CN101949932A - IgA (Immunoglobulin A) antibody detection reagent kit (colloidal gold method) for EB (Epstein-Barr) viruses and preparation method thereof - Google Patents
IgA (Immunoglobulin A) antibody detection reagent kit (colloidal gold method) for EB (Epstein-Barr) viruses and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an IgA (Immunoglobulin A) antibody detection reagent kit (a colloidal gold method) for EB (Epstein-Barr) viruses and a preparation method thereof. The reagent kit comprises recombination antigen EB-NA1 coated by a nitrocellulose membrane detection line, a goat-anti-mouse IgG antibody coated on a quality control line and a mouse-anti-human IGA monoclonal antibody marked by colloidal gold and coated on a gold mark pad. The preparation method comprises the steps of: preparing a reaction membrane and a mouse-anti-human IGA monoclonal antibody gold combo pad, cutting and assembling to prepare the product. The invention has the advantages that: the IgA antibody detection reagent kit for the EB viruses has the characteristics of fast, simple and convenient detection, and high accuracy and sensitivity; the integrated operation time only requires 20 minutes to judge and read results; the colloidal gold is used for fast detecting test paper; a multi-epitope recombination antigen is used as a raw material; the method has the characteristics of simple and convenient operation, low cost, good specificity, high sensitivity, single portion detection and easy popularization; and the detection and control effect to the EB viruses is obvious.
Description
Technical field
The present invention relates to a kind of antibody assay kit, specifically a kind of kit and preparation method who uses colloidal gold method detection Epstein-Barr virus IgA antibody.
Background technology
Epstein-Barr virus (Epstein-Barr virus, EBV) be unique Lymphocryptovirus that can cause the human infection in the herpetoviridae γ subfamily, Epstein in 1964 and Barr etc. at first find from the cellular incubation of burkitt lymphoma, so the called after Epstein-Barr virus is the human tumor virus that is identified the earliest.
The EB antibody that adopts enzyme linked immunological absorption (ELISA) detection method to detect in the blood serum is the most commonly used, existing commercial kit, for example detect the antibody titer of IgA2VCA antibody, the early stage complex antigen of Epstein-Barr virus (EBV 2EA) IgG in the nasopharyngeal carcinoma serum with ELISA method and indirect immunoperoxidase dyeing (IEA) method, under the prerequisite of susceptibility identical (88.3%), ELISA method specificity (98.3%) obviously is better than IEA method (53.2%).Be that the susceptibility that detects nasopharyngeal carcinoma serum with the ELISA method under 97.7% the prerequisite also is significantly higher than the susceptibility of measuring EBV 2EA IGA antibody titer (IGA 2EA IEA) with the IEA method in specificity.But operations such as ELISA method complicated operation, detection time are long, need dilute testing sample, incubation, separation, washing and colour developing probably need with more than two hours.
Summary of the invention
In order to address the above problem, the invention provides a kind of colloidal gold method of using and detect kit of Epstein-Barr virus IgA antibody and preparation method thereof, the Epstein-Barr virus NA1 IgA antibody that is used for qualitative detection serum (slurry) sample adopts this method to detect, only just can sentence read result with 20min.Colloidal gold fast detecting test paper strip is a raw material with the recombinant antigen of multi-epitope, and it is easier, with low cost to have an operation, and specificity is good, and highly sensitive characteristics can single part of detection, is easy to popularize, and plays very great function for the detection and the control of Epstein-Barr virus.
Technical scheme of the present invention is:
One, raw material:
The employed detection line coating antigen of this kit material is: reorganization EB NA1 antigen.
The employed colloid gold label raw material of this kit is: mouse-anti people IgA monoclonal antibody.
The used nature controlling line raw material of this kit is: sheep anti-mouse igg antibody.
Two, ingredient requirement:
1, recombined EB virus antigen
(1) outward appearance: colourless transparent liquid;
(2) concentration and purity requirement: concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount is only deposited a band under 10 μ l conditions;
2, sheep anti-mouse igg antibody
(1) outward appearance: colourless transparent liquid;
(2) concentration requirement: concentration is greater than 4mg/ml;
3, mouse-anti people IgA monoclonal antibody
(1) outward appearance: colourless transparent liquid;
(2) concentration and purity requirement: concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount is deposited two bands under 10 μ l conditions;
Three, manufacturing condition determines
1, the reaction film bag is by the optimization of condition:
1.1 the detection line bag is by the selection of concentration and colloid gold label mouse-anti people IgA dilution ratio:
Carry out mark according to above-mentioned definite good flag condition, its volume is concentrated into 1/10 of original volume, original volume is with the collaurum volume calculation, again with different dilution ratio dilution concentrates, soak gold mark pad with dilution, the 1.5ml/ bar, dry back with different detection line bags by concentration, 0.1 the package amount bag of μ l/mm is matched by good EB reaction film, detects internal control serum.Experimental program such as table 1, experimental result such as table 2,3:
The design of table 1 experimental program
Positive internal control product of table 2 and minimum detectability
The negative internal control product of table 3
By above experiment as can be known, two groups of experimental results of A3B3 and A3B4 are more satisfactory.
The concentrate dilution ratio is 1: 5, and the detection line bag is 1.0mg/ml during to 1.2mg/ml by concentration, and EB-IgA reaction film detection line color developing effect is better, and sensitivity, and specificity is better.Select the A3B3 combinations of pairs at this.That is:
Collaurum bond dilution is added to 1/2 of original volume, and original volume soaks gold mark pad 1.5mL/ bar with the collaurum volume calculation, is golden bond pad preparation condition behind the colloid gold label.
The detection line bag is 1.0mg/ml by concentration, 0.1 μ l/mm specking amount.
1.2 the nature controlling line bag is by the selection of concentration:
Sheep anti-mouse igg antibody is made into variable concentrations, with reorganization EB-NA1 1.0mg/ml, with 0.1 μ l/mm specking amount, be coated on respectively on the nature controlling line and detection line position of nitrocellulose filter, after carrying out drying,, carry out testing result table 4 with internal control serum with the golden bond pad combo for preparing
Table 4 nature controlling line testing result
By table 4 result as can be known: when nature controlling line concentration is 1.0mg/ml, 1.5mg/ml, nature controlling line show line than its concentration a little less than, and when detecting the strong positive sample nature controlling line to show line more weak, this competition mainly due to detection line and nature controlling line causes.When nature controlling line concentration is 3.0mg/ml when above, it is thicker and irregular that nature controlling line shows line.
Comprehensive above experimental result, we choose 2.0mg/ml as the bag of nature controlling line by concentration.
The final bag of reaction film by condition is for this reason:
The specking amount is 0.1 μ L/mm,
The nature controlling line bag is by concentration: sheep anti-mouse igg antibody is 2.0mg/ml
The detection line bag is 1.0mg/ml by concentration: EB-NA1
2, the selection of colloid gold particle:
2.1 principle: prepare collaurum according to gold chloride under the condition of boiling and trisodium citrate generation redox reaction.Can change and control the grain size of collaurum by the additional proportion of regulating gold chloride and trisodium citrate.
2.2 operation: add 1% citric acid three sodium solution that 0.1mL 10% gold chloride boils, adds different volumes under stirring condition in the 100mL distilled water, boiled 5 minutes, the colloid gold particle of preparation different condition treats that its natural cooling returns back to original volume.With the collaurum of mouse-anti people IgA monoclonal antibody mark variable grain, and then detect with enterprise's internal control product.The results are shown in Table 2:
Selection experiment---the particle diameter selection (1) of table 5.1% trisodium citrate consumption
Reaction film proportioning with the four kinds of colloid gold label mouse-anti people IgA monoclonal antibodies of preparation and the EB-Ag that recombinates wrap quilt detects with internal control serum again.The results are shown in Table 6
Table 6. internal control serum testing result----particle diameter is selected (2)
Interpretation of result: by table 6 result as can be known, the collaurum of No. 3 system gold bar spare systems, color is scarlet, limpid, no muddiness and floating thing, its specificity of the collaurum behind the mark, susceptibility are better.So select system gold bar spare to be: 0.01% gold chloride 100ml+1.8ml, 1% trisodium citrate.
3, the optimization of colloid gold label mouse-anti people IgA monoclonal antibody condition:
3.1 principle:,, form red collaurum mouse-anti people IgA monoclonal antibody bond with the mouse-anti people IgA monoclonal antibody stable bond of gold grain trickle in the collaurum and purifying according to the colloidal gold-labeled method characteristic.
3.2 the selection of colloid gold label optimum pH: adopt ocular estimate.Get several 1.5mL test tubes, add the 1mL collaurum respectively; Use 0.1mol/L K
2CO
3Or 5mol/L hydrochloric acid is adjusted to 3,4,5,6,7,8,9,10 respectively with pH; Get 96 hole ELISA Plate, respectively above-mentioned collaurum is got 100 μ L from low to high respectively by pH and added in the hand-hole triplicate; Every hole adds the mouse-anti people IgA monoclonal antibody that 3 μ L concentration are 1mg/mL respectively, mixes, and places 15min under the room temperature; It is 10%NaCl solution that every hole adds 20 μ L concentration respectively, and mixing was placed 2 hours under the room temperature; Observing colloid gold change color, record keeps red minimum pH (X).Adjustment pH is X-1.0, X-0.5, X, X+0.5, X+1.0, repeats above step, keeps red minimum pH value to be the optimal pH of mark.Result such as table 7:
Conclusion: by table 7 result as can be known, when the pH value is 8.5, promptly add 0.1mol/LK
2CO
3During 20 μ l/ml, colloid gold label mouse-anti people IgA's is effective, good stability, and therefore selected pH value 8.5 promptly adds 0.1mol/LK
2CO
320 μ l/ml are the optimum pH of colloid gold label mouse-anti people IgA.
3.3 the selection of mouse-anti people IgA monoclonal antibody optimum mark amount:
3.3.1 minimum mark amount: salt precipitation method and gradient method.At first adopt salt precipitation method to select the minimum mark amount of mouse-anti people IgA monoclonal antibody, select the optimum mark amount of mouse-anti people IgA monoclonal antibody then with the intersection matching method.Get 96 hole ELISA Plate, add the collaurum 100 μ L of best pH respectively, repeat 3 times; Each hole adds the albumen of different amounts successively, and mixing is placed 15min under the room temperature; Add 20 μ L 10%NaCl, place 2h under the room temperature; Color still keeps the red minimum albumen consumption of mark that is.Result such as table 8:
The experimental result that table 8. mouse-anti people IgA monoclonal antibody minimum mark amount is selected
By table 8 experimental result as can be known: the minimum mark amount of mouse-anti people IgA monoclonal antibody is 5 μ g/mL.
3.3.2 the optimum mark amount is selected: with the K of 0.1mol/L
2CO
3Solution is transferred pH to 8.5, adds 10 μ g-25 μ g/mL marks then respectively with mouse-anti people IgA monoclonal antibody, stirs 40 minutes, and adding 10% bovine serum albumin(BSA) to final concentration again is 1%, stirs 15 minutes.12000rpm, 4 ℃ centrifugal 40 minutes, careful abandoning supernatant adds collaurum bond dilution to 1/2 of original volume.Soak gold, after the drying, with the suitableeest bag by the condition bag by the pairing of good EB-IgA reaction film, detect its specificity and sensitivity.The results are shown in Table 9:
The experimental result that table 9. mouse-anti people IgA monoclonal antibody minimum mark amount is selected
By table 9 result as can be known: under pH value 8.5 conditions, when mouse-anti people IgA monoclonal antibody label concentration was 15 μ g/mL, the specificity and the susceptibility of test strips were better, and sensitivity is higher.Therefore the flag condition of determining is: every milliliter of collaurum adds 0.1mol/L solution of potassium carbonate 20 μ L, and mouse-anti people IgA monoclonal antibody label concentration is that 15 μ g/mL are the final production process conditions.
The selection of 4EB-IgA reaction film and gold mark pad drying condition
Reaction film and gold mark pad with drying finishes are prepared into test strips, enclose in the aluminium foil bag, place 37 ℃ to preserve 14 days.Regularly detect.
Suitable drying condition, not only influence the sensitivity of kit, and being related to the stability of kit. the result shows, at dry environment is under 37 ℃ of conditions, the test strips of preparation in dry 3 hours, its susceptibility, stability are all better, so drying 3 hours was the drying condition that reaction film and golden bond pad are produced under 37 ℃ of conditions of selection.
Four, Epstein-Barr virus IgA antibody assay kit (colloidal gold method) and preparation method
Epstein-Barr virus IgA antibody assay kit (colloidal gold method) comprises the sheep anti-mouse igg antibody of bag quilt on the recombinant antigen EB-NA1, nature controlling line of nitrocellulose filter detection line bag quilt and the mouse-anti people IgA monoclonal antibody by colloid gold label that gold mark pad is gone up the bag quilt.Reorganization EB NA1 antigen is colourless transparent liquid, and concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount is only deposited a band under 10 μ l conditions, and bag is 1.0mg/ml by concentration; Sheep anti-mouse igg antibody is a colourless transparent liquid, and concentration is greater than 4mg/ml, and bag is 2.0mg/ml by concentration; Mouse-anti people IgA monoclonal antibody is a colourless transparent liquid, and concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount exists two bands, label concentration 15 μ g/ml under 10 μ l conditions;
The preparation method of Epstein-Barr virus IgA antibody assay kit (colloidal gold method) may further comprise the steps:
(1) preparation of reaction film: is 1.0mg/ml to bag by concentration with the recombined EB virus antigen diluent with phosphate buffer, it is 2.0mg/ml by concentration that sheep anti-mouse igg antibody is diluted to bag, with a film machine two kinds of coating buffers are drawn respectively on the nitrocellulose filter, after wrapping the nitrocellulose filter drying of quilt, preserved;
(2) preparation of mouse-anti people IgA monoclonal antibody gold bond pad: with label concentration is that the mouse-anti people IgA monoclonal antibody of 10-23 μ g/ml and collaurum carry out coupling and make colloid gold label mouse-anti people IgA monoclonal antibody solution, with the rotating speed is the centrifugal 40min of 12000r/min, abandon supernatant, add collaurum bond dilution to 1/2 of original volume, then with mixed liquid dipping gold mark pad, be prepared into EB-IgA gold bond pad, EB-IgA gold bond pad is put into kept dry;
(3) cutting assembling: reaction film, EB-IgA gold bond pad, robust fibre filter paper, sample pad are assembled into reaction plate, are cut into the 4mm test strips with cutting cutter again, after being installed, make product in the aluminium foil bag of packing into.
The drying condition that reaction film and gold mark pad are produced is under 37 ℃ of conditions dry 3 hours.
When detecting positive sample, the Epstein-Barr virus NA1 IgA antibody in the sample can combine with the mouse-anti people IgA monoclonal antibody of colloid gold label, forms immune complex, because chromatography effect compound and sample flow forward in nitrocellulose filter inside.When compound combines with the EB NA1 of bag quilt during through the T line, form " Au-mouse-anti people IgA monoclonal antibody-EB NA1-IgA-EB NA1 " and aggegation develops the color.Remaining colloid gold label mouse-anti people IgA monoclonal antibody combines and the aggegation colour developing with the sheep anti-mouse igg antibody of C line place bag quilt, when detecting negative sample, does not contain Epstein-Barr virus NA1 IgA antibody in the sample, causes to form immune complex, then can only develop the color at C line place.
Beneficial effect of the present invention is: Epstein-Barr virus IgA antibody assay kit has quick, easy, accurate and highly sensitive characteristics, the whole operation time only need 20 minutes just can sentence read result.Colloidal gold fast detecting test paper strip is a raw material with the recombinant antigen of multi-epitope, and it is easier, with low cost to have an operation, and specificity is good, and highly sensitive characteristics can single part of detection, is easy to popularize, and are obvious for the detection and the control effect of Epstein-Barr virus.
Description of drawings
Fig. 1 is the technological process of production figure of the embodiment of the invention
Embodiment
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for description and interpretation the present invention, and be not used in qualification the present invention.
Embodiment 1
As shown in Figure 1, the production technology of Epstein-Barr virus IgA antibody assay kit (colloidal gold method) finished product is carried out according to following steps.
1, ingredient requirement:
1.1 recombined EB virus antigen
(1) outward appearance: colourless transparent liquid;
(2) concentration and purity requirement: concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount is only deposited a band under 10 μ l conditions;
1.2 sheep anti-mouse igg antibody
(1) outward appearance: colourless transparent liquid;
(2) concentration requirement: concentration is greater than 4mg/ml;
1.3 mouse-anti people IgA monoclonal antibody
(1) outward appearance: colourless transparent liquid;
(2) concentration and purity requirement: concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount is only deposited a band under 10 μ l conditions;
2. liquid dosage:
2.10.05M the preparation of PBS damping fluid:
(1) standard recipe: according to 100mL amount meter.
Na
2HPO
4·12H
2O 1.450g
NaH
2PO
4·2H
2O 0.148g
NaCL 0.850g
Purified water 80.0mL
Purified water is settled to 100.0ml
(2) compound method:
Accurately take by weighing Na according to the standard recipe content
2HPO
412H
2O, NaH
2PO
42H
2O, NaCl add purified water 80.00mL, after stirring made abundant dissolving, using pH meter to measure liquid pH value should be in 7.30～7.50 scope, and the conductivity of diluting solution that detection is joined with the purified water twice should be at 9000 μ s/cm～13000 μ s/cm, be settled to 100.00mL with purified water, mix.2～8 ℃ of preservations are standby, the term of validity 14 days.
The preparation of 2.21% chlorauric acid solution:
(1) standard recipe: according to 100mL amount meter.
Gold chloride 1.0g
Purified water is settled to 100.0ml
(2) compound method:
Get the gold chloride that a 1g/ props up, open bottle; Measure the 2ml purified water and pour in the container, get purified water the gold chloride dissolving and the reagent bottle that will hold gold chloride are cleaned up, cleaning fluid is poured in the container, add purified water again to 100ml; Cover tight bottle cap, fully rocked 15 minutes, mix, wrap with aluminium foil, 2～8 ℃ of preservations are standby, the term of validity 12 months.
2.30.1M solution of potassium carbonate preparation:
(1) standard recipe: according to 20mL amount meter.
K
2CO
3 0.276g
Purified water 20.0mL
(2). compound method:
Measure treat dosing volume 80% purified water in reagent bottle, accurately take by weighing K2CO3 according to the standard recipe content and add in the reagent bottle, add purified water to 20.0mL, make abundant dissolving.2～8 ℃ of preservations are standby, the term of validity 14 days.
The preparation of 2.410% bovine serum albumin solution:
(1) standard recipe: according to 10mL amount meter.
Bovine serum albumin(BSA) 1.00g
Purified water 10.00mL
(2). compound method:
Measure treat dosing volume 80% purified water in reagent bottle, accurately take by weighing bovine serum albumin(BSA) according to the standard recipe content and add in the reagent bottle, treat to dissolve fully, add purified water to 10.00mL, make abundant dissolving, instant joining.
2.5 the preparation of collaurum bond dilution:
(1) standard recipe: according to 100mL amount meter.
Trishydroxymethylaminomethane 0.1211g
Hydrochloric acid is an amount of
Sucrose 2.00g
Bovine serum albumin(BSA) 1.00g
Tween-20 1.00mL
Purified water 80.0mL
Be settled to 100.0mL with purified water
(2) compound method:
Accurately take by weighing trishydroxymethylaminomethane in container according to the standard recipe content, add purified water 80mL, after stirring makes abundant dissolving, dripping hydrochloric acid makes its pH value be modulated to 9.0 then, again load weighted other recipe ingredients are added in the above-mentioned solution successively, fully after the stirring and dissolving, measuring Tween-20 according to prescription with sample injector adds in the reagent bottle, use purified water to be settled to 100.00mL, batch pH value of using pH meter to measure liquid is 8.60～8.80, and the conductivity of solution that detection is joined should be at 2300 μ s/cm～3000 μ s/cm.2～8 ℃ of preservations are standby, the term of validity 14 days.
2.6 the preparation of sample dilution:
(1) standard recipe: according to 100mL amount meter.
NaH
2PO
4·2H
2O 0.06g
Na
2HPO
4·12H
2O 0.58g
Sodium chloride 0.85g
Purified water 80.0mL
Purified water is settled to 100.0mL with purified water
(2) preparation method: accurately take by weighing NaH according to the standard recipe content
2PO
42H
2O and Na
2HPO
412H
2O adds purified water 80.00mL, stirs to make abundant dissolving back add 0.85g sodium chloride, and fully mixing is settled to 100.00mL, and batch pH value of using pH meter to measure liquid is 7.30～7.50,4～30 ℃ of preservations, the term of validity 14 months.
2.7 the preparation of collaurum:
(1) standard recipe: according to 100mL amount meter.
1% chlorauric acid solution 1.0mL
1% citric acid three sodium solution 1.8mL
Purified water 100.0mL
Be settled to 100.0mL with purified water
(2) preparation method:
Accurately measure 1% chlorauric acid solution 1.0mL according to the standard recipe content, join in the 99mL purified water, heat while stirring to boiling; After 5 minutes, accurately measure 1% citric acid three sodium solution of 1.8ml, rapid, disposable joining in the container continued heated and boiled 5min.The adding purified water returns to original volume after being cooled to room temperature.2～8 ℃ of preservations are standby, the term of validity 14 days.
2.8EB-IgA the preparation of kit detection line coating buffer:
(1) standard recipe: according to 10mL amount meter.
0.05M the PBS damping fluid is an amount of
EB-NA1 10mg
Be settled to 10.00mL with 0.05M PBS
(2) preparation method:
Accurately measure reorganization EB-NA1 10mg according to the standard recipe content, join in the 0.05M PBS damping fluid of respective volume.Abundant mixing 15min.Instant joining.
2.9EB-IgA the preparation of kit nature controlling line coating buffer:
(1) standard recipe: according to 10mL amount meter.
0.05M the PBS damping fluid is an amount of
Sheep anti-mouse igg antibody 20mg
0.05M the PBS damping fluid is settled to final volume 10.0mL
(2) preparation method:
Accurately measure sheep anti-mouse igg antibody 20mg according to the standard recipe content, join in the 0.05MPBS damping fluid of respective volume, fully mixing 15min is settled to final volume 10.0mL with the 0.05MPBS damping fluid, the final concentration that makes sheep anti-mouse igg antibody is 2.0mg/ml, instant joining.
2.10 the preparation of colloid gold label mouse-anti people IgA monoclonal antibody:
(1) related reagent standard consumption: according to 100mL amount meter.
Collaurum 200mL
0.1M solution of potassium carbonate 4.0mL
Collaurum bond dilution 100.0mL
Mouse-anti people IgA monoclonal antibody 3.0mg
10% bovine serum albumin solution 20.0mL
Collaurum bond dilution is concentrated into final volume 100.0mL
(2) preparation method:
The collaurum of measuring the main formula ormal weight according to reagent standard consumption accurately adds the 0.1M solution of potassium carbonate of main formula ormal weight in triangular flask, mixing left standstill 10 minutes.Accurately measure the mouse-anti people IgA monoclonal antibody of main formula ormal weight, stir fast down, mouse-anti people IgA monoclonal antibody is dropwise joined in the triangular flask, room temperature left standstill 40 minutes.Accurately measure 10% bovine serum albumin solution of main formula ormal weight, dropwise join in the triangular flask under stirring fast, room temperature left standstill 15 minutes.12000rpm, 4 ℃ centrifugal 40 minutes, careful abandoning supernatant, it is standby to 1/2,2～8 ℃ of preservations of original volume to add collaurum bond dilution, the term of validity 14 days.
Embodiment 2
The preparation one of kit
(1) preparation of reaction film: is 1.0mg/ml to bag by concentration with the recombined EB virus antigen diluent with phosphate buffer, it is 2.0mg/ml by concentration that sheep anti-mouse igg antibody is diluted to bag, with a film machine two kinds of coating buffers are drawn respectively on the nitrocellulose filter, after wrapping the nitrocellulose filter drying of quilt, preserved;
(2) preparation of mouse-anti people IgA monoclonal antibody gold bond pad: with label concentration is that the mouse-anti people IgA monoclonal antibody of 10 μ g/ml and collaurum carry out coupling and make colloid gold label mouse-anti people IgA monoclonal antibody solution, with the rotating speed is the centrifugal 40min of 12000r/min, abandon supernatant, add collaurum bond dilution to 1/2 of original volume, soak gold mark pad with mixed liquor with the 1.5ml/ bar then, be prepared into EB-IgA gold bond pad, EB-IgA gold bond pad is put into kept dry;
(3) cutting assembling: reaction film, EB-IgA gold bond pad, robust fibre filter paper, sample pad are assembled into reaction plate, are cut into the 4mm test strips with cutting cutter again, after being installed, make product in the aluminium foil bag of packing into.
The drying condition that reaction film and gold mark pad are produced is under 37 ℃ of conditions dry 3 hours.
Embodiment 3
The preparation two of kit
(1) preparation of reaction film: is 1.0mg/ml to bag by concentration with the recombined EB virus antigen diluent with phosphate buffer, it is 2.0mg/ml by concentration that sheep anti-mouse igg antibody is diluted to bag, with a film machine two kinds of coating buffers are drawn respectively on the nitrocellulose filter, after wrapping the nitrocellulose filter drying of quilt, preserved;
(2) preparation of mouse-anti people IgA monoclonal antibody gold bond pad: with label concentration is that the mouse-anti people IgA monoclonal antibody of 23 μ g/ml and collaurum carry out coupling and make colloid gold label mouse-anti people IgA monoclonal antibody solution, with the rotating speed is the centrifugal 40min of 12000r/min, abandon supernatant, add collaurum bond dilution to 1/2 of original volume, soak gold mark pad with mixed liquor with the 1.5ml/ bar then, be prepared into EB-IgA gold bond pad, EB-IgA gold bond pad is put into kept dry;
(3) cutting assembling: reaction film, EB-IgA gold bond pad, robust fibre filter paper, sample pad are assembled into reaction plate, are cut into the 4mm test strips with cutting cutter again, after being installed, make product in the aluminium foil bag of packing into.
The drying condition that reaction film and gold mark pad are produced is under 37 ℃ of conditions dry 3 hours.
Embodiment 4
The preparation three of kit
(1) preparation of reaction film: is 1.0mg/ml to bag by concentration with the recombined EB virus antigen diluent with phosphate buffer, it is 2.0mg/ml by concentration that sheep anti-mouse igg antibody is diluted to bag, with a film machine two kinds of coating buffers are drawn respectively on the nitrocellulose filter, after wrapping the nitrocellulose filter drying of quilt, preserved;
(2) preparation of mouse-anti people IgA monoclonal antibody gold bond pad: with label concentration is that the mouse-anti people IgA monoclonal antibody of 15 μ g/ml and collaurum carry out coupling and make colloid gold label mouse-anti people IgA monoclonal antibody solution, with the rotating speed is the centrifugal 40min of 12000r/min, abandon supernatant, add collaurum bond dilution to 1/2 of original volume, soak gold mark pad with mixed liquor with the 1.5ml/ bar then, be prepared into EB-IgA gold bond pad, EB-IgA gold bond pad is put into kept dry;
(3) cutting assembling: reaction film, EB-IgA gold bond pad, robust fibre filter paper, sample pad are assembled into reaction plate, are cut into the 4mm test strips with cutting cutter again, after being installed, make product in the aluminium foil bag of packing into.
The drying condition that reaction film and gold mark pad are produced is under 37 ℃ of conditions dry 3 hours.
Embodiment 5
Sample detection
1. detect the requirement of sample
Serum sample is according to a conventional method by the vein collection.Plasma sample can adopt heparin, sodium citrate, EDTA to handle.The sample of measuring in 5 days can be placed 4 ℃ of preservations.Sample is placed on-20 ℃ and can preserves 3 months at least.Sample is avoided haemolysis or multigelation.Muddy or have the sample of precipitation should be centrifugal or filter clarification after detect again.
2. the method for inspection
From the original packing aluminium foil bag, take out reagent card, be placed on the horizontal operation table top horizontal and carry out sample labeling.Get 10 μ l serum or plasma samples with sample injector, directly join in the well, drip 2 of sample dilutions again.Sentence read result in 15-20 minute, assay is invalid after 20 minutes.
3. assay
(1), feminine gender: only red stripes occurs in the aggegation of C line.
(2), the positive a: red stripes occurs at T line and C line respectively.
(3), invalid: no C line appearance or T line and C line all do not occur, and show that experiment is invalid.
This product yin and yang attribute coincidence rate, accuracy, sensitivity etc. all meet the quality standard requirement, constant product quality in the effect phase.Rheumatoid factor, hepatitis B, syphilis can not cause interference to this product.
Embodiment 6
The selection of reaction time and application of sample amount
Produce the EB-IgA kit, its reaction time and application of sample amount are determined.The results are shown in Table 10:
The selection experimental result of table 10 application of sample amount and interpretation time
By table 10 experimental result as can be known: what of application of sample amount can influence test strips and produce correct reaction result, and the application of sample amount is low omission or sample can be occurred and launch not enoughly, and the application of sample amount is easy to generate false positive when high.By above experiment, consider the different users on using error and the convenience of use, selecting the application of sample amount is that 10 μ L serum add 100 μ L sample dilutions again, the 15-20min interpretation is an application of sample interpretation condition.
Embodiment 7
The test of the different anti-coagulants sample of serum and blood plasma contrast verification
Collection blood is with different anti-coagulants heparin, sodium citrate, EDTA processing blood sample and prepare serum, test strips with above-mentioned definite working condition preparation, detect the sample after handling, the result shows: the blood sample that anti-coagulants heparin, sodium citrate, EDTA handle does not have influence to testing result, detects consistent with serum.Therefore this kit can be used for the detection of serum and plasma sample.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment put down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1.EB viral IgA antibody assay kit comprises the sheep anti-mouse igg antibody of bag quilt on the recombinant antigen EB-NA1, nature controlling line of nitrocellulose filter detection line bag quilt and the mouse-anti people IgA monoclonal antibody by colloid gold label that gold mark pad is gone up the bag quilt, it is characterized in that:
Described recombined EB virus antigen is colourless transparent liquid, and concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount is deposited a band under 10 μ l conditions;
Described sheep anti-mouse igg antibody is a colourless transparent liquid, and concentration is greater than 4mg/ml;
Described mouse-anti people IgA monoclonal antibody is a colourless transparent liquid, and concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount is two bands under 10 μ l conditions.
2. the preparation method of Epstein-Barr virus IgA antibody assay kit according to claim 1 is characterized in that may further comprise the steps:
(1) preparation of reaction film: is 1.0mg/ml to bag by concentration with the recombined EB virus antigen diluent with phosphate buffer, it is 2.0mg/ml by concentration that sheep anti-mouse igg antibody is diluted to bag, with a film machine two kinds of coating buffers are drawn respectively on the nitrocellulose filter, after wrapping the nitrocellulose filter drying of quilt, preserved;
(2) preparation of mouse-anti people IgA monoclonal antibody gold bond pad: with label concentration is that the mouse-anti people IgA monoclonal antibody of 10-23 μ g/ml and collaurum carry out coupling and make colloid gold label mouse-anti people IgA monoclonal antibody solution, with the rotating speed is the centrifugal 40min of 12000r/min, abandon supernatant, add collaurum bond dilution to 1/2 of original volume, then with mixed liquid dipping gold mark pad, be prepared into EB-IgA gold bond pad, EB-IgA gold bond pad is put into kept dry;
(3) cutting assembling: reaction film, EB-IgA gold bond pad, robust fibre filter paper, sample pad are assembled into reaction plate, are cut into the 4mm test strips with cutting cutter again, after being installed, make product in the aluminium foil bag of packing into.
3. the preparation method of Epstein-Barr virus IgA antibody assay kit according to claim 2, the drying condition that it is characterized in that described step (1) and (2) is 37 ℃ of dryings 3 hours.
4. the preparation method of Epstein-Barr virus IgA antibody assay kit according to claim 2 is characterized in that the label concentration of the mouse-anti people IgA monoclonal antibody of described step (2) is 15 μ g/ml.
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| CN2010102551462A CN101949932A (en) | 2010-08-17 | 2010-08-17 | IgA (Immunoglobulin A) antibody detection reagent kit (colloidal gold method) for EB (Epstein-Barr) viruses and preparation method thereof |
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