CN102539768B - EB (Epstein-Barr) virus Zta IgA (Immunoglobulin A) antibody colloidal gold detection kit and preparation method thereof - Google Patents

EB (Epstein-Barr) virus Zta IgA (Immunoglobulin A) antibody colloidal gold detection kit and preparation method thereof Download PDF

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CN102539768B
CN102539768B CN201110425868.2A CN201110425868A CN102539768B CN 102539768 B CN102539768 B CN 102539768B CN 201110425868 A CN201110425868 A CN 201110425868A CN 102539768 B CN102539768 B CN 102539768B
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epstein
reaction film
barr virus
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CN102539768A (en
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王胜岚
向柱方
李峰
宋小冬
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ZHONGSHAN BIO-TECH Co Ltd
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ZHONGSHAN BIO-TECH Co Ltd
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Abstract

The invention discloses an EB (Epstein-Barr) virus Zta IgA (Immunoglobulin A) antibody colloidal gold detection kit and a preparation method thereof. The kit comprises a bottom plate, wherein a reaction film provided with a detection line and a quality control line is stuck to the bottom plate; a gold mark mat and a sample mat are stuck to one end of the reaction film which is close to the detection line in sequence in a laminated way; an absorbing mat is stuck to one end of the reaction film which is close to the quality control line in a laminated way; the detection line on the reaction film is coated with a recombinant EB virus Zta antigen; the quality control line is coated with a goat anti-mouse IgG antibody; and the gold mark mat is coated with a mouse anti-human IgA monoclonal antibody marked with colloidal gold. The kit has the advantages of rapid, simple, convenient detection, high accuracy, high sensitivity, no need of auxiliary instrument, direct observation of results with naked eyes, wide application range, single detection, easiness for popularizing and stable product quality in the effective period of the kit. The detection result of the kit is not interfered with a rheumatoid factor, a hepatitis B virus antibody, a treponema pallidum antibody, a human immunodeficiency virus antibody, a cytomegalovirus antibody and a herpes simplex virus antibody positive specimen.

Description

EB virus Zta IgA antibody colloidal gold detection kit and preparation method thereof
[technical field]
The present invention relates to a kind of antibody assay kit, be specifically related to a kind of gold-immunochromatographyreagent reagent for assay box that detects Epstein-Barr virus Zta IgA antibody and preparation method thereof, belong to medical biotechnology field.
[background technology]
Epstein-Barr virus (Epstein-barr virus, EBV) be a kind of gamma herpes viruses of having a liking for human B lymphocyte, the main bone-marrow-derived lymphocyte of invading, has affinity to people's bone-marrow-derived lymphocyte, epithelial cell (comprising Blasius' duct, pharynx and uterine neck etc.) and gland cell.In International Union Against Cancer's annual meeting of the World Health Organization (WHO) in 1997, EBV is classified as to first kind carcinogenic substance.Zta albumen is the early stage activity factor of Epstein-Barr virus of being expressed by immediate early gene BZLF1, by latent infection, to be converted into the product of solubility infection phase when early stage immediately, that Epstein-Barr virus enters the essential active element of cracking replication status, in virus, from latent infection, change into and dissolve to infect, and play an important role in the process such as virus genomic transcript and expression of solubility infection phase.Early protein Zta role in Epstein-Barr virus Carcinogenesis, more and more receives researcher's concern in recent years immediately.Current research finds that ebv infection is as relevant in nasopharyngeal carcinoma, lymph cancer etc. to multiple human tumor.
The method of existing detection Epstein-Barr virus mainly contains:
Real-time quantitative PCR method
This method is mainly used in the quantitative of epstein barr virus dna.With fluorescence quantifying PCR method, detect epstein barr virus dna in nasopharyngeal carcinoma (NPC) patient's blood plasma, recall rate reaches 96%, and confirm radiotherapy in research subsequently after in patient's blood plasma of tumor regression epstein barr virus dna also decline rapidly.Adopt Real-time quantitative PCR technique, utilize a pair of Auele Specific Primer and specificity fluorescent probe, in conjunction with round pcr and detection technique of fluorescence, realize the quantitative detection to Epstein-Barr virus.But diagnose in early days medium sensitivity low, specificity is similar to FA IgA.
Indirect immunofluorescence/immunoenzyme
Traditionally, by the indirect immunofluorescence containing virolymphocyte smear, detecting Epstein-Barr virus specific antibody, abroad also using at present, is mainly to detect VCA IgA antibody and FA IgA antibody, needs to use fluorescent microscope.China, from the eighties, has promoted the use of immunoenzyme (EIA method) and has detected VCA IgA antibody and FA IgA antibody, yet, between this method exists and criticizes, quality differs greatly, the shortcomings such as visual inspection is short of objectivity, and index is thicker, are progressively replaced by euzymelinked immunosorbent assay (ELISA) at present.
Euzymelinked immunosorbent assay (ELISA) (ELISA)
Euzymelinked immunosorbent assay (ELISA) (ELISA) detects each antibody-like in serum or blood plasma, adopts the antigen of the Epstein-Barr virus replicative cycle different phase expression of genetic recombination.It is Epstein-Barr virus solubility infection bone-marrow-derived lymphocyte the most widely that domestic application obtains, the eb early antigen FA that propagation produces while starting, and propagation synthetic Epstein-Barr virus shell antigen VCA of later stage.But ELISA method complicated operation, detection time is long, need to dilute testing sample, the operation such as incubation, separation, washing and colour developing.
[summary of the invention]
The object of the invention is to overcome the deficiencies in the prior art, a kind of Epstein-Barr virus ZtaIgA antibody colloidal gold detection kit is provided, select be the early stage activity factor Zta of Epstein-Barr virus specific antigen be recombined EB virus zta antigen as detection line envelope antigen, be applicable to the Epstein-Barr virus Zta IgA antibody in qualitative detection human serum.This kit both can be used for the auxiliary diagnosis of the nasopharyngeal carcinoma that ebv infection is relevant, also can be used for people at highest risk's screening and risk assessment, for disease is found and early treatment provides possibility early.
The present invention also provides a kind of method of preparing Epstein-Barr virus Zta IgA antibody colloidal gold detection kit.
Technical scheme of the present invention is:
A kind of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit, comprise base plate, on described base plate, be pasted with the reaction film with detection line (T line) and nature controlling line (C line), described reaction film superposes successively and is pasted with gold mark pad near one end of detection line, sample pad, described reaction film is pasted with absorption pad near one end stack of nature controlling line, it is characterized in that: on described reaction film, detection line has been coated with recombined EB virus Zta antigen, on described reaction film, nature controlling line has been coated with sheep anti-mouse igg antibody, has been coated with the mouse-anti people IgA monoclonal antibody of colloid gold label on described gold mark pad.
Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, is characterized in that
The coated concentration of described recombined EB virus Zta antigen is 1.2mg/ml, described recombined EB virus Zta antigen is colourless transparent liquid, concentration is greater than 2mg/ml, by SDS-PAGE (SDS PAGA), measures, and applied sample amount is only deposited a band under 10 μ l conditions;
The coated concentration of described sheep anti-mouse igg antibody is 2.0mg/ml;
The label concentration of described mouse-anti people IgA monoclonal antibody is 30 μ g/ml, and described mouse-anti people IgA monoclonal antibody is colourless transparent liquid, and concentration is greater than 2mg/ml, with SDS PAGA, measures, and applied sample amount is deposited two bands under 10 μ l conditions.
Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, is characterized in that described base plate is polyvinyl chloride plate (PVC plate).
Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, is characterized in that described reaction film is nitrocellulose filter (NC film).
A preparation method for Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, is characterized in that comprising the following steps:
The preparation of a, reaction film: reaction film is attached on base plate, with damping fluid, by recombined EB virus Zta antigen diluent, to coated concentration, be 1.2mg/ml, it is 2.0mg/ml that sheep anti-mouse igg antibody is diluted to coated concentration, on reaction film, rule respectively recombined EB virus Zta antigen detection line and sheep anti-mouse igg antibody nature controlling line, save backup after dry;
The preparation of b, gold mark pad: smear glass fibre element film with the immersion of colloid gold label mouse-anti people IgA monoclonal antibody solution and make gold mark pad, save backup after being dried;
The processing of c, sample pad: Epstein-Barr virus Zta antigen is smeared to glass fibre with original content, then two glass fibre of smearing are superposeed be made into sample pad completely;
D, assembling cutting: reaction film, gold mark pad, absorption pad, sample pad are assembled into reaction plate, then are cut into reagent strip with cutting cutter, after being installed, packing in aluminium foil bag is generate a reagent box.
The preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, is characterized in that damping fluid in described step a is that pH value is the mixed solution of 6.8 phosphate buffered solution (PBS), sucrose and Sodium azide (NaN3).
The preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, is characterized in that the label concentration of colloid gold label mouse-anti people IgA monoclonal antibody solution in described step b is 30 μ g/ml.
The preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, is characterized in that in described step b that gold mark liquid is by 55～60 μ l/m 2evenly smear.
The preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, is characterized in that the drying condition of described reaction film and gold mark pad is to be dried 3 hours under 37 ℃ of conditions.
The preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit as above, it is characterized in that the collaurum bond dilution in described step b is the potpourri of trishydroxymethylaminomethane, hydrochloric acid, sucrose, polyvinylpyrrolidone (PVP), bovine serum albumin(BSA), polyoxyethylene 20 sorbitan monolaurate (polysorbas20), purified water, stand-by in 2～8 ℃ of preservations.
In the present invention, manufacturing condition is definite as follows:
1, the selection of colloid gold particle
According to gold chloride under the condition of boiling and trisodium citrate generation redox reaction, prepare collaurum.By regulating the additional proportion of gold chloride and trisodium citrate to change and control the grain size of collaurum.During preparation, in 100ml distilled water, add 1% chlorauric acid solution 1ml to boil, under stirring condition, add 1% citric acid three sodium solution of different amounts, continue to boil 5 minutes to prepare the colloid gold particle of different sizes, naturally cooling stand-by.By the colloid gold label mouse-anti people IgA monoclonal antibodies of different sizes, (described enterprises quality-control product, as 6 parts of quality-control products that enterprise oneself manufactures, comprises P1, P2, P3, N1, N2, N3 to take enterprises quality-control product.Following P1, P2, P3 are respectively strong positive sample, middle positive sample, weak positive sample; N1, N2, the negative sample of N3) for research material detects, the results detailed in Table 1, shown in table 2.
Choice experiment---the selection (1) of colloid gold particle size of table 1:1% trisodium citrate consumption
(following number percent is weight percentage)
Figure BDA0000121143310000061
Table 2: the selection (2) of inner quality control product testing result---colloid gold particle size
Figure BDA0000121143310000062
Interpretation of result: table 1, table 2 can be found out, No. 2 colloidal gold solution color is purplish red, bright, without muddiness and floating thing, its susceptibility of colloidal gold antibody after mark, specificity are best, therefore select the process conditions of preparing collaurum to be: to add 1% chlorauric acid solution 1ml to boil in 100ml distilled water, 1% citric acid three sodium solution that adds 1.4ml under stirring condition, continues to boil 5 minutes, naturally cooling stand-by.
2, the optimization of colloid gold label mouse-anti people IgA monoclonal anti body technology:
Collaurum is electronegative hydrophobic sol solution because electrostatic interaction forms, and collaurum is electronegative under weak base environment, can form firmly and be combined with the positive charge group of mouse-anti people IgA monoclonal antibody, forms stable mouse-anti people IgA monoclonal antibody gold label.
The selection of 2.1 colloid gold label optimum PHs
Get 8 1.5ml centrifuge tubes, add respectively 1ml collaurum, with the HCl of 5mol/L and the K of 0.1mol/L 2cO 3pH is adjusted into respectively to 3,4,5,6,7,8,9,10.Every pipe adds the mouse-anti people IgA monoclonal antibody of 50 μ g, mixes, and room temperature is placed 10 minutes.Every pipe adds 100 μ l 10%NaCl solution, mixes, and room temperature is placed 2 hours, and observing colloid gold change color, records the minimum PH (X) that collaurum hue preserving is constant.Adjustment PH is X-1.0, X-0.5, X, X+0.5, X+1.0, repeats above step, and the minimum pH value that collaurum hue preserving is constant is the optimum PH of mark.The results detailed in Table 3, shown in table 4.
Table 3: the selection of colloid gold label optimum PH (1)
Figure BDA0000121143310000071
Table 4: the selection of colloid gold label optimum PH (2)
Interpretation of result: can find out from table 3, table 4, when pH value is 9.0, the good stability of golden mark mouse-anti people IgA monoclonal antibody, therefore the optimum PH of selected colloid gold label is 9.0, adds the K of 0.1mol/L 2cO 3amount be 25 μ l/ml.
2.2 mouse-anti people IgA monoclonal antibodies are the selection of suitable label concentration
Get 8 1.5ml centrifuge tubes, add respectively 1ml collaurum, with the K of 0.1mol/L 2cO 3pH is adjusted into respectively to 9.0.Every pipe adds mouse-anti people IgA monoclonal antibody 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, mixes, and room temperature is placed 10 minutes.Every pipe adds 100 μ l 10%NaCl solution, mix, room temperature is placed 2 hours, observing colloid gold change color, record the minimum mouse-anti people IgA monoclonal antibody addition that collaurum hue preserving is constant, the minimum antibody addition that collaurum hue preserving is constant is the suitableeest label concentration of mouse-anti people IgA monoclonal antibody of mark.Shown in 5.
Table 5: mouse-anti people IgA monoclonal antibody is the selection of suitable label concentration
Figure BDA0000121143310000081
Interpretation of result: as can be seen from Table 5, at pH value, be under 9.0 conditions, when the mouse-anti people IgA monoclonal anti bulk concentration adding is 30 μ g/ml, the nondiscolouring of golden mark mouse-anti people IgA monoclonal antibody, therefore the selected the suitableeest label concentration of mouse-anti people IgA monoclonal antibody is 30 μ g/ml.
3, the selection of the coated concentration of detection line and colloid gold label mouse-anti people IgA dilution ratio
According to the above-mentioned golden marking process mark mouse-anti people IgA monoclonal antibody determining, its volume is concentrated into 1/10 of original volume, then with different dilution ratio dilution concentrates, with dilution, presses 55～60 μ l/m 2oil gidling mark pad, drying for standby.With the coated concentration of different detection lines, by the package amount of 0.15 μ l/mm, be coated with nitrocellulose filter (NC film) detection line.By gold mark pad and the pairing of line NC film, (the self-ordained one group of sample for kits for evaluation performance index of reference material Shi Ben enterprise of described enterprise, comprising: positive quality control product P1-P10 to detect enterprise's reference material, negative quality-control product N1-N10, minimum detectability L1, L2, L3), experimental program as
Table 6, experimental result is as shown in table 7, table 8.
Table 6: experimental design scheme
Figure BDA0000121143310000091
The positive inner quality control product of table 7:10 part and minimum detectability quality-control product testing result
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 L2
A1 ++ + ++ + ++ +++ ± ++ ++ +++ -
A2 ++ + ++ + + ++ ± ++ + ++ -
A3 ++ + ++ ± + ++ ± + + ++ -
A4 ++ + ++ - + ++ - + + ++ -
B1 +++ ++ +++ + ++ +++ ± ++ ++ +++ ±
B2 +++ ++ +++ + ++ +++ ± ++ ++ +++ ±
B3 +++ + ++ + ++ +++ ± ++ ++ ++ ±
B4 ++ + ++ ± + ++ ± + + ++ -
C1 +++ ++ +++ + ++ +++ + ++ ++ +++ +
C2 +++ ++ +++ + ++ +++ + ++ ++ +++ +
C3 +++ ++ +++ + ++ +++ + ++ ++ +++ +
C4 +++ ++ +++ + ++ +++ + ++ ++ +++ ±
D1 +++ +++ +++ + ++ +++ + +++ ++ +++ +
D2 +++ ++ +++ + ++ +++ + ++ ++ +++ +
D3 +++ ++ +++ + ++ +++ + ++ ++ +++ +
D4 +++ ++ +++ + ++ +++ + ++ ++ +++ ±
E1 +++ +++ +++ + ++ +++ ++ +++ ++ +++ +
E2 +++ +++ +++ + ++ +++ ++ +++ ++ +++ +
E3 +++ ++ +++ + ++ +++ + ++ ++ +++ +
E4 +++ ++ +++ + ++ +++ + ++ ++ +++ +
The negative inner quality control product of table 8:10 part testing result
N1 N2 N3 N4 N5 N6 N7 N8 N9 N10
A1 - - - - - - - - - -
A2 - - - - - - - - - -
A3 - - - - - - - - - -
A4 - - - - - - - - - -
B1 - - - - - - - - - -
B2 - - - - - - - - - -
B3 - - - - - - - - - -
B4 - - - - - - - - - -
C1 - - ± - - - - ± - -
C2 - - - - - - - ± - -
C3 - - - - - - - - - -
C4 - - - - - - - - - -
D1 - - ± - - - - + - -
D2 - - ± - - - - ± - -
D3 - - - - - - - ± - -
D4 - - - - - - - ± - -
E1 - - + - - - - + - -
E2 - - ± - - - - + - -
E3 ± ±
E4 ±
interpretation of result: can find out the highly sensitive of C3 combination by table 7, table 8 result, specificity is good, therefore the coated concentration of detection line is decided to be to 1.2mg/ml, draws NC film detection line by the package amount of 0.15 μ l/mm; Colloid gold label mouse-anti people IgA dilution ratio is decided to be to 1: 8, and collaurum bond dilution is added to 80% of original volume, and original volume calculates with collaurum volume.The preparation condition of gold mark pad is that the collaurum bond after dilution soaks and smears by 55～60 μ l/m2.
4, the selection of the coated concentration of nature controlling line
Sheep anti-mouse igg antibody is made into variable concentrations, with recombined EB virus Zta antigen 1 .2mg/ml, package amount with 0.15 μ l/mm is coated on respectively on the nature controlling line and detection line position of NC film, after dry, support the use with the gold mark pad preparing, with enterprises quality-control product, detect, the results detailed in Table 9.
Table 9: nature controlling line testing result
Figure BDA0000121143310000121
Interpretation of result: as can be seen from Table 9, when nature controlling line antibody concentration >=2mg/ml, reaction arrives maximal value, is 2mg/ml so select nature controlling line antibody concentration.
In sum, NC film finally coated condition be:
Package amount is 0.15 μ l/mm;
Detection line is coated with concentration: recombined EB virus Zta antigen is 1.2mg/ml;
Nature controlling line is coated with concentration: sheep anti-mouse igg antibody is 2.0mg/ml.
5, coating buffer formula determines
Preparing different coating buffers is 1.2mg/ml by recombined EB virus Zta antigen diluent to coated concentration, it is 2.0mg/ml that sheep anti-mouse igg antibody is diluted to coated concentration, on NC film, with 0.15 μ l/mm, draw T line and C line, after dry, mark pad assembling slitting with gold, application of sample end near T line is immersed in purified water, chromatography, observes C toe-in fruit as table 10.
The following number percent of table 10:(is weight percentage)
Figure BDA0000121143310000131
Interpretation of result: the C toe-in fruit that No. 3, coating buffer is best, therefore select 10mM/L PBS, PH6.82% sucrose is as the end formulation of coating buffer.
6, T line-spacing NC film lower end distance determines
The distance of T line-spacing NC film lower end is made as respectively to 5mm, 7mm and 10mm, carries out the line of T line, after being dried, pad and be assembled into slitting with gold mark, with enterprises quality-control product, detect, according to sensitivity, select optimum distance, result is as shown in table 11.
Table 11:
T line-spacing NC film lower edge distance 5mm 7mm 10mm
Sensitivity Cannot detect All meet All meet
Reaction background Clear More clear Darker
Interpretation of result: determine that T line-spacing NC film lower edge optimum distance is 7mm.
Because C line, T distance between centers of tracks affect not quite sensitivity, therefore be combination plastic clip Design Orientation 4mm attractive in appearance.
7, sample pad antigen working concentration determines
Epstein-Barr virus Zta antigen diluent is become to smear to glass fibre after different concentration and be made into sample pad, be assembled into after reagent strip with other assemblies, with inner quality control product, detect, according to testing result, determine the suitableeest antigen working concentration, result is as shown in table 12.
Table 12:
Extension rate P1 P2 P3 N1 N2
Former times +++ ++ + - -
2 times +++ ++ + - ±
4 times +++ ++ + ± ±
6 times +++ +++ ++ ± ±
8 times +++ +++ ++ ± ±
Interpretation of result: according to experimental result, select Epstein-Barr virus Zta antigen to smear on glass fibre with former times, make sample pad.
8, the reaction time determines
By above-mentioned confirmation parameter, prepare reagent strip, with observing different time after inner quality control product application of sample, determine the reaction time of reagent strip according to the reaction of reagent strip, testing result is as shown in table 13.
Table 13:
Reaction time 5min 10min 15min 20min 25min 30min 35min
P1 ++ +++ +++ +++ +++ +++ +++
P2 + ++ ++ ++ ++ ++ ++
P3 - ± + + + + +
N1 - - - - - - -
N2 - - - - - - ±
Interpretation of result: can find out from experimental result, between reaction 15-30 minute, detection line color tends towards stability substantially, and limit of identification is unchanged, therefore will the reaction time be decided to be 15-30 minute.Finally selecting the suitableeest reading result time is 20min, tries not at the above reading result of 30min.
9, process certification and optimization
According to aforementioned definite parameter, prepare reagent strip, by inner quality control product examine test agent bar performance, technique is carried out to preliminary identification, testing result is as shown in table 14.
Table 14:
P1 P2 P3 N1 N2
+++ ++ + - -
Interpretation of result: according to interpretation of result, yin and yang attribute result all meets, but the time that result occurs is oversize, just has very faint band to occur, therefore reply technique is optimized after 20min.
For Optimization Technology, attempt improving the concentration of sample pad Epstein-Barr virus Zta antigen, employing is two-layer by smearing on the sample pad pad of Epstein-Barr virus Zta antigen, improves the amount of Epstein-Barr virus Zta antigen, when pasting sample pad, two sample pad of cutting, wherein one than another wide 1～2mm, first paste narrow, then by wide alignment PVC plate bottom, cover ground floor sample pad and be adjacent to, assembling slitting
With internal reference product, detect, result is as shown in Table 15.
Table 15:
P1 P2 P3 N1 N2
+++ ++ + - -
Interpretation of result: yin and yang attribute result all meets, and it is clear to develop the color, and developing time is suitable.
The present invention is than prior art, and its beneficial effect is:
It is quick, easy, accurate and highly sensitive to detect, the whole running time only needs 20 minutes just can sentence read result, do not need supplementary instrument, can directly visual inspection result, applied widely, can single part of detection, be easy to popularize, this kit is for detection of Epstein-Barr virus Zta IgA antibody, can reduce the loss of Epstein-Barr virus with the antibody combined detection of eb nuclear antigen 1 (NA1) IgA, for examining the morning of Epstein-Barr virus, early controls, detects and control successful.
Kit yin and yang attribute coincidence rate of the present invention, accuracy, sensitivity etc. all meet quality standard requirement, constant product quality in the term of validity.Rheumatoid factor, hepatitis B virus antibody, syphilis helicoid antibody, human immune defect virus antibody, CMV antibody, herpes simplex virus antibody positive sample can not cause interference to this kit testing result.
[accompanying drawing explanation]
Fig. 1 is the structural drawing of reagent strip in kit of the present invention.
[embodiment]
Below in conjunction with specific embodiment, the present invention is described in further detail:
As shown in Figure 1, a kind of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit, comprise base plate 1, on base plate 1, be pasted with the reaction film 2 with detection line 3 and nature controlling line 4, at reaction film 2, near one end of detection line 3, superpose successively and be pasted with gold mark pad 5, sample pad 6, at reaction film 2, near one end stack of nature controlling line 4, be pasted with absorption pad 7, on described reaction film 2, detection line 3 has been coated with the recombined EB virus Zta antigen that concentration is 1.2mg/ml, nature controlling line 4 has been coated with the sheep anti-mouse igg antibody that concentration is 2.0mg/ml, the label concentration that has been coated with colloid gold label on described gold mark pad 5 is the mouse-anti people IgA monoclonal antibody of 30 μ g/ml.
Described base plate 1 is PVC plate, and described reaction film 2 is NC film.Embodiment 1: the preparation of kit
1. reagent preparation
(1) preparation of 0.05M PBS damping fluid (by 100ml amount):
Accurately take Na 2hPO 412H 2o 1.450g, NaH 2pO 42H 2o 0.148g, NaCl0.850g, add purified water 80.00mL, stirs and make, after fully dissolving, by purified water, to be settled to 100.00mL, mixes, and 2～8 ℃ save backup, the term of validity 14 days.
The preparation of (2) 1% chlorauric acid solutions (by 100ml amount):
Take 1g gold chloride, add purified water 80ml it is dissolved, then add purified water and be settled to 100ml, mix, with aluminium foil, wrap, 2～8 ℃ save backup, the term of validity 12 months.
(3) 0.1M solution of potassium carbonate preparation (by 20ml amount):
Measure the purified water of 16ml in reagent bottle, accurately take the K of 0.276g 2cO 3add in reagent bottle, add purified water and be settled to 20mL, make abundant dissolving, 2～8 ℃ save backup, the term of validity 14 days.
The preparation of (4) 10% bovine serum albumin solutions (by 20ml amount):
Measure the purified water of 16ml in reagent bottle, the bovine serum albumin(BSA) that accurately takes 2.0g adds in reagent bottle, treats to dissolve completely, adds purified water and is settled to 20.0mL, makes abundant dissolving, instant joining.
(5) preparation of collaurum bond dilution (by 100ml amount):
Take the trishydroxymethylaminomethane of 0.1211g in container, add purified water 80mL, after stirring makes fully to dissolve, then dripping appropriate hydrochloric acid adjusting pH value is 9.0, take again sucrose 2.00g, PVP 1.00g, bovine serum albumin(BSA) 2.00g adds in above-mentioned solution successively, fully, after stirring and dissolving, the Tween-20 that measures 0.5ml adds in reagent bottle, by purified water, be settled to 100.0mL again, measuring pH value is between 8.60～8.80.2～8 ℃ save backup, the term of validity 14 days.
(6) preparation of collaurum (by 100ml amount):
Measure 1% chlorauric acid solution 1mL, join in 99mL purified water, heat while stirring to boiling; After 5min, measure 1% citric acid three sodium solution of 1.4mL, rapid, disposable joining in container, continues heating and boils 5min.After being cooled to room temperature, add purified water to be settled to 100ml, 2～8 ℃ save backup, the term of validity 14 days.
(7) preparation of detection line coating buffer (by 10ml amount):
Measure recombined EB virus Zta antigen 1 2mg and add in container, with 0.05M PBS damping fluid, be settled to 10ml, fully mix 15min, the final concentration of recombined EB virus Zta antigen is 1.2mg/ml, instant joining.
(8) preparation of nature controlling line coating buffer (by 10ml amount):
Measure sheep anti-mouse igg antibody 20mg, with 0.05M PBS damping fluid, be settled to final volume 10mL, fully mix 15min, the final concentration of sheep anti-mouse igg antibody is 2.0mg/mL, instant joining.
(9) preparation of colloid gold label mouse-anti people IgA monoclonal antibody solution (by 80ml amount):
Measure 100ml collaurum in triangular flask, add 2.5ml 0.1M solution of potassium carbonate, mix, standing 10min, measure the mouse-anti people IgA monoclonal antibody of 3.0mg, under rapid stirring, dropwise join in triangular flask, the standing 40min of room temperature, measures 10% bovine serum albumin solution 10.0ml, under rapid stirring, dropwise join in triangular flask, the standing 15min of room temperature, in 10000rpm, 4 ℃ of centrifugal 30min, abandoning supernatant, add 80.0ml collaurum bond dilution to 80%, 2～8 ℃ of original volume to save backup, the term of validity 14 days.
2. the preparation of kit
The preparation of a, NC film: NC film is attached on PVC plate, with detection line coating buffer and nature controlling line coating buffer, with package amount 0.15 μ l/mm rule respectively on reaction film recombined EB virus Zta antigen detection line and sheep anti-mouse igg antibody nature controlling line, dryly under 37 ℃ of conditions after 3 hours, save backup;
The preparation of b, gold mark pad: press 55～60 μ l/m with colloid gold label mouse-anti people IgA monoclonal antibody solution 2immersion is smeared glass fibre element film and is made gold mark pad, under 37 ℃ of conditions, is dried and saves backup after 3 hours;
The processing of c, sample pad: Epstein-Barr virus Zta antigen is smeared to glass fibre with original content, then two glass fibre of smearing are superposeed be made into sample pad completely;
D, assembling cutting: line PVC plate is laid on work top, gold mark pad is covered to NC film 1～2mm stack sticks on NC film and PVC plate in detection line one end, sample pad being covered to gold mark pad 1～2mm stack sticks on gold mark pad and PVC plate again, finally absorption pad is covered to NC film 1～2mm stack sticks on NC film and PVC plate in nature controlling line one end, be about to reaction film, gold mark pad, absorption pad, sample pad is assembled into reaction plate, on sample pad and absorption pad, after pasting protective film, with cutting cutter, be cut into the wide test strips of 3.5mm, after being installed, packing in aluminium foil bag is generate a reagent box.
Embodiment 2: the selection of reaction time and application of sample amount, embodiment is shown in table 16 with result.
Table 16:
Figure BDA0000121143310000201
Interpretation of result: can find out from table 16 experimental result, application of sample amount how much can affects test strips and produce correct reaction result, application of sample amount is low there will be undetected, easy generation false positive when application of sample amount is high.By above experimental result, consider the stability of testing result and the convenience of operation, selecting application of sample amount is that 10 μ l serum add 100 μ l sample dilutions again, interpretation in 15-25 minute is application of sample interpretation condition.
Embodiment 3: pattern detection
(1) preparation of sample dilution (by 100ml amount):
Take the NaH of 0.06g 2pO 42H 2the Na of O and 0.58g 2hPO 412H 2o, adds purified water 80.0mL, stirs the NaCl that adds 0.85g after making fully to dissolve, and fully mixes, and is settled to 100mL, measures pH value and is 7.30～7.50,4～30 ℃ and saves backup, the term of validity 14 months.
(2) detect collection and the preservation of sample:
Serum sample is gathered by vein according to a conventional method.The sample of measuring in 5 days can be placed 4 ℃ of preservations.Sample is placed on 20 ℃ and at least can preserves 3 months.Sample is avoided haemolysis or multigelation.Muddy or have the sample of precipitation to enter centrifugal or filter after detect again after clarification.
(3) detect:
From original packing aluminium foil bag, take out reagent card, be placed on horizontal operation table top horizontal and carry out sample labeling, with sample injector, getting 10 μ l blood serum samples, being directly added in sample pad, drip 2 of sample dilutions, in 1525 minutes for there are two bands of aubergine nature controlling line and detection line in sentence read result again.
(4) Analysis of test results:
Positive: to occur two bands of aubergine nature controlling line and detection line.
Negative: only to occur an aubergine nature controlling line band.
Invalid: do not occur aubergine nature controlling line band, show incorrect operating process or reagent rotten damage, it is invalid to test, and after 25 minutes, assay is also invalid.

Claims (9)

1. the preparation method of an Epstein-Barr virus Zta IgA antibody colloidal gold detection kit, this kit comprises base plate (1), on described base plate (1), be pasted with the reaction film (2) with detection line (3) and nature controlling line (4), one end of the close detection line (3) of described reaction film (2) superposes successively and is pasted with gold mark pad (5), sample pad (6), described reaction film (2) is pasted with absorption pad (7) near one end stack of nature controlling line (4), the upper detection line (3) of described reaction film (2) has been coated with recombined EB virus Zta antigen, the upper nature controlling line (4) of described reaction film (2) has been coated with sheep anti-mouse igg antibody, on described gold mark pad (5), be coated with the mouse-anti people IgA monoclonal antibody of colloid gold label, the preparation method who it is characterized in that described kit comprises the following steps:
The preparation of a, reaction film: reaction film is attached on base plate, with damping fluid, by recombined EB virus Zta antigen diluent, to coated concentration, be 1.2mg/ml, it is 2.0mg/ml that sheep anti-mouse igg antibody is diluted to coated concentration, on reaction film, rule respectively recombined EB virus Zta antigen detection line and sheep anti-mouse igg antibody nature controlling line, save backup after dry;
The preparation of b, gold mark pad: smear glass fibre element film with the immersion of colloid gold label mouse-anti people IgA monoclonal antibody solution and make gold mark pad, save backup after being dried;
The processing of c, sample pad: two sheet glass fibers are superposeed be made into sample pad completely;
D, assembling cutting: reaction film, gold mark pad, absorption pad, sample pad are assembled into reaction plate, then are cut into reagent strip with cutting cutter, after being installed, packing in aluminium foil bag is generate a reagent box.
2. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1, is characterized in that
The coated concentration of described recombined EB virus Zta antigen is 1.2mg/ml;
The coated concentration of described sheep anti-mouse igg antibody is 2.0mg/ml;
The label concentration of described mouse-anti people IgA monoclonal antibody is 30 μ g/ml.
3. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1, is characterized in that described base plate (1) is polyvinyl chloride panel.
4. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1, is characterized in that described reaction film (2) is nitrocellulose filter.
5. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1, is characterized in that the damping fluid in described step a is the mixed solution of phosphate buffered solution, sucrose and Sodium azide.
6. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1, is characterized in that the label concentration of colloid gold label mouse-anti people IgA monoclonal antibody solution in described step b is 30 μ g/ml.
7. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1, is characterized in that in described step b that gold mark liquid is by 55～60 μ l/m 2evenly smear.
8. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1, is characterized in that the drying condition of described reaction film and gold mark pad is to be dried 3 hours under 37 ℃ of conditions.
9. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1, it is characterized in that the collaurum bond dilution in described step b is the potpourri of trishydroxymethylaminomethane, hydrochloric acid, sucrose, polyvinylpyrrolidone, bovine serum albumin(BSA), polyoxyethylene 20 sorbitan monolaurate, purified water, stand-by in 2～8 ℃ of preservations.
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