CN104297472B - EB (epstein-barr) virus NA1-IgA antibody detection reagent and preparation method thereof - Google Patents
EB (epstein-barr) virus NA1-IgA antibody detection reagent and preparation method thereof Download PDFInfo
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- CN104297472B CN104297472B CN201410472951.9A CN201410472951A CN104297472B CN 104297472 B CN104297472 B CN 104297472B CN 201410472951 A CN201410472951 A CN 201410472951A CN 104297472 B CN104297472 B CN 104297472B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides an EB (epstein-barr) virus NA1-IgA antibody detection reagent. The EB virus NA1-IgA antibody detection reagent at least comprises a reaction membrane, an antigen pad and a gold label pad, wherein a detection line and a quality control line are labeled on the reaction membrane; the detection line contains a rat anti-human IgA monoclonal antibody; the quality control line contains biotin; the antigen pad contains a recombinant EB virus NA1 antigen labeled with the biotin; and the gold label pad contains an avidin compound containing a colloid gold label. The detection reagent provided by the invention has the characteristics of rapidness, simplicity and convenience, accuracy and high sensitivity.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of antibody test reagent.
The invention further relates to include the test kit and preparation method of the detectable.
Background technology
Epstein-Barr virus (epstein-barr virus, EBv), also known as ebb virus (Human herpesvirus
4(HHV-4)).Epstein and Barr is in 1964 first successfully by Burkitt african children lymphomas cell by external
Suspension culture and build strain, and in plant cell smear is built with electron microscopic observation to simplex virus particles, it is believed that the virus is various evils
One of cause of disease of property tumor (such as nasopharyngeal carcinoma), the epithelial cell and bone-marrow-derived lymphocyte in its main infection human oropharyngeal portion.In South China
All detected with the presence of Epstein-Barr virus genome in square nasopharyngeal carcinoma patient groups mostly.Epstein-Barr virus are widely distributed, more in sporadic, also
Prevalence can be caused.Epstein-Barr virus are very close with Nasopharyngeal Cancer, and the virus specific antigen that Epstein-Barr virus are formed in course of infection can
To divide into early antigen (EA), viral capsid antigen (VCA), core associated tumor antigen (EBNA) and membrane antigen (MA).Detection
The corresponding antibodies reaction of these antigens, contributes to the diagnosis and treatment of EBV relevant diseases.
Viral capsid antigen (VCA) is initially can detect that in the patients serum of infection EBV with very strong immunogenicity
VCA-IgM, afterwards IgM antibody be gradually reduced to the level that cannot be detected, almost simultaneously VCA-IgG gradually increases, and can be just
Exist in ordinary person's body throughout one's life.If this negative, EBV infection can be excluded.
Core associated tumor antigen (EBNA) can be divided into six kinds, and wherein NA1 antigens are unique a kind of related in all Epstein-Barr virus
The virus protein all expressed in tumor cell, occurs in all cores for continuing infected cell, and its immunogenicity expression is relative
Later, only anti-NA1 antibody is formed after several weeks or several months.One obvious positive test result (the second titre stage) shows once had
Infection.If VCA tests are positive, titre is 1：160 or higher, with reference to negative or p+ anti-EBNA1 tests, mean that one kind
Acute, new or recurrent infection.
Therefore, the detection of Epstein-Barr virus NA1 antigens for nasopharyngeal carcinoma early diagnosiss and development Index for diagnosis have extremely important
Meaning.
The detectable of detection Epstein-Barr virus NA1-IgA is mainly using Enzyme-linked Immunosorbent Assay (ELISA) method, Real- at present
Time quantitative PCR methods etc., but have that complex operation, detection sensitivity be relatively low, the defect of detection time length.
Gold colloidal is in the reducing agent such as effect such as white phosphorus, ascorbic acid, sodium citrate, tannic acid by gold chloride (HAuCl4)
Under, a certain size gold grain is can be grouped to, and as electrostatic interaction becomes a kind of stable colloidal state, is formed electronegative
Hydrophobic sol solution, therefore claim gold colloidal.Gold colloidal is negatively charged under mild alkaline conditions, can be with the positive charge group shape of protein molecule
Into firm combination, as this combination is electrostatical binding, so not affecting the biological nature of protein.Gold colloidal except with egg
White matter is combined in addition, can be to be combined with many other biomacromolecules, such as SPA, PHA, ConA etc..According to gold colloidal some
Physical behavior, such as high electron density, granular size, shape and color reaction, add immunity and the biological characteristicses of conjugate, because
And make gold colloidal be widely used in the fields such as immunology, histology, pathology and cytobiology.
Immuno-gold labeling technology (Immunogold labelling techique) mainly make use of gold grain to have high electricity
The characteristic of sub- density, marks at protein binding in gold, under the microscope visible dark brown coloured particles, when these labels are matched somebody with somebody accordingly
When assembling at body in a large number, naked eyes red color visible or pink speckle, thus for qualitative or semiquantitative tachysynthesises detection side
In method.
The antigen or antibody of specificity are fixed on film by colloidal gold method detectable with ribbon, colloid gold label reagent
(antibody or monoclonal antibody) adsorbs on pad, after sample to be checked is added in the sample pad of test strips one end, by hair
Spy moves forward, and reacts to each other after dissolving the colloid gold label reagent on pad, then is moved to fixed antigen or anti-
During the region of body, there is specific binding and be trapped in the conjugate of thing to be checked and gold marked reagent, be gathered in detection band again therewith
On, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, using very convenient.
Chinese patent application CN101949932A provides the detection examination that a kind of colloidal gold method detects Epstein-Barr virus IgA antibody
Agent, which adopts Epstein-Barr virus NA1 antigen coat detection lines, the anti-human IgA monoclonal antibodies labelling gold colloidal of Mus to be coated with sheep anti-mouse igg antibody
Nature controlling line.Chinese patent application CN102539768A provides a kind of Epstein-Barr virus Zta IgA antibody gold-immunochromatographyreagent reagent for assay, and which is adopted
With Epstein-Barr virus Zta antigen coat detection lines, the anti-human IgA monoclonal antibodies labelling gold colloidal of Mus, nature controlling line is coated with sheep anti-mouse igg antibody.
Both the above colloidal gold method detectable adopts Epstein-Barr virus antigen coat detection line, and the antigen of detection line can be with inspection
All antibody responses such as Epstein-Barr virus IgG, IgA, IgM in test sample sheet, as IgG is Immunoglobulin in Serum main constituent, about
The 75% of Immunoglobulin in Serum total content is accounted for, is detect target IgA antibody 4～7 times, can significant contention Interference Detection mesh
The combination of mark IgA and coating Epstein-Barr virus antigen, causes testing result sensitivity to reduce with specificity.
The content of the invention
It is an object of the present invention to provide a kind of Epstein-Barr virus NA1-IgA antibody test reagents, with the anti-human IgA Dan Ke of Mus
Grand antibody draws detection line, and introduces biotin-avidin system, the whole IgA antibodies in Direct Acquisition detection sample, Ke Yiyou
The interference for avoiding the antibody such as IgG, IgM of effect, improves sensitivity and the specificity of detection.
Further object is that providing a kind of Epstein-Barr virus NA1-IgA antibody assay kits.
The present invention also provides the preparation method of the detectable.
According to an aspect of the present invention, a kind of Epstein-Barr virus NA1-IgA antibody test reagents, at least include
One reaction film, drawing on the reaction film has detection line and nature controlling line, and the detection line contains the anti-human IgA monoclonals of Mus
Antibody；The nature controlling line contains biotin；
One antigen pad, the antigen pad contain by the recombined EB virus NA1 antigens of biotin labeling；
One gold standard pad, the gold standard pad contain the Avidin complex of colloid gold label.
Described Epstein-Barr virus NA1-IgA antibody test reagents, wherein preferably, the detection line to be coated with concentration is
The anti-human IgA monoclonal antibodies line of Mus of 1.4mg/ml, the nature controlling line are rule with being coated with biotin of the concentration as 2.2mg/ml,
In the Avidin complex of the colloid gold label label concentration of Avidin be 25 μ g/ml, the restructuring of the biotin labeling
In Epstein-Barr virus NA1 antigens, the label concentration of biotin is 30 μ g/ml.
Described Epstein-Barr virus NA1-IgA antibody test reagents, further include one for loading the sample pad of detection sample.
According to a further aspect in the invention, there is provided a kind of detection kit, which includes described Epstein-Barr virus NA1-IgA antibody
Detectable, and explanation used in connection with, auxiliary detection instrument, reagent and/or the merchandise sales information for concurrently providing.
In accordance with a further aspect of the present invention, there is provided prepare the Epstein-Barr virus NA1-IgA antibody test reagents described in claim 1
Method, including：
1) preparation of line NC films：Detection line is drawn on NC films with the anti-human IgA monoclonal antibodies of Mus of coating concentration；With bag
Nature controlling line is drawn on NC films by the biotin of concentration；
2) preparation of gold standard pad：The Avidin that prepared by the Avidin of label concentration and colloidal gold conjugate colloid gold label is multiple
Compound, immersion smear glass fibre element film preparation gold standard pad；
3) preparation of antigen pad：The biotin of label concentration and recombined EB virus NA1 antigens are carried out being coupled biology is obtained
The recombined EB virus NA1 antigenic compounds of plain labelling, immersion smear glass fibre element film preparation antigen pad.
Method of the present invention, it preferably includes following step：
(1) preparation of line NC films：Mus anti-human IgA monoclonal antibodies are diluted to coating concentration with phosphate buffer is
1.4mg/ml, it is 2.2mg/ml that biotin is diluted to coating concentration, two kinds of coating buffers is drawn on NC films with gold mark pen machine,
Save backup after drying；
(2) preparation of gold standard pad：The Avidin that label concentration is 25 μ g/ml is carried out being coupled prepared gold colloidal with gold colloidal
The Avidin complex of labelling, is centrifuged 30 minutes with rotating speed as 10000 revs/min, abandons supernatant, adds colloidal gold conjugate dilution
Then liquid press 60 μ l/cm with diluent to the 80% of original volume2Immersion is smeared glass fibre element film and makes gold standard pad, after being dried
Save backup；
(3) preparation of antigen pad：The biotin that label concentration is 30 μ g/ml is carried out into idol with recombined EB virus NA1 antigens
Connection is obtained the recombined EB virus NA1 antigenic compounds of biotin labeling, and mixed liquor is pressed 60 μ l/cm2Glass fibre is smeared in immersion
Plain film makes antigen pad, saves backup after being dried；
(4) cutting assembling：Line NC films, gold standard pad, antigen pad, absorbent paper, sample pad are assembled into into Sptting plate, then with cutting
Bar machine is cut into the test strips of 4mm, after being installed, loads in aluminium foil bag and makes detectable.
In method of the present invention, the wherein preparation of NC films, gold standard pad and antigen pad, drying condition is that 37 DEG C of dryings 3 are little
When.
Method of the present invention, wherein step 1) in, the coating concentration of detection line is 1.4mg/ml, by 0.15 μ l/mm
Package amount coating NC film detection lines；Step 2) in the preparation method of gold colloidal include：1% chlorine is added in 100ml distilled waters
Auric acid solution 1.0ml boils, and adds 1% citric acid three sodium solution of 1.4ml under agitation, continues to boil 5 minutes, natural
Cooling is stand-by；The pH of colloid gold label Avidin be 9.0～10.0, Avidin concentration be 25 μ g/ml, colloid gold label Avidin
Dilution ratio is 1:8, the preparation condition of gold standard pad is colloidal gold conjugate after dilution by 60 μ l/cm2Immersion is smeared；Step 3)
In, the labelled amount of biotin is 30 μ g/ml, and nature controlling line biotin concentration is 2.2mg/ml.
The present invention adopts colloidal gold immunity chromatography, and Mus anti-human IgA monoclonal antibodies, biotin is solid with ribbon respectively
It is scheduled on NC films, the Avidin of golden labelling adsorbs in gold standard pad, and the recombined EB virus NA1 Antigen adsorptions of biotin labeling are anti-
On former pad, after the testing sample containing Epstein-Barr virus NA1 antibody is added in the sample pad of test strips, by capillarity to reach
It is dynamic, dissolve the Avidin of the colloid gold label on the recombined EB virus NA1 antigens and gold standard pad of the biotin labeling in antigen pad
After react to each other, then be moved to be fixed on NC films the anti-human IgA monoclonal antis body region of Mus react to be formed golden labelled immune be combined
Thing and be trapped, be gathered in detection band.By the Epstein-Barr virus in the colloid gold label analyte detection human serum that can estimate or blood plasma
NA1-IgA antibody.
The present invention draws detection line with the anti-human IgA monoclonal antibodies of Mus, and Direct Acquisition detects the whole IgA antibodies in sample,
The interference of the antibody such as IgG, IgM can be effectively avoided, sensitivity and the specificity of detection is improved.
The present invention introduces biotin-avidin system (BAS), and this is a kind of new bio reaction amplification system.Avidin
Combination and between biotin has high affinity, and its reaction is in high specificity.Avidin combines the affine normal of biotin
Number can be million times of antigen-antibody reaction, and the two combines to form the dissociation constant very little of complex, in irreversible reaction；
And acid, alkali, denaturant, protein resolvase and organic solvent do not affect which to combine.Therefore, BAS in actual applications, is produced
The stability of thing is high, so as to reduce operating error, improves the degree of accuracy for determining.Additionally, each avidin molecule has four lifes
Thing element binding site, therefore, BAS has multistage amplification so as to using when can be greatly enhanced the sensitive of detection method
Degree.
Beneficial effects of the present invention are：Epstein-Barr virus NA1-IgA antibody assay kits (colloidal gold method) are with quick, simple
Just, accurately and the characteristics of sensitivity is high, only need 20 minutes can sentence read result the whole operation time, it is not necessary to supplementary instrument, can
Directly visual results, applied widely, can single part of detection, it is easy to popularizes, for Epstein-Barr virus detection and control effect it is bright
It is aobvious.
Brief description
Fig. 1 is the technological process of production and detection principle diagram of detectable of the present invention.
Specific embodiment
Below in conjunction with the accompanying drawings, by specific description of embodiments of the present invention, describe in detail but do not limit the present invention.
Material source：Following material is unless stated otherwise by commercially available commercially available.
1 detectable preparation condition of embodiment is screened
1. the selection of colloid gold particle
1.1 principle：Redox reaction is occurred according to gold chloride under boiling conditions and trisodium citrate and prepares gold colloidal.It is logical
Overregulate the additional proportion of gold chloride and trisodium citrate the granular size of gold colloidal is varied and controlled.
1.2 preparation method
In 100ml distilled waters, add 1% chlorauric acid solution 1ml to boil, add different amounts of 1% lemon under agitation
Lemon three sodium solutions of acid, continue to boil 5 minutes to prepare different size of colloid gold particle, and natural cooling is stand-by.Use different size
Colloid gold label Avidin, detected with enterprises quality-control product as research material, the results detailed in Table 1, table 2.
The selection (1) of the choice experiment of 1.1% trisodium citrate consumption of table --- colloid gold particle size
2. inner quality control product testing result of table --- the selection (2) of colloid gold particle size
Interpretation of result：Table 1, table 2 as can be seen that No. 2 colloidal gold solution colors are purplish red, bright, without muddy and floating thing, mark
Preferably, therefore the process conditions that selection prepares gold colloidal are gold colloidal Avidin its sensitivity, specificity after note：It is double in 100ml
Add 1% chlorauric acid solution 1.0ml to boil in steaming water, add 1% citric acid three sodium solution of 1.4ml under agitation, after
Continuous to boil 5 minutes, natural cooling is stand-by.2. the optimization of colloid gold label Avidin technique
2.1 principle：Gold colloidal electronegative hydrophobic sol solution as electrostatic interaction is formed, gold colloidal is in mild alkaline conditions lower band
Negative charge, can form firm combination with the positive charge group of Avidin, form stable Avidin gold label.
The selection of 2.2 colloid gold label optimum PHs
Several 1.5ml centrifuge tubes are taken, 1.0ml gold colloidals are separately added into, with the HCl and 0.1mol/L of 5.0mol/L
K2CO3PH is adjusted to 3,4,5,6,7,8,9,10 respectively.Often pipe adds the Avidin of 50 μ g, mixes, and room temperature is placed 10 minutes.
Often pipe adds 100 μ l 10%NaCl solution, mixes, and room temperature is placed 2 hours, observing colloid gold color change, records gold colloidal face
Color keeps constant minimum pH (X).Adjustment pH is X-0.8, X-0.4, X, X+0.4, X+0.8, repeats above step, gold colloidal face
Color keeps constant minimum pH value to be the optimum pH of labelling.The results detailed in Table 3, table 4.
The selection (1) of 3. colloid gold label optimum PH of table
Interpretation of result：Can be seen that when pH value is 9.0～10.0 from table 3, table 4, the stability of golden labelling Avidin
It is good, therefore the optimum pH for selecting colloid gold label is 9.0, that is, add the K of 0.1mol/L2CO3 29μl/ml。
The selection of the most suitable labelled amount of 2.3 Avidins
Several 1.5ml centrifuge tubes are taken, 1ml gold colloidals are separately added into, with the K of 0.1mol/L2CO3PH is adjusted to respectively
9.0.Often manage and Avidin is added by 5 μ g, 10 μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g, 35 μ g, 40 μ g respectively successively, mix, room temperature
Place 10 minutes.Often pipe adds 100 μ l 10%NaCl solution, mixes, and room temperature is placed 2 hours, observing colloid gold color change,
The constant minimum Avidin addition of record gold colloidal color keep, the constant minimum Avidin addition of gold colloidal color keep
The as most suitable labelled amount of the Avidin of labelling.The results detailed in Table 5.
The selection of the most suitable labelled amount of 5. Avidin of table
Interpretation of result：As can be seen from Table 5, when the Avidin concentration for adding is 30～40 μ g/ml, golden labelling Avidin
Invariant color, therefore it is 30 μ g/ml to select the most suitable labelled amount of Avidin.
3. the selection of detection line coating concentration and colloid gold label Avidin dilution ratio
According to the above-mentioned golden marking process labelling Avidin for determining, by the 1/10 of its volume concentration to original volume, then with
Different dilution ratio dilution concentrated solutions, presses 60 μ l/cm with diluent2Apply gold standard pad, drying for standby；Restructuring EB to former times is sick
Biotin is added by the concentration of 30 μ g/ml in malicious NA1 antigens, is mixed, mixed liquor is pressed into 60 μ l/cm2Antigen pad is applied, drying is standby
With.Package amount coating nitrocellulose filter (NC film) detection line of the concentration by 0.15 μ l/mm is coated with different detection lines.By gold mark
Pad, antigen pad and line NC film pairings, detect inner quality control serum, experimental program such as table 6, experimental result such as table 7, table 8.
6. EXPERIMENTAL DESIGN scheme of table
7.10 parts of positive internal quality-control products of table and minimum detectability quality-control product testing result
8.10 parts of negative internal quality-control product testing results of table
Interpretation of result：Can be seen that by table 7,8 result of table the sensitivity of D2 combinations is high, specificity is good, therefore by detection line
Coating concentration be set to 1.4mg/ml, by 0.15 μ l/mm package amount coating NC film detection lines；Will be colloid gold label Avidin dilute
The ratio of releasing is set to 1:8, i.e. colloidal gold conjugate diluent are added to the 80% of original volume, and original volume is calculated with gold colloidal volume.
The preparation condition of gold standard pad be dilution after colloidal gold conjugate by 60 μ l/cm2Immersion is smeared.
4. the selection of the most suitable labelled amount of biotin labeling recombined EB virus NA1 antigens
Several 1.5ml centrifuge tubes are taken, 1ml recombined EB virus NA1 antigens are separately added into.Often manage successively respectively by 5 μ g, 10
μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g, 35 μ g, 40 μ g add biotin, mix, and mixed liquor is pressed 60 μ l/cm2Antigen pad is applied, is done
It is dry standby.Gold standard pad and line NC films are made according to the above-mentioned gold standard pad processing technology for determining, detection line coating technique, by gold
Mark pad, antigen pad and line NC film pairings, detect inner quality control serum.The results are shown in Table 9, table 10.
9.10 parts of positive internal quality-control products of table and minimum detectability quality-control product testing result
10.10 parts of negative internal quality-control product testing results of table
Interpretation of result：Can be seen that when the biotin concentration for adding is 25～30 μ g/ml by table 9,10 result of table,
The sensitivity of antigen pad is high, and specificity is good, therefore it is 30 μ g/ml to select the most suitable labelled amount of biotin.
5 nature controlling lines are coated with the selection of concentration
Biotin is made into into variable concentrations, IgA monoclonal antibodies 1.4mg/ml anti-human with Mus, with the coating of 0.15 μ l/mm
Amount is coated in the detection line and Quality Control line position of NC films respectively, is supported the use with the gold standard pad for preparing, with inside after being dried
Quality-control product is detected, the results detailed in Table 11.
11 nature controlling line testing result of table
Interpretation of result：As can be seen from Table 11, as C lines antibody concentration >=2.2mg/ml, it is reacted to up to maximum, so
Most suitable nature controlling line biotin concentration is selected to be 2.2mg/ml.
In sum, NC films are finally coated with condition and are：
Package amount is 0.15 μ l/mm；
Detection line is coated with concentration：The anti-human IgA monoclonal antibodies of Mus are 1.4mg/ml；
Nature controlling line is coated with concentration：Biotin is 2.2mg/ml.
6. the selection of line NC films and gold standard pad drying condition
Suitable drying condition, not only affects the sensitivity of test kit, and is related to the stability of test kit.As a result table
It is bright, under the conditions of dry environment is 37 DEG C, the test strips for preparing for 3 hours are dried, its sensitivity, stability are preferable, therefore select
3 hours are dried under the conditions of 37 DEG C for line NC films and the drying condition of gold standard pad production.
2. preparation of reagents of embodiment
1. ingredient requirement
1.1 recombined EB virus NA1 antigens
Manufacturer：Hongkong Shennong Co., Ltd.
Trade name：Recombined EB virus VCA antigens, recombined EB virus NA1 antigens
Lot number:20140126E
Outward appearance：Colourless transparent liquid；
Concentration and purity requirement：Concentration be more than 2.0mg/ml, with SDS-PAGA determine, applied sample amount under the conditions of 10 microlitres only
Deposit a band.
The anti-human IgA antibody of 1.2 Mus
Manufacturer：Hangzhou Long Ji Bioisystech Co., Ltd
Trade name：The anti-human IgA antibody of Mus
Lot number:20131105
Outward appearance：Colourless transparent liquid；
Concentration and purity requirement：Concentration is more than 2.0mg/ml, is determined with SDS-PAGA, and applied sample amount is deposited under the conditions of 10 microlitres
Two bands.
2. liquid dosage
2.10.05M the preparation of PBS：
(1) standard recipe：According to 100ml gauge.
(2) compound method
Na is weighed accurately according to standard recipe content2HPO4·12H2O、NaH2PO4·2H2O, NaCl add purified water
80.00mL, after stirring makes fully dissolving, should be in the range of 7.30～7.50, with purified water two using pH meter measurement liquid pH value
The electrical conductivity of the matched somebody with somebody solution of dilution detection should be settled to 100.00mL with purified water in 9000 μ s/cm～13000 μ s/cm again, mix
Close uniform.2～8 DEG C save backup, 14 days effect duration.
The preparation of 2.21% chlorauric acid solution：
(1) standard recipe：According to 100mL gauge.
(2) compound method：
The gold chloride of a 1g/ is taken, bottle is opened；Measure 2mL purified water to pour in container；Purified water is taken gold chloride
Dissolving and the reagent bottle for holding gold chloride is cleaned up, washer bottle is poured in container, then plus purified water to 100mL；Lid
Tight bottle cap, fully rocks 15 minutes, and mix homogeneously is wrapped with aluminium foil, and 2～8 DEG C save backup, 12 months effect duration.
2.30.1M solution of potassium carbonate is prepared：
(1) standard recipe：According to 20mL gauge.
K2CO3 0.276g
Purified water 20.0mL
(2) compound method：
Measure and treat that the purified water with liquid product 80%, in reagent bottle, accurately weighs K according to content in standard recipe2CO3Plus
Enter in reagent bottle, plus purified water is to 20mL, make fully dissolving.2～8 DEG C save backup, 14 days effect duration.
The preparation of 2.410% bovine serum albumin solution：
(1) standard recipe：According to 20mL gauge.
Bovine serum albumin 2.00g
Purified water 20.00mL
(2) compound method：
Measure and treat that the purified water with liquid product 80%, in reagent bottle, accurately weighs Sanguis Bovis seu Bubali according to standard recipe content pure
During albumen adds reagent bottle, wait to be completely dissolved, plus purified water is to 20.0mL, make fully dissolving, it is instant to match somebody with somebody.
The preparation of 2.5 colloidal gold conjugate diluents：
(1) standard recipe：According to 100mL gauge.
(2) compound method：
Trishydroxymethylaminomethane is weighed accurately in container according to standard recipe content, plus purified water 80mL, stirring makes
After fully dissolving, then Deca hydrochloric acid makes its pH modulate to 9.0, then load weighted other recipe ingredients is sequentially added above-mentioned molten
In liquid, after being sufficiently stirred for dissolving, tween 20 is measured according to formula sample injector and is added in reagent bottle, be settled to using purified water
100.0mL, the pH value for measuring liquid using pH meter are 8.60～8.80.2～8 DEG C save backup, 14 days effect duration.
The preparation of 2.6 Sample dilutions：
(1) standard recipe：According to 100.0mL gauge.
(2) compound method
NaH is weighed accurately according to standard recipe2PO4·2H2O and Na2HPO4·12H2O, plus purified water 80.0mL, stirring make
0.85g Sodium Chloride being added after fully dissolving, is fully mixed, is settled to 100mL, the pH value for measuring liquid using pH meter is 7.30～
7.50,4～30 DEG C of preservations, 14 months effect duration.
The preparation of 2.7 gold colloidals：
(1) standard recipe：According to 100.0mL gauge.
(2) preparation method
1% chlorauric acid solution 1mL is accurately measured according to standard recipe content, is added in 99mL purified water, in heating
Stir to boiling；1% trisodium citrate of 1.4mL after 5 minutes, is accurately measured, rapidly, is disposably added in container, is continued
Heated and boiled 5min.Purified water is added to recover to original volume after being cooled to room temperature.2～8 DEG C save backup, 14 days effect duration.
2.8 detection lines are coated with the preparation of liquid：
(1) standard recipe：By 10mL gauge.
2) preparation method：
Anti-human IgA monoclonal antibodies 12mg of Mus are accurately measured according to standard recipe content, the 0.05M of respective volume is added to
In PBS.15min is mixed fully, it is instant to match somebody with somebody.
The preparation of 2.9 nature controlling line coating buffers：
(1) standard recipe：According to 10mL gauge
(2) preparation method：
Biotin 22mg is accurately measured according to standard recipe content, is added in the 0.05M PBSs of respective volume,
15min is fully mixed, and final volume 10mL is settled to 0.05M PBSs, is made the final concentration of 2.2mg/mL of biotin, i.e.,
With matching somebody with somebody.
The preparation of 2.10 colloid gold label Avidins：
(1) related actual standard consumption：According to 80mL gauge.
(2) preparation method：
The gold colloidal of main formula ormal weight is measured in triangular flask according to reagent standardized amount, main formula regulation is accurately added
The 0.1M solution of potassium carbonate of amount, mixes, and stands 10min.The Avidin of main formula ormal weight is measured accurately, under quick stirring, will
Avidin is added dropwise in triangular flask, is stored at room temperature 40min.10% bovine serum albumin of main formula ormal weight is measured accurately
Solution, is added dropwise in triangular flask under quick stirring, is stored at room temperature 15min.10000rpm, 4 DEG C of centrifugation 30min, carefully abandons
Supernatant is removed, adds colloidal gold conjugate diluent to save backup to the 80% of original volume, 2～8 DEG C, 14 days effect duration.
It is prepared by embodiment 3.EB virus N A1-IgA antibody assay kits (colloidal gold method)
(1) preparation of line NC films：Mus anti-human IgA monoclonal antibodies are diluted to coating concentration with phosphate buffer is
1.4mg/mL, it is 2.2mg/mL that biotin is diluted to coating concentration, is drawn two kinds of coating buffers on NC films respectively with a film machine,
Preserve after coated NC films are dried；
(2) preparation of Avidin gold standard pad：The Avidin that label concentration is 30 μ g/mL is carried out into coupling with gold colloidal
Colloid gold label biotin solution, is centrifuged 30min by 10000r/min of rotating speed, abandons supernatant, adds colloidal gold conjugate dilution
Then liquid press 60 μ l/m with mixed liquor to the 80% of original volume2Immersion is smeared and makes gold standard pad, by gold standard pad kept dry；
(3) preparation of biotin recombined EB virus NA1 antigen pads：By biotin that label concentration is 30 μ g/ml and restructuring
Epstein-Barr virus NA1 antigens carry out the group Epstein-Barr virus NA1 antigenic compounds for being coupled prepared biotin labeling, and mixed liquor is pressed 60 μ l/cm2
Immersion is smeared glass fibre element film and makes antigen pad, saves backup after being dried.
(4) cutting assembling：Line NC films, gold standard pad, antigen pad, crude fibre filter paper, sample pad are assembled into into Sptting plate,
4mm test strips are cut into cutting machine, after being installed, are loaded in aluminium foil bag and is made product.
The drying condition of line NC films, gold standard pad and antigen pad production is dry 3 hours under the conditions of 37 DEG C.
4. sample detection of embodiment
1. the requirement of sample is detected
Serum sample is according to a conventional method by venous collection.Plasma sample can be adopted at heparin, sodium citrate, EDTA
Reason.The sample determined in 5 days can place 4 DEG C of preservations.Sample is placed on -20 DEG C and at least can preserve 3 months.Sample avoid haemolysis or
Person's multigelation.Sample that is muddy or having precipitation will enter centrifugation or be detected after clarifying after filtering again.
2. the method for inspection
Reagent card is taken out from original packing aluminium foil bag, horizontal is placed on horizontal countertop and is carried out sample labeling.With plus
Sample device takes 10 μ l serum or plasma sample, is added directly in well, then Deca Sample dilution 2 drips.In 15-25 minutes
Sentence read result, after 25 minutes, assay is invalid.
3. assay
(1) it is, positive：There is aubergine band in quality control region and detection zone.
(2) it is, negative：Only there is an aubergine band in quality control region.
(3) it is, invalid：There is not aubergine band in quality control region (C), shows that incorrect operating process or reagent go bad and damages
Bad, it is invalid to test
This product yin and yang attribute coincidence rate, elaboration, sensitivity etc. meet quality criteria requirements, and in the effect phase, product quality is steady
It is fixed.Rheumatoid factor, hepatitis B virus antibody, syphilis helicoid antibody, HIV antibody, cytomegaloviruses
Antibody, herpes simplex virus antibody positive specimen will not be interfered to this product testing result.
4. the selection of response time and sample-adding amount
Production Epstein-Barr virus NA1 IgA antibody detection kits (colloidal gold method), was carried out really to its response time and sample-adding amount
It is fixed.The results are shown in Table 12：
12. sample-adding amount of table and the choice experiment result of interpretation time
Interpretation of result：Sample-adding amount many be can be seen that from 12 experimental result of table, and I haven't seen you for ages that to affect test strips to produce correct anti-
Result is answered, sample-adding amount is low to occur missing inspection, and false positive is easily produced when sample-adding amount is high.By above experimental result, it is contemplated that inspection
The stability of result and the convenience of operation being surveyed, sample-adding amount being selected for 10 μ l serum and is added 90 μ l Sample dilutions, 15-25 divides
Clock interpretation is sample-adding interpretation condition.
5 serum of embodiment and blood plasma difference anticoagulant specimen contrast verification test
Collection serum difference anti-coagulant heparin, sodium citrate, EDTA are processed and blood sample and are prepared serum, with above-mentioned true
Sample after ELISA test strip process prepared by fixed working condition, as a result shows：At anti-coagulant heparin, sodium citrate, EDTA
Blood sample is managed on testing result without impact, it is consistent with Virus monitory result.Therefore, this test kit can be used for serum and blood plasma sample
This detection.
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, although with reference to the foregoing embodiments
The present invention is described in detail, it will be apparent to those skilled in the art which still can be remembered to foregoing embodiments
The technical scheme of load is modified, or carries out equivalent to which part technical characteristic, and these modifications and replacement all should belong to
In the scope of the present invention.
Claims (7)
1. a kind of Epstein-Barr virus NA1-IgA antibody test reagents, consist of
One reaction film, drawing on the reaction film has detection line and nature controlling line, and the detection line contains the anti-human IgA monoclonal antis of Mus
Body；The nature controlling line contains biotin；
One antigen pad, the antigen pad contain by the recombined EB virus NA1 antigens of biotin labeling；
One gold standard pad, the gold standard pad contain the Avidin complex of colloid gold label；
One is used to load the sample pad of detection sample；
One crude fibre filter paper；
Wherein described detection line to be coated with the line of Mus of the concentration as 1.4mg/ml anti-human IgA monoclonal antibodies, the nature controlling line with
Coating concentration is the >=biotin of 2.2mg/ml line, and in the Avidin complex of the colloid gold label, the labelling of Avidin is dense
Spend for 30～40 μ g/ml, in the recombined EB virus NA1 antigens of the biotin labeling, the label concentration of biotin is 25～30 μ
g/ml。
2. a kind of detection kit, which includes the Epstein-Barr virus NA1-IgA antibody test reagents described in claim 1.
3. the method for preparing the Epstein-Barr virus NA1-IgA antibody test reagents described in claim 1, comprises the steps of：
(1) preparation of line NC films：Mus anti-human IgA monoclonal antibodies are diluted to coating concentration with phosphate buffer is
1.4mg/ml, it is >=2.2mg/ml that biotin is diluted to coating concentration, and two kinds of coating buffers are drawn to NC films with gold mark pen machine
On, save backup after being dried；
(2) preparation of gold standard pad：The Avidin that label concentration is 30～40 μ g/ml is carried out being coupled prepared gold colloidal with gold colloidal
The Avidin complex of labelling, is centrifuged 30 minutes with rotating speed as 10000 revs/min, abandons supernatant, adds colloidal gold conjugate dilution
Then liquid press 60 μ l/cm with diluent to the 80% of original volume2Immersion is smeared glass fibre element film and makes gold standard pad, after being dried
Save backup；
(3) preparation of antigen pad：The biotin that label concentration is 25～30 μ g/ml is carried out into idol with recombined EB virus NA1 antigens
Connection is obtained the recombined EB virus NA1 antigens of biotin labeling, and mixed liquor is pressed 60 μ l/cm2Glass fibre element film system is smeared in immersion
Into antigen pad, save backup after being dried；
(4) cutting assembling：Line NC films, gold standard pad, antigen pad, crude fibre filter paper, sample pad are assembled into into Sptting plate, then with cutting
Bar machine is cut into the test strips of 4mm, after being installed, loads in aluminium foil bag and makes detectable.
4. the method described in claim 3, wherein drying condition is 37 DEG C and does in the preparation of line NC films, gold standard pad and antigen pad
Dry 3 hours.
5. the method described in claim 3, wherein step 1) in, the coating concentration of detection line is 1.4mg/ml, by 0.15 μ l/mm
Package amount coating NC film detection lines.
6. the method described in claim 3, wherein step 2) in the preparation method of gold colloidal include：Add in 100ml distilled waters
Enter 1% chlorauric acid solution 1.0ml to boil, add 1% citric acid three sodium solution of 1.4ml under agitation, continue to boil 5 points
Clock, natural cooling are stand-by；The pH of colloid gold label Avidin be 9.0～10.0, Avidin concentration be 30 μ g/ml, gold colloidal mark
The Avidin complex dilution ratio of note is 1:8, the preparation condition of gold standard pad is that the Avidin of the colloid gold label after dilution is multiple
Compound presses 60 μ l/cm2Immersion is smeared.
7. the method described in claim 3, wherein step 3) in, will be biotin that label concentration is 30 μ g/ml sick with restructuring EB
Malicious NA1 antigens carry out the recombined EB virus NA1 antigens for being coupled prepared biotin labeling, and mixed liquor is pressed 60 μ l/cm2Immersion is smeared
Glass fibre element film makes antigen pad, saves backup after being dried；
Nature controlling line biotin concentration is 2.2mg/ml.
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