CN102539768A - 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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CN102539768A
CN102539768A CN2011104258682A CN201110425868A CN102539768A CN 102539768 A CN102539768 A CN 102539768A CN 2011104258682 A CN2011104258682 A CN 2011104258682A CN 201110425868 A CN201110425868 A CN 201110425868A CN 102539768 A CN102539768 A CN 102539768A
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epstein
antibody
reaction film
barr virus
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CN102539768B (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 the medical biotechnology field.
[background technology]
(Epstein-barr virus is a kind of gamma herpes viruses of having a liking for human B lymphocyte EBV) to Epstein-Barr virus, mainly invades bone-marrow-derived lymphocyte, and people's bone-marrow-derived lymphocyte, epithelial cell (comprising Blasius' duct, pharynx and uterine neck etc.) and gland cell had affinity.Classify as first kind carcinogenic substance to EBV in International Union Against Cancer's annual meeting of the World Health Organization (WHO) in 1997.Zta albumen is the early stage activity factor of Epstein-Barr virus of being expressed by immediate early gene BZLF1; It is the product that is converted into solubility infection phase when early stage immediately by latent infection; Be that Epstein-Barr virus gets into the essential active element of cracking replication status; Change dissolving in virus into from latent infection and 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 canceration process more and more received researcher's concern in recent years immediately.Present discovers that ebv infection is relevant with multiple human tumor such as nasopharyngeal carcinoma, lymph cancer etc.
The existing method that detects Epstein-Barr virus mainly contains:
The Real-time quantitative PCR method
This method is mainly used in the quantitative of epstein barr virus dna.Detect epstein barr virus dna in nasopharyngeal carcinoma (NPC) patient's the blood plasma with fluorescence quantifying PCR method, recall rate reaches 96%, and confirms that in research subsequently epstein barr virus dna also descends rapidly in patient's blood plasma of tumor regression after the radiotherapy.Adopt the Real-time quantitative PCR technique, utilize a pair of Auele Specific Primer and specificity fluorescent probe,, realize detection by quantitative Epstein-Barr virus in conjunction with round pcr and detection technique of fluorescence.But diagnose medium sensitivity low in early days, specificity is similar with FA IgA.
Indirect immunofluorescence/immunoenzyme
Traditionally, detecting the Epstein-Barr virus specific antibody with the IIF that contains the virolymphocyte smear, abroad also using at present, mainly is to detect VCA IgA antibody and FA IgA antibody, needs to use fluorescent microscope.China promoted the use of immunoenzyme (EIA method) and has detected VCA IgA antibody and FA IgA antibody from the eighties, yet; Mass discrepancy was bigger between this method existence was criticized; Shortcomings such as visual inspection is short of objectivity, and index is thicker are progressively replaced by ELISA at present.
ELISA (ELISA)
ELISA (ELISA) detects each antibody-like in serum or the blood plasma, adopts the antigen of the Epstein-Barr virus replicative cycle different phase expression of genetic recombination.Domesticly use to such an extent that be Epstein-Barr virus solubility infection bone-marrow-derived lymphocyte the most widely, the eb early antigen FA that is produced during the propagation beginning, and synthetic Epstein-Barr virus shell antigen VCA of propagation later stage.But ELISA method complicated operation, detection time is long, need dilute testing sample, operation such as incubation, separation, washing and colour developing.
[summary of the invention]
The objective of the invention is to overcome the deficiency of prior art; A kind of Epstein-Barr virus ZtaIgA antibody colloidal gold detection kit is provided; Select for use be the early stage activity factor Zta of Epstein-Barr virus specific antigen be recombined EB virus zta antigen as the detection line envelope antigen, be applicable to the Epstein-Barr virus Zta IgA antibody in the qualitative detection human serum.This kit both can be used for the auxiliary diagnosis of the relevant nasopharyngeal carcinoma of ebv infection, 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 for 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; Be pasted with the reaction film that has detection line (T line) and nature controlling line (C line) on the said base plate, said reaction film superposes successively near an end of detection line and is pasted with gold mark pad, sample pad; Said reaction film adds near an endlap of nature controlling line and is pasted with absorption pad; It is characterized in that: detection line has encapsulated recombined EB virus Zta antigen on the said reaction film, and nature controlling line has encapsulated sheep anti-mouse igg antibody on the said reaction film, has encapsulated the mouse-anti people IgA monoclonal antibody of colloid gold label on the said gold mark pad.
Aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit is characterized in that
The concentration that encapsulates of said recombined EB virus Zta antigen is 1.2mg/ml; Said recombined EB virus Zta antigen is colourless transparent liquid; Concentration is measured with SDS-PAGE (SDS PAGA) greater than 2mg/ml, and applied sample amount is only deposited a band under 10 μ l conditions;
The concentration that encapsulates of said sheep anti-mouse igg antibody is 2.0mg/ml;
The label concentration of said mouse-anti people IgA monoclonal antibody is 30 μ g/ml, and said mouse-anti people IgA monoclonal antibody is a colourless transparent liquid, and concentration is measured with SDS PAGA greater than 2mg/ml, and applied sample amount is deposited two bands under 10 μ l conditions.
Aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit is characterized in that said base plate is polyvinyl chloride plate (a PVC plate).
Aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit is characterized in that said reaction film is nitrocellulose filter (a NC film).
A kind of preparation method of aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit is characterized in that may further comprise the steps:
The preparation of a, reaction film: reaction film is attached on the base plate; Use damping fluid that recombined EB virus Zta antigen diluent is 1.2mg/ml to encapsulating concentration; Sheep anti-mouse igg antibody is diluted to encapsulate concentration be 2.0mg/ml; On reaction film, rule respectively recombined EB virus Zta Detection of antigen line and sheep anti-mouse igg antibody nature controlling line, dry back is preserved subsequent use;
The preparation of b, gold mark pad: smear the plain film of spun glass with colloid gold label mouse-anti people IgA monoclonal antibody solution soaking and process gold mark pad, dry back is preserved subsequent use;
The processing of c, sample pad: Epstein-Barr virus Zta antigen is smeared to spun glass with original content, and two spun glass of smearing are superposeed fully is made into sample pad again;
D, assembling cutting: reaction film, gold mark pad, absorption pad, sample pad being assembled into reaction plate, being cut into reagent strip with cutting cutter again, after being installed, is the generate a reagent box in the aluminium foil bag of packing into.
The preparation method of aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit is characterized in that damping fluid among the said step a is that pH value is the mixed solution of 6.8 PBS (PBS), sucrose and Sodium azide (NaN3).
The preparation method of aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit is characterized in that the label concentration of colloid gold label mouse-anti people IgA monoclonal anti liquid solution among the said step b is 30 μ g/ml.
The preparation method of aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit is characterized in that gold mark liquid is by 55～60 μ l/m among the said step b 2Evenly smear.
The preparation method of aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit, the drying condition that it is characterized in that said reaction film and gold mark pad is under 37 ℃ of conditions dry 3 hours.
The preparation method of aforesaid Epstein-Barr virus Zta IgA antibody colloidal gold detection kit; It is characterized in that the collaurum bond dilution among the said step b is the potpourri of trishydroxymethylaminomethane, hydrochloric acid, sucrose, polyvinylpyrrolidone (PVP), bovine serum albumin(BSA), polyoxyethylene 20 sorbitan monolaurate (polysorbas20), purified water, for use in 2～8 ℃ of preservations.
The confirming as follows of manufacturing condition among the present invention:
1, the selection of colloid gold particle
Prepare collaurum according to gold chloride under the condition of boiling and trisodium citrate generation redox reaction.Change and control the grain size of collaurum through the additional proportion of regulating gold chloride and trisodium citrate.During preparation, in the 100ml distilled water, add 1% chlorauric acid solution 1ml and 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, natural cooling is for use.With the colloid gold label mouse-anti people IgA monoclonal antibody of different sizes, (said enterprises quality-control product is 6 parts of quality-control products that enterprise oneself manufactures, and comprises P1, P2, P3, N1, N2, N3 with the 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 result sees table 1 for details, shown in the table 2.
Selection experiment---the selection (1) of colloid gold particle size of table 1:1% trisodium citrate consumption
(following number percent is weight percentage)
Table 2: inner quality-control product testing result---the selection (2) of colloid gold particle size
Figure BDA0000121143310000062
Interpretation of result: table 1, table 2 can be found out; No. 2 the colloidal gold solution color is purplish red, bright, no muddiness and floating thing, and its susceptibility of the colloidal gold antibody behind the mark, specificity are best; So select the process conditions of preparation collaurum to be: in the 100ml distilled water, to add 1% chlorauric acid solution 1ml and boil; Under stirring condition, add 1% citric acid three sodium solution of 1.4ml, continued to boil 5 minutes, natural cooling is for use.
2, the optimization of colloid gold label mouse-anti people IgA monoclonal anti body technology:
Collaurum forms stable mouse-anti people IgA monoclonal antibody gold label because electrostatic interaction forms electronegative hydrophobic sol solution, and collaurum is electronegative under the weak base environment, can form firm combining with the positive charge group of mouse-anti people IgA monoclonal antibody.
2.1 the selection of colloid gold label optimum PH
Get 8 1.5ml centrifuge tubes, add the 1ml collaurum respectively, with the HCl of 5mol/L and the K of 0.1mol/L 2CO 3PH is adjusted into 3,4,5,6,7,8,9,10 respectively.Every pipe adds the mouse-anti people IgA monoclonal antibody of 50 μ g, mixing, and room temperature was placed 10 minutes.Every pipe adds 100 μ l 10%NaCl solution, mixing, and room temperature was placed 2 hours, observing colloid gold change color, the minimum PH (X) that record collaurum color remains unchanged.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 the collaurum color remains unchanged is the optimum PH of mark.The result sees for details shown in table 3, the table 4.
Table 3: the selection of colloid gold label optimum PH (1)
Table 4: the selection of colloid gold label optimum PH (2)
Figure BDA0000121143310000072
Interpretation of result: can find out that from table 3, table 4 when pH value was 9.0, the good stability of golden mark mouse-anti people IgA monoclonal antibody so the optimum PH of selected colloid gold label is 9.0, promptly added the K of 0.1mol/L 2CO 3Amount be 25 μ l/ml.
2.2 mouse-anti people IgA monoclonal antibody is the selection of right label concentration
Get 8 1.5ml centrifuge tubes, add the 1ml collaurum respectively, with the K of 0.1mol/L 2CO 3PH is adjusted into 9.0 respectively.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, mixing, and room temperature was placed 10 minutes.Every pipe adds 100 μ l 10%NaCl solution; Mixing; Room temperature was placed 2 hours; Observing colloid gold change color, the minimum mouse-anti people IgA monoclonal antibody addition that record collaurum color remains unchanged, the minimum antibody addition that the collaurum color remains unchanged is the righttest label concentration of mouse-anti people IgA monoclonal antibody of mark.The result sees for details shown in the table 5.
Table 5: mouse-anti people IgA monoclonal antibody is the selection of right label concentration
Figure BDA0000121143310000081
Interpretation of result: can find out from table 5; At pH value is under 9.0 conditions; When the mouse-anti people IgA monoclonal anti bulk concentration that adds was 30 μ g/ml, the nondiscolouring of golden mark mouse-anti people IgA monoclonal antibody was so the selected the righttest label concentration of mouse-anti people IgA monoclonal antibody is 30 μ g/ml.
3, detection line encapsulates the selection of concentration and colloid gold label mouse-anti people IgA dilution ratio
According to above-mentioned definite good golden marking process mark mouse-anti people IgA monoclonal antibody, its volume is concentrated into 1/10 of original volume, with different dilution ratio dilution concentrates, press 55～60 μ l/m again with dilution 2Oil gidling mark pad, drying for standby.Encapsulate concentration with different detection lines and encapsulate nitrocellulose filter (NC film) detection line by the package amount of 0.15 μ l/mm.With gold mark pad and the pairing of line NC film, (said enterprise is the self-ordained one group of sample that is used for the kits for evaluation performance index of this enterprise with reference to article, comprising: positive quality control product P1-P10 with reference to article to detect enterprise; Negative quality-control product N1-N10, minimum detectability L1, L2; L3), experimental program as
Table 6, experimental result is shown in table 7, table 8.
Table 6: experimental design scheme
Figure BDA0000121143310000091
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 +++ ++ +++ + ++ +++ + ++ ++ +++ +
[0060]The negative inner quality-control product testing result of table 8:10 part
? 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 ? ? ? ? ? ? ? ± ? ?
[0062]Interpretation of result: can find out the highly sensitive of C3 combination through table 7, table 8 result, specificity is good, so the concentration that encapsulates of detection line is decided to be 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 1: 8, and promptly collaurum bond dilution is added to 80% of original volume, and original volume is with the collaurum volume calculation.The preparation condition of gold mark pad is smeared for the collaurum bond after diluting soaks by 55～60 μ l/m2.
4, nature controlling line encapsulates the selection of concentration
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; Dry back and the supporting use of gold mark pad for preparing are detected with the enterprises quality-control product, and the result sees table 9 for details.
Table 9: nature controlling line testing result
Figure BDA0000121143310000121
Interpretation of result: can find out that from table 9 when nature controlling line AC >=2mg/ml, reaction arrives maximal value, is 2mg/ml so select the nature controlling line AC.
In sum, the NC film finally the condition of encapsulating be:
Package amount is 0.15 μ l/mm;
Detection line encapsulates concentration: recombined EB virus Zta antigen is 1.2mg/ml;
Nature controlling line encapsulates concentration: sheep anti-mouse igg antibody is 2.0mg/ml.
5, coating buffer determination of formula
Preparing different coating buffers is 1.2mg/ml with recombined EB virus Zta antigen diluent to encapsulating concentration; Sheep anti-mouse igg antibody is diluted to encapsulate concentration be 2.0mg/ml; On the NC film, draw T line and C line with 0.15 μ l/mm, dry back and gold mark pad assembling slitting will be immersed near the application of sample end of T line in the purified water; Chromatography is observed C toe-in fruit like table 10.
Table 10: (following number percent is weight percentage)
Figure BDA0000121143310000131
Interpretation of result: the C toe-in fruit that No. 3, coating buffer is best, so select 10mM/L PBS, PH6.82% sucrose is as the end formulation of coating buffer.
6, T line-spacing NC film lower end distance confirms
The distance of T line-spacing NC film lower end is made as 5mm, 7mm and 10mm respectively, carries out the line of T line, dry back is assembled into slitting with gold mark pad, detects with the enterprises quality-control product, selects optimum distance according to sensitivity, and the result is as shown in table 11.
Table 11:
T line-spacing NC film lower edge distance 5mm 7mm 10mm
Sensitivity Can't detect All meet All meet
The reaction background Clear More clear Darker
Interpretation of result: confirm that T line-spacing NC film lower edge optimum distance is 7mm.
Because C line, T distance between centers of tracks are to the sensitivity influence not quite, so be combination plastic clip Design Orientation 4mm attractive in appearance.
7, sample pad antigen working concentration confirms
Epstein-Barr virus Zta antigen diluent is become to smear to spun glass after the different concentration and be made into sample pad, dress up reagent strip with other component groups after, detect with inner quality-control product, confirm the righttest antigen working concentration according to testing result, the 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 for use Epstein-Barr virus Zta antigen to smear on the spun glass with former times, process sample pad.
8, the reaction time confirms
Prepare reagent strip by above-mentioned affirmation parameter, with observing different time behind the inner quality-control product application of sample, confirm 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 that from experimental result between reaction 15-30 minute, the detection line color tends towards stability basically, therefore the limit of identification no change will be decided to be 15-30 minute the reaction time.Select at last the rightlyyest to read as a result that the time is 20min, try not more than 30min, to read the result.
9, process certification and optimization
Prepare reagent strip according to aforementioned definite parameter, with inner quality-control product detectable bar performance, technology is carried out 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 is the result all meet, but the time that the result occurs is oversize, just has very faint band to occur behind the 20min, so reply technology is optimized.
Be to optimize technology, attempt improving the concentration of sample pad Epstein-Barr virus Zta antigen, employing will be smeared on the sample pad pad of Epstein-Barr virus Zta antigen two-layer; Improve the amount of Epstein-Barr virus Zta antigen, when pasting sample pad, two sample pad of cutting; Wherein one than another wide 1～2mm, paste earlier narrow, again will wide alignment PVC plate bottom, covering ground floor sample pad is adjacent to; The assembling slitting
Detect with the internal reference article, the result is as shown in Tble 15.
Table 15:
P1 P2 P3 N1 N2
+++ ++ + - -
Interpretation of result: yin and yang attribute is the result all meet, 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:
Detect quick, easy, accurate and highly sensitive, but the whole operation time only need 20 minutes just 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 used to detect Epstein-Barr virus Zta IgA antibody, with the loss of eb nuclear antigen 1 (NA1) IgA antibody combined detection can reduction Epstein-Barr virus, early controls, detects and to control effect obvious for examining the morning of Epstein-Barr virus.
Kit yin and yang attribute coincidence rate of the present invention, accuracy, sensitivity etc. all meet the 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, cytomegalovirus antibody, herpes simplex virus antibody positive sample can not cause interference to this kit testing result.
[description of drawings]
Fig. 1 is the structural drawing of reagent strip in the kit of the present invention.
[embodiment]
Below in conjunction with specific embodiment the present invention is done further explain:
As shown in Figure 1; A kind of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit comprises base plate 1, on base plate 1, is pasted with the reaction film 2 that has detection line 3 and nature controlling line 4; Superpose successively near an end of detection line 3 at reaction film 2 and to be pasted with gold mark pad 5; Sample pad 6 adds near an endlap of nature controlling line 4 at reaction film 2 and to be pasted with absorption pad 7, and to have encapsulated concentration be the recombined EB virus Zta antigen of 1.2mg/ml to detection line 3 on the said reaction film 2; It is the sheep anti-mouse igg antibody of 2.0mg/ml that nature controlling line 4 has encapsulated concentration, and the label concentration that has encapsulated colloid gold label on the said gold mark pad 5 is the mouse-anti people IgA monoclonal antibody of 30 μ g/ml.
Said base plate 1 is the PVC plate, and said reaction film 2 is the NC film.Embodiment 1: the preparation of kit
1. reagent preparation
(1) preparation of 0.05M PBS damping fluid (by the 100ml amount):
Accurately take by weighing Na 2HPO 412H 2O 1.450g, NaH 2PO 42H 2O 0.148g, NaCl0.850g add purified water 80.00mL, after stirring makes abundant dissolving, are settled to 100.00mL with purified water, mix, and 2～8 ℃ of preservations are subsequent use, the term of validity 14 days.
The preparation of (2) 1% chlorauric acid solutions (by the 100ml amount):
Take by weighing the 1g gold chloride, add purified water 80ml and make its dissolving, add purified water again and be settled to 100ml, mix, wrap with aluminium foil, 2～8 ℃ of preservations are subsequent use, the term of validity 12 months.
(3) 0.1M solution of potassium carbonate preparation (by the 20ml amount):
The purified water of measuring 16ml accurately takes by weighing the K of 0.276g in reagent bottle 2CO 3Add in the reagent bottle, add purified water and be settled to 20mL, make abundant dissolving, 2～8 ℃ of preservations are subsequent use, the term of validity 14 days.
The preparation of (4) 10% bovine serum albumin solutions (by the 20ml amount):
The purified water of measuring 16ml is in reagent bottle, and the bovine serum albumin(BSA) that accurately takes by weighing 2.0g adds in the reagent bottle, treats to dissolve fully, adds purified water and is settled to 20.0mL, makes abundant dissolving, instant joining.
(5) preparation of collaurum bond dilution (by the 100ml amount):
The trishydroxymethylaminomethane that takes by weighing 0.1211g adds purified water 80mL in container, after stirring makes abundant dissolving; Dripping appropriate hydrochloric acid adjusting pH value then is 9.0; Take by weighing sucrose 2.00g, PVP 1.00g, bovine serum albumin(BSA) 2.00g again and add successively in the above-mentioned solution, fully after the stirring and dissolving, the Tween-20 of measuring 0.5ml adds in the reagent bottle; Be settled to 100.0mL with purified water again, measuring pH value is between 8.60～8.80.2～8 ℃ of preservations are subsequent use, the term of validity 14 days.
(6) preparation of collaurum (by the 100ml amount):
Measure 1% chlorauric acid solution 1mL, join in the 99mL purified water, heat while stirring to boiling; Behind the 5min, measure 1% citric acid three sodium solution of 1.4mL, rapid, disposable joining in the container continued heated and boiled 5min.The adding purified water is settled to 100ml after being cooled to room temperature, and 2～8 ℃ of preservations are subsequent use, the term of validity 14 days.
(7) preparation of detection line coating buffer (by the 10ml amount):
Measure recombined EB virus Zta antigen 1 2mg and add in the container, be settled to 10ml with 0.05M PBS damping fluid, abundant mixing 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 the 10ml amount):
Measure sheep anti-mouse igg antibody 20mg, be settled to final volume 10mL with 0.05M PBS damping fluid, abundant mixing 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 anti liquid solution (by the 80ml amount):
Measure the 100ml collaurum in triangular flask, add 2.5ml 0.1M solution of potassium carbonate, mixing leaves standstill 10min; Measure the mouse-anti people IgA monoclonal antibody of 3.0mg, dropwise join in the triangular flask under stirring fast, room temperature leaves standstill 40min; Measure 10% bovine serum albumin solution 10.0ml, dropwise join in the triangular flask under stirring fast, room temperature leaves standstill 15min; In 10000rpm, 4 ℃ of centrifugal 30min, abandoning supernatant; It is subsequent use to 80%, 2～8 ℃ of preservations of original volume to add 80.0ml collaurum bond dilution, the term of validity 14 days.
2. the preparation of kit
The preparation of a, NC film: the NC film is attached on the PVC plate; With package amount 0.15 μ l/mm on reaction film, rule respectively recombined EB virus Zta Detection of antigen line and sheep anti-mouse igg antibody nature controlling line, after under 37 ℃ of conditions dry 3 hours, preserve subsequent use with detection line coating buffer and nature controlling line coating buffer;
The preparation of b, gold mark pad: press 55～60 μ l/m with colloid gold label mouse-anti people IgA monoclonal anti liquid solution 2Immersion is smeared the plain film of spun glass and is processed gold mark pad, after under 37 ℃ of conditions dry 3 hours, preserves subsequent use;
The processing of c, sample pad: Epstein-Barr virus Zta antigen is smeared to spun glass with original content, and two spun glass of smearing are superposeed fully is made into sample pad again;
D, assembling cutting: the PVC plate of will ruling is tiled on the work top; Gold mark pad is covered NC film 1～2mm stack at detection line one end to stick on NC film and the PVC plate; Again sample pad being covered gold mark pad 1～2mm stack sticks on gold mark pad and the PVC plate; At last absorption pad is covered NC film 1～2mm stack at nature controlling line one end and stick on NC film and the PVC plate, soon reaction film, gold mark pad, absorption pad, sample pad are assembled into reaction plate, behind pasting protective film on sample pad and the absorption pad, are cut into the wide test strips of 3.5mm with cutting cutter; After being installed, in the aluminium foil bag of packing into the generate a reagent box.
Embodiment 2: the selection of reaction time and application of sample amount, embodiment is shown in table 16 with the result.
Table 16:
Figure BDA0000121143310000201
Interpretation of result: can find out that from table 16 experimental result the test strips that how much can influence of application of sample amount produces correct reaction result, the application of sample amount is low omission can to occur, and the application of sample amount is easy to generate false positive when high.Through above experimental result, consider the stability of testing result and the convenience of operation, selecting the application of sample amount is that 10 μ l serum add 100 μ l sample dilutions again, interpretation in 15-25 minute is an application of sample interpretation condition.
Embodiment 3: pattern detection
(1) preparation of sample dilution (by the 100ml amount):
Take by weighing the NaH of 0.06g 2PO 42H 2The Na of O and 0.58g 2HPO 412H 2O adds purified water 80.0mL, stirs to make abundant dissolving back add the NaCl of 0.85g, and fully mixing is settled to 100mL, and measuring pH value is that 7.30～7.50,4～30 ℃ of preservations are subsequent use, the term of validity 14 months.
(2) collection and the preservation of detection sample:
Serum sample is pressed conventional method by the vein collection.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 deposition will advance centrifugal or filter the back clarification after detect again.
(3) detect:
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 blood serum samples, directly be added on the sample pad with sample injector; Drip 2 of sample dilutions again, sentence read result is for two bands of aubergine nature controlling line and detection line occurring in 1525 minutes.
(4) testing result analysis:
Positive: as two bands of aubergine nature controlling line and detection line to occur.
Negative: as an aubergine nature controlling line band only to occur.
Invalid: aubergine nature controlling line band do not occur, show the rotten damage of incorrect operating process or reagent, it is invalid to test, and assay is also invalid after 25 minutes.

Claims (10)

1. Epstein-Barr virus Zta IgA antibody colloidal gold detection kit; Comprise base plate (1); Be pasted with the reaction film (2) that has detection line (3) and nature controlling line (4) on the said base plate (1), said reaction film (2) superposes successively near an end of detection line (3) and is pasted with gold mark pad (5), sample pad (6); Said reaction film (2) adds near an endlap of nature controlling line (4) and is pasted with absorption pad (7); It is characterized in that: said reaction film (2) is gone up detection line (3) and has been encapsulated recombined EB virus Zta antigen, and said reaction film (2) is gone up nature controlling line (4) and encapsulated sheep anti-mouse igg antibody, has encapsulated the mouse-anti people IgA monoclonal antibody of colloid gold label on the said gold mark pad (5).
2. Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1 is characterized in that
The concentration that encapsulates of said recombined EB virus Zta antigen is 1.2mg/ml;
The concentration that encapsulates of said sheep anti-mouse igg antibody is 2.0mg/ml;
The label concentration of said mouse-anti people IgA monoclonal antibody is 30 μ g/ml.
3. Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1 is characterized in that said base plate (1) is the polyvinyl chloride plate.
4. Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 1 is characterized in that said reaction film (2) is a nitrocellulose filter.
5. the preparation method of the described Epstein-Barr virus Zta of claim 1 an IgA antibody colloidal gold detection kit is characterized in that may further comprise the steps:
The preparation of a, reaction film: reaction film is attached on the base plate; Use damping fluid that recombined EB virus Zta antigen diluent is 1.2mg/ml to encapsulating concentration; Sheep anti-mouse igg antibody is diluted to encapsulate concentration be 2.0mg/ml; On reaction film, rule respectively recombined EB virus Zta Detection of antigen line and sheep anti-mouse igg antibody nature controlling line, dry back is preserved subsequent use;
The preparation of b, gold mark pad: smear the plain film of spun glass with colloid gold label mouse-anti people IgA monoclonal antibody solution soaking and process gold mark pad, dry back is preserved subsequent use;
The processing of c, sample pad: Epstein-Barr virus Zta antigen is smeared to spun glass with original content, and two spun glass of smearing are superposeed fully is made into sample pad again;
D, assembling cutting: reaction film, gold mark pad, absorption pad, sample pad being assembled into reaction plate, being cut into reagent strip with cutting cutter again, after being installed, is the generate a reagent box in the aluminium foil bag of packing into.
6. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 5 is characterized in that the damping fluid among the said step a is the mixed solution of PBS, sucrose and Sodium azide.
7. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 5 is characterized in that the label concentration of colloid gold label mouse-anti people IgA monoclonal anti liquid solution among the said step b is 30 μ g/ml.
8. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 5 is characterized in that gold mark liquid is by 55～60 μ l/m among the said step b 2Evenly smear.
9. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 5, the drying condition that it is characterized in that said reaction film and gold mark pad is under 37 ℃ of conditions dry 3 hours.
10. the preparation method of Epstein-Barr virus Zta IgA antibody colloidal gold detection kit according to claim 5; It is characterized in that the collaurum bond dilution among the said step b is the potpourri of trishydroxymethylaminomethane, hydrochloric acid, sucrose, polyvinylpyrrolidone, bovine serum albumin(BSA), polyoxyethylene 20 sorbitan monolaurate, purified water, for use in 2～8 ℃ of preservations.
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