy is poor.
The second object of the present invention is to provide the application of the kit of detection HIV-1 p24 antigen, to solve existing detection
Not only accuracy is poor for technology, but also detection process very complicated, time-consuming and laborious problem.
The present invention is achieved in the following ways: a kind of kit detecting HIV-1 p24 antigen, including ELISA Plate,
Sample diluting liquid, enzyme conjugates, negative control object, positive control, substrate solution, developing solution, terminate liquid and cleaning solution；
The ELISA Plate is coated with by mouse anti-HIV-1 p24 monoclonal coated antibody, the mouse anti-HIV-1 p24 monoclonal packet
It is 2-C6 antibody by antibody, the 2-C6 antibody is that the hybridoma cell strain 2-C6 that deposit number is CGMCC No.12981 secretes
It obtains；The package amount of the mouse anti-HIV-1 p24 monoclonal coated antibody is 4 μ g/mL；
Contain biotin labeling mouse anti-HIV-1 p24 monoclonal antibody, the biotin labeling in the sample diluting liquid
Mouse anti-HIV-1 p24 monoclonal antibody is the 11-A4 antibody of biotin labeling；The 11-A4 antibody is that deposit number is CGMCC
What the hybridoma cell strain 11-A4 of No.12982 secreted；The biotin labeling mouse anti-HIV-1 p24 monoclonal antibody
Content is 2 μ g/mL；
The hybridoma cell strain 2-C6 and hybridoma cell strain 11-A4 is in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, preservation day are on September 2nd, 2016.
It is the slow of 0.025% horseradish peroxidase-labeled Streptavidin that the enzyme conjugates, which is containing volume by volume concentration,
Rush dilution；The negative control object is to detect the human serum that HIV-1 p24 antigen is feminine gender；
The positive control is the dilution of -1 p24 antigen positive reference substance of Recombinant HIV containing 200IU/mL；
The developing solution is the citrate buffer solution containing tetramethyl benzidine；
The substrate solution is the phosphate buffer containing hydrogen peroxide urea；
The terminate liquid is the H that concentration is 2mol/L2SO4Solution, and
The cleaning solution is 20 times of concentrated phosphoric acid salt buffers.
Every liter of substrate solution configures as follows in the present invention: by 35-37g Na2HPO4·12H2O, 10-11g lemon
Lemon acid, 0.5-0.6g hydrogen peroxide urea, 0.8-1.0g 2- chloroacetamide are dissolved in water for injection, with water for injection constant volume
To 1000mL.
Every liter of developing solution configures as follows in the present invention: by 0.17-0.19g EDTA-2Na, 1-2g lemon
Acid, 0.2-0.3g tetramethyl benzidine, 0.8-1.2g 2- chloroacetamide are dissolved in water for injection, are settled to water for injection
1000mL。
Heretofore described enzyme conjugates is that horseradish peroxidase-labeled Streptavidin is dissolved in buffer diluent
The solution of formation, every liter of buffer diluent configure as follows: by 12-13g Tris, 9-11g BSA, 1-1.2g enzyme
Stabilizer, 10-12mL Qula are logical, 1-1.5mL Proclin 300,0.2-0.3mL mass concentration ratio are that 4% fast green is dissolved in
In water for injection, 1000mL, Tris salt acid for adjusting pH value to 7.5 are settled to water for injection.
Heretofore described sample diluting liquid, which refers to, is dissolved in buffering for biotin labeling mouse anti-HIV-1 p24 monoclonal antibody
Solution is formed in liquid, every liter of buffer configures as follows: by the casein-sodium of Tris, 1-2g of 11-12.5g, 10-
The safron that Tween-20,8-12mL mass percent concentration of 15mL is 10% extracts element and the thimerosal of 1-1.5g is dissolved in
In water for injection, 1000mL, Tris salt acid for adjusting pH value to 7.5 are settled to water for injection.
The phosphate concentrated cleaning solution that the cleaning solution in the present invention is 20 times.
The application of kit as described above in preparation detection HIV-1 p24 antigenic agents in the present invention.
Application of the kit of the present invention in qualitative or quantitative detection or diagnosis HIV-1 p24 antigen.
The design principle of kit provided by the invention are as follows: it is measured using biotin-avidin system double antibody sandwich method,
I.e. by the monoclonal antibody solid phase of anti-HIV-1 p24 in the sample dilution that the antibody containing biotin labeling on microwell plate, is added
Liquid and sample to be tested；After incubation, resist if containing in HIV-1 p24 antigen, the antigen and liquid phase and solid phase ELISA Plate in sample
Body combines the compound for forming Ab-Ag- (AbB) sandwich sample, and is fixed on the surface of ELISA Plate；Wash away it is unbonded at
Point, the enzyme conjugates of the Streptavidin containing horseradish peroxidase-labeled is added, utilizes the strong knot of biotin and avidin
With joint efforts, horseradish peroxidase solid phase is developed the color in the substrate solution and developing solution on ELISA Plate, adding enzyme, after terminating reaction,
OD value is measured with microplate reader, determines the positive and negative of reaction；And it can determine the content of HIV-1 p24 antigen in sample.
The innovation of the invention consists in that being come by using the p24 antigen fragment from HIV-1B hypotype and HIV-1E hypotype
Immune mouse, and number and time interval by improving immune mouse, it is anti-thus just to have prepared high-titer advantage monoclonal
Body, and by screening, finishing screen selects 2 plants of good monoclonal antibodies of conformability, and not only sensibility with higher, can make
The antibody that sensibility, the accuracy of kit are improved significantly, and are screened can be with multiple types inhibition of HIV lysate
Reaction, can more fully detect different types of inhibition of HIV；In addition, the specific ELISA Plate coating of the present invention and preparation condition,
The coated antibody on ELISA Plate is set to keep good immunologic competence；Optimal screening in sample diluting liquid and enzyme conjugates reagent
The Tris-Hcl of special ratios is added in the buffer diluent of enzyme conjugates as buffer, by enzyme stabilizers, substantially increases
The stability of enzyme, while also substantially prolonging the validity period of kit.As it can be seen that not only being operated using kit provided by the invention
Simply, and testing result is accurate, clear.It proves through a large number of experiments, detects HIV-1 p24 antigen using the kit,
Antigen sensitive can be made to have reached 1.25IU/mL, be significantly better than the detection sensitivity of available reagent box.
Kit provided by the invention can save backup in the environment of 4 DEG C of refrigerator.
Kit provided by the invention and detection method can not only detect HIV-p24 antigen, can also be to the HIV positive
The early stage auxiliary antidiastole and the reaction of the 4th generation HIV-1 antigen/antibody ELISA reagent tests positive of Mothers baby
When, anti-HIV-1 confirms the auxiliary diagnosis of negative patient；And evaluation and the sieve of external inverase of inverase therapeutic effect
Choosing etc., it is easy to operate, easy to use, with important practical application value.
Detailed description of the invention
Fig. 1 is HIV-1 p24 antigen measuring schematic diagram of the present invention.
Fig. 2 is the standard curve of HIV-1 p24 antigen quantitative detection of the present invention.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material used in embodiment, reagent etc., unless otherwise specified,
Commercially obtain.Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Embodiment 1 is immunized mouse and obtains monoclonal coated antibody and monoclonal antibody
(1) mouse is immunized
6-8 week old female Balb/c mouse is selected, with the p24 antigen fragment from HIV-1BC hypotype or HIV-1AE hypotype
Immune Balb/c mouse, the number that mouse is immunized be 5 times (traditional immunization method is 3 times), and multiple spot is subcutaneously, muscle, abdominal cavity exempt from
Epidemic disease, only, time interval gradually increases 50ug/, from being spaced 15 days to being spaced 40 days, finally attacks 2 times (traditional attack is primary),
Antigen is 30ug/, 2 days after last 1 attack mouse, takes mice spleen for cell fusion, prepares monoclonal antibody hybridoma
Cell strain.
(2) preparation of immune spleen cell and myeloma cell
It learns from else's experience immune Balb/c mouse, extracts eyeball bloodletting.Mouse is put to death, sterile to win spleen, spleen leaching is produced in grinding
Bar cell suspension, washing adjustment cell concentration is 1~5 × 107/ mL is spare.
The myeloma cell Sp2/0 through multiple proportions passage is collected, is counted after washed spare.
(3) cell fusion
Myeloma cell is mixed with mouse boosting cell, by immune spleen cell and ready homology myeloma cell Sp2/
0 presses (2~10): 1 is mixed in 50mL centrifuge tube, and 1000rpm is centrifuged 10 minutes, abandons supernatant, taps tube wall, makes the cell of tube bottom
Precipitating mixes such as paste, is slowly added to 50%PEG4000 about 1mL, and side edged shakes, and adds, is remake with 1.5 minutes in 1 minute
Afterwards, it is slowly dropped into serum-free medium about 10mL in 5 minutes, the effect of PEG is terminated, needed for being added after washed removal fusion agent
Amount be containing mass concentration ratio 20% fetal calf serum 1640 cell culture fluid of HAT, point kind in 96 well culture plate holes, 100 μ
The hole L/.37 DEG C, 5%CO2Culture in incubator.
(4) the selection culture of hybridoma
The selection culture solution of HAT 1640 containing the fetal calf serum that mass concentration ratio is 20%, HAT selection culture 1~2 week, not
Though the lymphocyte of fusion has hypoxanthine-guanine-phosphoribosyltransferase, itself cannot long-term surviving in vitro
Also gradually dead；Only the hybridoma of myeloma cell and bone-marrow-derived lymphocyte can be proliferated, and have myeloma cell can be unlimited
The characteristic of proliferation can survive and be proliferated in HAT culture medium, i.e., hybridoma is selected to turn out.
(5) screening of hybridoma
HAT 1640 selects culture solution to select the myeloma cell-bone-marrow-derived lymphocyte fused cell turned out, not all
This kind of fused cell all secrete expected antibody, need to carry out the screening of specific antibody.1 μ g/mL p24 antigen coated enzyme
Target, 50 holes μ L/, 4 DEG C overnight.1 × PBST washing lotion, board-washing 3 times, 100 holes μ L/, 3% skimmed milk power is closed, and 37 DEG C of incubations 1 are small
When.Board-washing 1 time, cells and supernatant to be checked is added, 50 holes μ L/, 37 DEG C are incubated for 1 hour in 1 × PBST washing lotion.1 × PBST is washed
Board-washing 4 times, HRP- sheep anti-mouse igg is added, 50 holes μ L/, 37 DEG C are incubated for 30 minutes in liquid.1 × PBST washing lotion board-washing 4 times, is added
Substrate solution: developing solution=1:1,50 holes μ L/, 37 DEG C are incubated for 30 minutes.2mol/L sulfuric acid terminates reaction.Microplate reader OD450nm is read
Take OD value, reference wavelength 630nm.Negative control uses 1640 culture medium, cutoff value=negative control mean value × 2.1.It will sieve
Choosing has the cell hole cloning as early as possible of antibody activity, freezes conservation as early as possible.
(6) cloning of hybridoma
Using limiting dilution assay, clone prepares feeder cells the previous day, collects mouse peritoneal emigrated cell using asepsis,
It counts, adjustment feeder cells concentration is 1 × 106/ mL is implanted into 100 μ L/ hole cells in every hole in 96 porocyte culture plates and hangs
Liquid, 37 DEG C, 5%CO2In incubator overnight.2nd day, cell to be cloned gently is blown down out of cultivation plate hole, calculates every milliliter
Viable count.Serial dilution cell suspension is at 10 cell/mL.Cell is added to the culture plate of existing feeder cells with dropper
It is interior, average every 100 μ L of Kong Jiayue.Start to change culture solution within 7th day, when cell covers with the 1/10 of bottom hole, starts to detect supernatant
In antibody activity.Cell taking-up in the monoclonal cell hole for having antibody activity measured in supernatant is placed in 24 well culture plates
Or continue to expand culture in 5mL culture bottle, the clone of gained is subjected to freezing conservation as early as possible.
(7) a large amount of preparations of monoclonal antibody
It induces method in vivo: taking Balb/c mouse, intraperitoneal injection 0.5mL white oil or norphytane first is pre-processed.
After 1-2 weeks, intraperitoneal inoculation hybridoma.Hybridoma is proliferated in mouse peritoneal, and is generated and secreted monoclonal and resist
Body.About 1-2 weeks, it is seen that mouse web portion expands.Ascites is extracted with syringe, can be obtained a large amount of monoclonal antibodies.
10 high cell strains of antibody activity: 2-C6 plants, 2-B5 plants, 11-A4 plants, 10-A9 plants, 5-D4 plants, 11- will be generated
C2 plants, 12-A1 plants, 9-F4 plants, 2-G6 plants and 2-E3 plants；Expand respectively and cultivates, 1 × 106/ mL injects Balb/c mouse peritoneal,
0.5mL/ is only.Aseptic aspiration ascites after 7~10 days.As measured antibody activity in (5).Above-mentioned 10 plants of monoclonal antibodies is pure using salting out method
Change, obtains 2-C6 antibody, 2-B5 antibody, 11-A4 antibody, 10-A9 antibody, 5-D4 antibody, 11-C2 antibody, 12-A1 antibody, 9-F4
Antibody, 2-G6 antibody and 2-E3 antibody.
(8) calibrating of monoclonal antibody
Above-mentioned 10 plants of monoclonal antibodies are purified using salting out method, monoclonal antibody marks HRP with improvement sodium periodate oxidizing process after purification, will
One monoclonal antibody carries out pairing screening as HRP detection antibody as coated antibody, another monoclonal antibody, the method is as follows: the 4 anti-p24 of μ g/mL are anti-
1 coated elisa plate of body, 50 holes μ L/, 4 DEG C overnight.1 × PBST washing lotion, board-washing 3 times, 100 holes μ L/, the closing of 3% skimmed milk power, 37
DEG C be incubated for 1 hour.Board-washing 1 time, p24 antigen 1 00ng/mL is added, 50 holes μ L/, 37 DEG C are incubated for 1 hour in 1 × PBST washing lotion.1×
Board-washing 4 times, anti-2,50 hole μ L/ of p24 antibody HRP- is added in PBST washing lotion, and 37 DEG C are incubated for 30 minutes.1 × PBST washing lotion, board-washing 4
It is secondary, substrate solution: developing solution=1:1 is added, 50 holes μ L/, 37 DEG C are incubated for 30 minutes.2mol/L sulfuric acid terminates reaction.Microplate reader
OD450Nm reads OD value, reference wavelength 630nm.Negative control use 1640 culture medium, cutoff value=negative control mean value ×
2.1.2 plants of good monoclonal antibodies of conformability are filtered out from above-mentioned ten strain antibody, wherein 1 plant is used to be coated with, 1 plant for marking
Measurement.
By screening above, measurement result as shown in Table 1 is obtained.
1 10 strain antibody of table matches screening test result two-by-two
Remarks: Ncx=0.065；OD is matched in the expression of overstriking data two-by-two in table450Value >=1.0
The preferable 2-C6 antibody of conformability can be selected as mouse anti-HIV-1 p24 monoclonal packet from the measurement result of table 1
It is used to be used as biotin labeling mouse anti-HIV-1 p24 monoclonal antibody by antibody, 11-A4 antibody, and 2-C6 antibody will be secreted
Hybridoma cell strain 2-C6 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is
CGMCC No.12981, preservation day are on September 2nd, 2016；The hybridoma cell strain 11-A4 for secreting 11-A4 antibody is preserved in
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are CGMCC No.12982, and preservation day is
On September 2nd, 2016.
(9) it will screen as mouse anti-HIV-1 p24 monoclonal coated antibody 2-C6 solid phase on ELISA Plate, 11-A4
Antibody is used to react with multiple types inhibition of HIV lysate as the monoclonal antibody of biotin labeling: HIV A/E is sub-
Type, subtype B, C hypotype, G hypotype.For concrete operation step referring to embodiment 3, testing result is as described in Table 2.
Antibody determination HIV hypotype prepared by table 2
The preparation of 2 kit of embodiment
1, the preparation of ELISA Plate
(1) each solution is configured:
Coating buffer: the Na of 1.59g is taken2CO3With the NaHCO of 2.93g3, after the dissolution of a small amount of water for injection, then use injection
Water is settled to 1000mL, pH value 9.6；
Cleaning solution (20 times of concentrated phosphoric acid salt buffers): 58.0g Na is weighed2HPO4·12H2O、5.93g NaH2PO4·
2H2O, the 2- chloroacetamide of 148g NaCl, 10mL Tween-20 and 1g, after being dissolved after mixing with water for injection, is settled to
1000mL, as 20 times of concentrated phosphoric acid salt buffers；PH value is 7.4；
Confining liquid: weighing the Tris of 12.11g, adds HCl tune pH value to 7.2,10g BSA, 20g sucrose, 20g casein-sodium
With 0.1g Sodium azide, after being dissolved after mixing with water for injection, it is settled to 1000mL.
(2) using the microwell plate in 96 holes as bottom plate, by mouse anti-HIV-1 p24 monoclonal coated antibody (2-C6 antibody) coating
It is 4 μ g/mL that liquid (0.05mol/L, CB, pH value 9.6), which is diluted to concentration, adds the 2 mercapto ethanol that mass concentration ratio is 1 ‰, packet
By microwell plate, sample-adding amount is 125 holes μ L/, and at 4 DEG C, coating is for 24 hours；Cleaning solution (pH value 7.4) 100mL is taken to be added later
1900mL distilled water is made into 1 times of cleaning solution (pH value 7.4), and the microwell plate after coating is washed with 1 × cleaning solution (pH value 7.4)
3 times；Confining liquid is added in micropore after washing, 150 holes μ L/ are placed at 4 DEG C, and closing is for 24 hours；The confining liquid in hole is discarded, is clapped
It is dry；Coating microwell plate after patting dry is put into the drying machine that drying temperature is 20 DEG C and drains 6 hours, is resisted to obtain the final product by mouse after dry
The coated ELISA Plate of HIV-1 p24 monoclonal coated antibody, the ELISA Plate vacuum seal after coating, sets 4 DEG C of preservations, spare.
2, the preparation of sample diluting liquid
(1) biotin labeling mouse anti-HIV-1 p24 monoclonal antibody is prepared: using biotin N-hydroxy-succinamide ester
(BNHS) labelling method prepares biotin labeling mouse anti-HIV-1 p24 monoclonal antibody, prepares 10mg/mL biology with anhydrous DMSO
Plain N-hydroxy-succinamide ester solution；With carbonate buffer solution (0.1mol/L, pH value 9.0) respectively by the anti-p24 monoclonal of mouse
Antibody (11-A4 antibody) is diluted to the antibody-solutions of 2mg/mL；The anti-p24 monoclonal antibody of mouse (11-A4 antibody) is pressed with BNHS
Volume ratio is 8:1 mixing, and lower reaction 4h is stirred at room temperature；Be fitted into bag filter, to 0.05mol/L, pH value be 7.2 PBS at 4 DEG C
Lower dialysed overnight obtains biotin labeling mouse anti-HIV-1 p24 monoclonal antibody (biotin labeling 11-A4 antibody；Bio-11-
A4)；
(2) buffer used in sample diluting liquid (pH 7.4) is prepared: the Tris of 1.211g is taken to add HCL tune pH value to 7.4,
The thimerosal of the casein-sodium of 0.1g, Tween-20,1mL safron T (mass percent concentration 10%) and 0.1g of 1mL,
After being first completely dissolved Tris with appropriate water for injection, concentrated hydrochloric acid tune pH value is added to 7.4, adds Tween-20 and other is molten
Agent finally injects water to 100 milliliters；
(3) Bio-11-A4 (1:1000 dilution) of 200 μ L is added in the buffer 100mL prepared in step (2), obtains
Sample diluting liquid.
3, the preparation of enzyme conjugates
(1) horseradish peroxidase-labeled Streptavidin: horseradish peroxidase is prepared with sodium periodate-glycol method
Labelled streptavidin.Specifically: it weighs 1mg horseradish peroxidase (HRP) and is dissolved in 0.1mL distilled water, under 4 DEG C of stirrings
The 0.06mol/L NaIO that 0.1mL newly matches is added4Solution is protected from light stirring 30 minutes at room temperature；0.16mol/L ethylene glycol is added
0.1mL, room temperature, which is protected from light, to be gently mixed 30 minutes；It is added 8ul 0.2mol/L carbonate buffer solution (pH value 9.5), makes the above aldehyde
The pH value for changing HRP is increased to 9.0-9.5, and 1mg Streptavidin is added immediately after, after mixing, is fitted into bag filter, right
0.05mol/L carbonate buffer solution is dialysed 4 hours, and the 5mg/mL NaBH that 40 μ L newly match is added4Liquid mixes, quiet at 4 DEG C
It sets 2 hours, above-mentioned liquid is fitted into bag filter, dialyse to 0.01mol/L, the PBS that pH value is 7.4,4 DEG C overnight；Up to horseradish
Peroxidase labelling Streptavidin (HRP-SAV)；
(2) it prepares buffer diluent used in enzyme conjugates: 12.11g Tris is taken, with hydrochloric acid tune pH value to 7.5,10g
BSA；1g enzyme stabilizers, 10mL Qula be logical, 1mL Proclin 300,0.2mL fast green (mass concentration ratio 4%)；It is water-soluble with injecting
Filling jetting is settled to 1000mL after solving above-mentioned solvent；
(3) 0.25mL HRP-SAV (1:4000 dilution) is added in 1000mL buffer diluent, after aseptic filtration, obtains enzyme
Conjugate.
4, the preparation of negative control object
Negative control is the serum of normal person, carries out detection qualification by the screening requirement of blood source, detection HIV-1 p24 antigen is
Feminine gender, 5 parts of mixing or more detect qualified normal human serum, and preservative is added, dispenses after aseptic filtration, 4 DEG C of preservations.
5, the preparation of positive control
1. configuring dilution: taking 2.9g disodium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, 4g junket
Albumen sodium, 10g BSA, 1mL Proclin-300 and 1.25mL gentamicin sulphate, after being dissolved with water for injection and are settled to
1000mL；
2. taking -1 p24 antigen positive reference substance of Recombinant HIV (CHINA virus prevention and control center virosis prevention and control institute
Professor Zeng Yi give) it is added in dilution and is diluted to 200IU/mL, after aseptic filtration, 4 DEG C are saved backup.
6, the preparation of developing solution
Weigh the 2- chloracetyl of 0.186g EDTA-2Na, 1.05g citric acid, 250mg tetramethyl benzidine (TMB) and 1g
EDTA-2Na, citric acid and a small amount of water for injection of 2- chloroacetamide are first dissolved, then TMB are first used to 2mL DMSO (diformazan by amine
Base sulfoxide) after dissolution, then it is added dropwise in solution, is finally settled to 1000mL, pH value 3.0 with water for injection.
7, the preparation of substrate solution
Take 36.82g Na2HPO4·12H2O, the 2- chloroacetamide of 10.21g citric acid, 0.6g hydrogen peroxide urea and 1g,
1000mL is settled to after being dissolved with injection water, the pH value of solution is 5.0.
8, the preparation of terminate liquid
The concentrated sulfuric acid of the 10mL water for injection of 80mL is diluted.
9, be equipped with a kit according to following combination: the sample of ELISA Plate portion, 3.5mL including 96 holes dilutes
Liquid, 7mL/ bottles of negative control object, 1mL/ bottles of positive control, 13mL/ bottles of enzyme conjugates, 50mL/ bottles of cleaning solution, 7mL/
Developing solution, 7mL/ bottles of the substrate solution, 6mL/ bottles of terminate liquid of bottle；The kit is placed in 4 DEG C of refrigerator preservations.
The qualitative and quantitative detecting method of 3 HIV-1 p24 antigen of embodiment
(1) kit prepared by embodiment 2 is placed on room temperature 30 minutes from refrigerator taking-up；
(2) it dilutes cleaning solution: 50mL cleaning solution being diluted to 1000mL with distilled water, obtains 1 times of cleaning solution；
(3) qualitative determination:
A, take ELISA Plate, sample to be tested corresponding aperture sequentially numbered, 1 hole of blank, every plate should set 3 hole of negative control (NC),
Two hole (10IU/mL) of positive control (PC).If measurement sample is cell culture supernatant, need to do feminine gender with cell culture fluid
Compare 3 holes；
B, prior to 25 μ L sample dilutions are added in each reacting hole, sample diluting liquid is not added in blank well；
C, according to number, by negative control object, the positive control (10IU/ after 100 μ L samples to be tested, 100 μ L dilution
ML it) is added separately in the reacting hole in step B added with sample diluting liquid, vibrates 30-60 seconds and mix, set 37 DEG C and be incubated for 1 hour；
Blank well is not added；
D, with cleaning solution board-washing 4-5 times after dilution, patting dry rear every hole addition 125 hole μ L/ of enzyme conjugates, (blank well is not
Add), it sets 37 DEG C and is incubated for 30 minutes
E, it with shown cleaning solution board-washing 4-5 times, pats dry rear every hole and substrate solution, colour developing liquid mixture is successively added (before use
Prepare) each hole 62.5 μ L/, first plus substrate solution or first plus developing solution；It mixes within oscillation 30-60 seconds, set 37 DEG C of incubations 30 minutes；
F, 50 hole μ L/ of terminate liquid is added；
G, within 5min, each hole absorbance is read with dual wavelength microplate reader (measurement wavelength 450nm, reference wavelength 630nm)
Value；
H, the light absorption value according to measured by negative control value, calculating critical value=negative control OD value average value+
0.095；It is feminine gender as the OD value of test sample is less than critical value；It is sun as the OD value of test sample is greater than or equal to critical value
Property.
I, its testing conditions are as follows: three hole negative controls are at least arranged in measurement every time, and two hole positive controls carry out quality control,
To ensure the reliability of each measurement result；And test need to meet following five conditions simultaneously every time, otherwise measurement experiment result
In vain, need total Test that need to redeterminate.
A, reagent blank OD value answers < 0.050 (before blank school zero).
B, the normal range (NR) of negative control: under normal circumstances, negative control OD value answers≤0.100.
C, under normal circumstances, positive control is proposed with p24 (10IU/mL) when qualitative determination.
D, the measurement result of repeated sample differs within 15%.
E, the linearly dependent coefficient R value of the standard curve of drafting it is necessary >=0.95.
With kit as described in example 2, the OD value of three negative controls is measured are as follows: NC1=0.025 NC2=0.028
NC3=0.031 NCx=0.028, then its critical value=0.028+0.095=0.123, if using measured by the kit
When the OD value of sample is greater than or equal to 0.123, can preliminary judgement sample HIV-1 p24 antigen be the positive, measured OD value is small
When 0.123, can preliminary judgement sample HIV-1 p24 antigen be the positive.The accuracy for improving detection can be repeated several times.
If qualitative detection sample is positive sample, if repetition measurement is still the positive, HIV-1 should need to be used with duplicate hole repetition measurement
P24 confirmatory reagent confirmation.The detection principle diagram of its HIV-1 p24 antigen is shown in Fig. 1.
(4) it quantitative determines:
A, ELISA Plate is taken, sample corresponding aperture is sequentially numbered, 1 hole of blank, every plate should set 3 hole of negative control, be serially diluted
Positive control 20IU/mL, 10IU/mL, 5IU/mL, 2.5IU/mL, 1.25IU/mL, each diluted concentration do two multiple holes；If
Measurement sample is cell culture supernatant, then needs to do 3 hole of negative control with cell culture fluid.
B, prior to 25 μ L sample dilutions are added in each reacting hole, sample diluting liquid is not added in blank well；
C, dilute positive control: Recombinant HIV -1p24 antigen of the positive control (PC) containing 200IU/mL uses negative control
It after object first dilutes positive control into 20IU/mL, then prepares and is serially diluted 4 concentration, dilution gradient and method are shown in Table 3, if fixed
Measuring fixed sample is cell culture supernatant, then needs to be carried out being serially diluted antigen with the cell culture supernatant being uninfected by, often
A diluted concentration does two multiple holes.
3 HIV-1p24 of table is serially diluted method
D, according to number, by 100 μ L samples to be tested (blank well is not added), negative control object and more than dilute after series sun
Property reference material be added on ELISA Plate have plus the reacting hole of sample diluting liquid in, vibrate 30-60 second and mix, it is 1 small to set 37 DEG C of incubations
When；
E, it pats dry rear every hole and 125 hole μ L/ (blank well is not added) of enzyme conjugates is added, set 37 DEG C and be incubated for 30 minutes；
F, with cleaning solution board-washing 4-5 times after dilution, pat dry that substrate solution is successively added in rear every hole, colour developing liquid mixture (faces
With preceding preparation) each hole 62.5 μ L/, first plus substrate solution or first plus developing solution；It mixes within oscillation 30-60 seconds, set 37 DEG C of incubations 30
Minute；
G, 50 hole μ L/ of terminate liquid is added；
H, within 5min, each hole absorbance is read with dual wavelength microplate reader (measurement wavelength 450nm, reference wavelength 630nm)
Value；The light absorption value of the HIV-1 p24 dilution series sample of its various concentration is shown in Table 4.
The HIV-1 p24 of 4 various concentration of table is serially diluted
I, by the positive antigen control being serially diluted shown in table 3 as a result, being cross with positive control dilution series concentration
Coordinate maps by ordinate of the average value of the OD value detected corresponding to abscissa concentration, it is as shown in Figure 2 to obtain standard curve；
When test sample, to cross from the scale of positive sample mean OD value on the y axis perpendicular to y-axis to standard curve, then oneself and mark
The crosspoint of directrix curve is crossed perpendicular to x-axis, reads HIV-1 p24 antigen concentration, the as HIV-1 in positive sample in x-axis
The antigenic content of p24；
J, its detection while the same qualitative detection of condition met.
Multiplicating can be used in the above qualitative and quantitative detection, to improve the accuracy of its detection.
Application of the kit of embodiment 4 in test sample
The HIV-1 p24 detection kit and detection method described in embodiment 3 prepared using embodiment 2 measures HIV-
1 p24 antigen National reference, the specific steps are as follows:
(1) 25 μ L sample dilutions will be added in each ELISA Plate reacting hole (containing Bio-11-A4)；
(2) by 100 μ L negative controls, sample to be tested (diluted various concentration HIV-1 p24, HIV-1 p24 antigen country
Sample in reference material) it is added in the reacting hole of sample diluting liquid, it vibrates 30-60 seconds and mixes, set 37 DEG C and be incubated for 1 hour；
(3) board-washing 4 times pat dry rear every hole and 125 hole μ L/ (blank well is not added) of enzyme conjugates are added, set 37 DEG C and be incubated for 30 points
Clock；
(4) board-washing 4 times, pat dry rear every hole be added substrate solution and colour developing liquid mixture (substrate solution: developing solution volume ratio is 1:
1, prepared before use is added within 10 minutes) totally 125 hole μ L/, it sets 37 DEG C and is incubated for 30 minutes；
(5) 50 hole μ L/ of terminate liquid is added.
Within (6) 5 minutes, each hole extinction is read with dual wavelength microplate reader (measurement wavelength 450nm, reference wavelength 630nm)
Angle value.
After adopting the above technical scheme, 20 HIV-1 p24 antigen negatives in measurement HIV-1 p24 antigen National reference
Reference material is all detected as negative (20/20)；10 HIV-1 p24 antigen positive reference materials, whole test positive (10/
10)；10 branch linear and sensitive reference material limits of identification are not higher than 1.25IU/mL；L10 (matrix serum) is negative reaction.L1-
L5 is after totally 5 sample measurements statistical analysis, linearly dependent coefficient (R value) >=0.95；Precision reference material is measured in parallel (10
Hole) after A value, statistical analysis, precision (CV value)≤15%.To the quantitative accurate of p24 antigen, good linearity.Specific detection data
As shown in table 5.
The kit measurement HIV-1 p24 antigen National reference result prepared by the present invention of table 5
Claims (5)
1. a kind of kit for detecting HIV-1 p24 antigen, which is characterized in that combined including ELISA Plate, sample diluting liquid, enzyme
Object, negative control object, positive control, substrate solution, developing solution, terminate liquid and cleaning solution；
The ELISA Plate is coated with by mouse anti-HIV-1 p24 monoclonal coated antibody, and the mouse anti-HIV-1 p24 monoclonal coating is anti-
Body is 2-C6 antibody, and the 2-C6 antibody is that the hybridoma cell strain 2-C6 that deposit number is CGMCC No.12981 secretes
It arrives；The package amount of the mouse anti-HIV-1 p24 monoclonal coated antibody is 4 μ g/mL；
Contain biotin labeling mouse anti-HIV-1 p24 monoclonal antibody in the sample diluting liquid, the biotin labeling mouse is anti-
HIV-1 p24 monoclonal antibody is the 11-A4 antibody of biotin labeling；The 11-A4 antibody is that deposit number is CGMCC
What the hybridoma cell strain 11-A4 of No.12982 secreted；The biotin labeling mouse anti-HIV-1 p24 monoclonal antibody
Content is 2 μ g/mL；
The hybridoma cell strain is that mouse, sieve are immunized by using the antigen fragment from HIV-1B hypotype or HIV-1E hypotype
Choosing preparation；
The sample diluting liquid refer to biotin labeling mouse anti-HIV-1 p24 monoclonal antibody is dissolved in formed in buffer it is molten
Liquid, every liter of buffer configure as follows: by the Tris of 11-12.5g, the casein-sodium of 1-2 g, 10-15 mL
Tween-20, the safron that 8-12 mL mass percent concentration is 10% extract element and the thimerosal of 1-1.5 g is dissolved in injection
With in water, 1000 mL, Tris salt acid for adjusting pH value to 7.5 are settled to water for injection；
The enzyme conjugates is the buffer diluent containing horseradish peroxidase-labeled Streptavidin；The enzyme conjugates is
Horseradish peroxidase-labeled Streptavidin is dissolved in the solution formed in buffer diluent；The horseradish peroxidase mark
Remember that volume percent content of the Streptavidin in enzyme conjugates is 0.025%；Every liter of buffer diluent is as follows
Configuration: 12-13 g Tris, 9-11 g BSA, 1-1.2 g enzyme stabilizers, 10-12 mL Qula are led to, 1-1.5mL Proclin
300,0.2-0.3 mL mass concentration ratio be 4% it is fast green be dissolved in water for injection, be settled to 1000 mL, institute with water for injection
State Tris salt acid for adjusting pH value to 7.5；
The negative control object is to detect the human serum that HIV-1 p24 antigen is feminine gender；
The positive control is the dilution containing -1 p24 antigen positive reference substance of Recombinant HIV；
The developing solution is the citrate buffer solution containing tetramethyl benzidine；
The substrate solution is the phosphate buffer containing hydrogen peroxide urea；
The terminate liquid is sulfuric acid solution, and
The cleaning solution is phosphate buffer.
2. the kit of detection HIV-1 p24 antigen according to claim 1, which is characterized in that every liter of substrate solution
It configures as follows: by 35-37 g Na2HPO4•12H2O, 10-11 g citric acid, 0.5-0.6 g hydrogen peroxide urea,
0.8-1.0 g 2- chloroacetamide is dissolved in water for injection, is settled to 1000 mL with water for injection.
3. the kit of detection HIV-1 p24 antigen according to claim 1, which is characterized in that every liter of developing solution
It configures as follows: by 0.17-0.19 g EDTA-2Na, 1-2 g citric acid, 0.2-0.3 g tetramethyl benzidine, 0.8-
1.2 g 2- chloroacetamides are dissolved in water for injection, are settled to 1000 mL with water for injection.
4. the kit of detection HIV-1 p24 antigen according to claim 1,2 or 3, which is characterized in that the positive is right
It is 200 IU/mL according to -1 p24 antigen positive reference substance content of Recombinant HIV in object；The concentration of the sulfuric acid solution is 2mol/L.
5. a kind of application of the kit described in claim 1 in preparation detection HIV-1 p24 antigenic agents.
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| CN112285346B (en) * | 2020-07-25 | 2024-02-13 | 四川大学华西医院 | High-sensitivity HIV p24 detection method based on quantum dot selective cation exchange reaction |
| CN113281503B (en) * | 2020-10-29 | 2022-01-04 | 杭州微策生物技术股份有限公司 | Reagent for splitting virus sample |
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