CN101149385A - Hepatitis C virus antibody quick diagnosis test paper and its preparation method - Google Patents
Hepatitis C virus antibody quick diagnosis test paper and its preparation method Download PDFInfo
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- CN101149385A CN101149385A CNA2007100600354A CN200710060035A CN101149385A CN 101149385 A CN101149385 A CN 101149385A CN A2007100600354 A CNA2007100600354 A CN A2007100600354A CN 200710060035 A CN200710060035 A CN 200710060035A CN 101149385 A CN101149385 A CN 101149385A
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Abstract
This invention discloses a kind of immunity chromatography test paper and its preparation method which can diagnose the type-C hepatitis virus fast, the said test paper includes the sampling tray, the gold size tray has labeled HCV mix antigen which links one end of the sampling tray closely, the pyroxylin film which links the other end of the gold size tray closely, and the suction sampling tray which links the other end of the pyroxylin film closely, the film encrusts the test (T) line and the quality control (C) line which are separate with each other, the sampling tray, the gold size tray, the film and the suction sampling tray are affixed in the plastic shoe plate to form the test paper, the said T line is the HCV mix antigen which is entrusted in the NC film, the said gold size tray has HCV recombination mix antigen, the said C line is the antibody of the HCV mix antigen entrusted in the film, it is used to test or clinical diagnosis of the HVC antigen, has the merits that the sensitive is high, the specificity is good, the operation is simple, the reaction is fast, it is fit to test in local and it is economical and useful.
Description
Technical field
The present invention relates to the diagnostic reagent of hepatitis C virus (HCV) antibody, particularly relate to and a kind ofly prepare a kind of hepatitis C virus antibody quick diagnosis test paper and preparation method thereof with dual-antigen sandwich method.
Background technology
Hepatitis C virus (HCV) is to be found at first from infected chimpanzee blood preparation by U.S. Choo etc. in 1989, mainly by blood, body fluid communication, accounts for 70% of post-transfusion hepatitis.According to World Health Organization's statistics, about 1.7 hundred million people's HCV infection are estimated in the whole world.Hepatitis C chronicity rate is 50%-85%, wherein has 20% can develop into liver fibrosis again, endangers very serious.China HCV antibody the carrier reach about 4,000 ten thousand.Up to the present hepatitis C had not both had effective vaccine, did not also have effective methods of treatment, and it is unique effective method that prevention is infected.HCV antibody is checked and hepatitis B surface antigen (HBsAg), human immunodeficiency virus (HIV) antibody and the inspection of syphilis (TP) antibody are four legal haematogenous examination projects of China, and meaning is very important.
Enzyme Linked Immunoadsorbent Assay (ELISA) method HCV antibody diagnosis is current HCV antibody inspection method the most commonly used, develops into for the 3rd generation, adopts Core, NS3, NS4 and the NS5 antigen hybrid packet of HCV to be detected by reaction plate.Criticize the data of examining according to the HCV antibody diagnosis ELISA reagent that country announces, domestic HCV antibody diagnosis ELISA kit was 6,000 7 million people's parts in 2006, with respect to the high infection rate of HCV, the population base of China and the improvement of sanitary condition, HCV antibody diagnosis demand also can improve in following 5-10 year by year.
But the ELISA diagnosis also has its weak point, detects the laboratory that needs specialty, professional's operation, and detection time is longer, and stability is relatively low.Another major issue is that sensitivity and specificity do not reach desirable requirement, main cause is that the ELISA reagent of HCV up to the present still uses indirect method rather than dual-antigen sandwich method to detect, because the Core epitope of HCV is rich in lysine and arginine, and can produce during horseradish peroxidase enzyme couplings such as (HRP) sterically hindered, reduce immunoreactive affinity, so can't detect with conventional double antigens sandwich ELISA method, limited dual-antigen sandwich method ELISA diagnostic reagent all needs antigen is carried out special processing, and up to the present still do not form real product [a kind of method that detects antibody of HCV, China Patent No.: 200510071806.0].
The immunochromatography colloidal gold method is novel immunological detection method, compares with ELISA, have easy to detect, quick, be suitable for on-the-spot detecting characteristics such as good stability.But see that totally the sensitivity of collaurum class reagent is lower slightly than ELISA.The research and development of HCV antibody diagnosis quick detection reagent have difficulty, compare ELISA reagent, and collaurum class reagent is more strict to the requirement of antigen, antibody, not only needs high-purity, more needs high stability and pair relationhip.The same with ELISA on the HCV antibody diagnosis, detection [the Chen M of reorganization hybrid antigen that needs multi-epitope to satisfy different subtype, different antibodies, Sallberg M, Sonnerborg A.et al.Limited humoral immunityin hepatitis C virus infection J.Gastroenterology.1999,116 (1): 135-43].
Up to the present,, continued to use the thinking of ELISA both at home and abroad, adopted indirect method to detect, in the sensitivity of product, generally all be lower than ELISA reagent though have a small amount of company to develop the HCV antibody diagnosing reagent kit with the immunochromatography colloidal gold technique.The HCV antibody fast diagnose test paper that also with the dual-antigen sandwich method is not the principle development occurs.Similar with the enzyme connection, lysine is the important molecule of albumen and collaurum combination equally, but except that lysine, the combination of collaurum and albumen also has two important molecule of tryptophane halfcystine, so be different from the enzyme connection of antigen and HRP, it is feasible using dual-antigen sandwich method to develop the HCV antibody rapid diagnosis reagent in theory, but the antigen of the pairing that need choose is grasped technology.There is the combination of twice specific antigen-antibody in dual-antigen sandwich method, can significantly improve the specificity and the sensitivity of product.The immunoglobulin G (IgG) that the while dual-antigen sandwich method not only can detect in the sample can detect immunoglobulin M in the sample (IgM), and indirect method can not, also can increase the sensitivity of detection, particularly for HCV, there is [Nikolaeva LI in IgM in the course of infection midium or long term, Blokhina NP, Tsurikova NN, et al.Virus-specific antibody titres in different phases of hepatitisC virus infection J.J Viral Hepat.2002,9 (6): 429-37.].
Summary of the invention
The object of the invention provides a kind of easy, quick, sensitivity and specificity height, be suitable for HCV antibody fast diagnose test paper that clinical diagnosis or examination use and preparation method thereof.
The present invention uses the double antigens sandwich immunochromatography technique to realize HCV antigen/antibody combination in the test sample is detected, the immune chromatography test paper of a kind of hepatitis C virus (HCV) antibody quick diagnosis is provided, comprise sample pad 1, closely be connected in the collaurum pad 2 that contains underlined HCV hybrid antigen of sample pad one end, with the close-connected NC of the other end of collaurum pad (cellulose nitrate) film 3 with closely be connected in the suction sample pad 4 of the NC film other end, the NC film is coated with detection T line (5) and the Quality Control C line 6 that is separated from each other, last sample pad, the collaurum pad, NC film and suction sample pad paste on the plastic support board 7 and form test paper, it is characterized in that, described T line is the HCV hybrid antigen that is coated on the NC film, described collaurum pad contains HCV reorganization hybrid antigen, and described C line is the antibody that is coated on the anti-HCV hybrid antigen on the NC film.
Described hepatitis C virus antibody quick diagnosis test paper, it is characterized in that, mark and bag are cAg (Core), non-structural protein (NS) 3, NS4 and the NS5 antigen of reorganization expression of HCV with HCV hybrid antigen composition, and antigen is genetic engineering single expression or two kinds and above amalgamation and expression albumen.The HCV hybrid antigen can be that Core, NS3 mix, or Core, NS3, NS4 mix, and Core, NS3, NS5 mix or four kinds of whole hybrid antigens.
Described hepatitis C virus antibody quick diagnosis test paper is characterized in that described upward sample pad is glass fibre membrane or nonwoven fabrics, inhales the sample pad and is made of absorbent filter.
Described hepatitis C virus antibody quick diagnosis test paper, it is characterized in that, the method for coating of antigen is: the solution that hybrid antigen is mixed with 1mg/ml with 0.01M pH 7.2 phosphate buffers (PBS), rule with the parameter of 1ul/cm in NC film bottom with spray film instrument, bag is wrapped by HCV antigen/antibody combination as the C line on NC film top simultaneously by the T line.After the line with the NC film drying room (temperature 20-25 ℃, humidity is less than 30%) dry 8-10 hour, standby.
Described hepatitis C virus antibody quick diagnosis test paper, it is characterized in that, the method of hybrid antigen mark colloid gold particle is: prepare the colloidal gold solution that diameter is 30-50nm with gold chloride-trisodium citrate reduction method, get 100ml collaurum liquid after preparation is finished and be placed in the beaker, use 0.2MK
2CO
3Transfer to pH8.0, press the 100ml colloidal gold solution and add 1mg HCV reorganization hybrid antigen, stirring at room 2 hours adds protective agent, sealing 20min, and centrifugal 30 minutes of 12000r/m abandons supernatant, redissolves to 100ml with the collaurum working fluid, presses 1ml solution shop 22cm
2Ratio be layered on equably on glass fibre membrane or the nonwoven fabrics, put drying room (temperature 20-25 ℃, humidity is less than 30%) drier 2-4 hour, make the collaurum pad, standby.
Described hepatitis C virus antibody quick diagnosis test paper, it is characterized in that, the assembly method of test strips is: in hothouse (temperature 20-25 ℃, humidity is less than 30%), get the plastic support board plate, paste at the middle part that the NC film that wraps quilt is placed on the plastic support board plate, pastes at NC film T line one side overlap joint collaurum pad (take collaurum pad 1/3rd), pastes sample pad (cost sample pad 1/10th) at collaurum pad opposite side overlap joint; Inhale sample pad (take inhale sample pad 1/10th) at NC film C line one side overlap joint; Paste one deck marking film topmost, will post plastic plate with cutter at last and be cut into 3mm or the wide test strips of 4mm.The test strips that cuts can reinstall in the plastic clip, forms HCV antibody diagnosing reagent card.
Described hepatitis C virus antibody quick diagnosis test paper, it is characterized in that, detection method is: with tested serum or blood plasma balance to the greenhouse, test strips or reagent card are kept flat, on last sample pad, add the 50-100ul test sample, the sample dissolution collaurum and on the NC film chromatography, the appearance situation of Direct observation C, T line in 30 minutes with the naked eye then, and judge testing result.
The invention has the beneficial effects as follows: utilization HCV hybrid antigen bag quilt, adopt dual-antigen sandwich method to prepare HCV antibody quick diagnosis immune chromatography test paper.Adopt dual-antigen sandwich method to make the performance of test paper improve a grade, be the HCV antibody diagnosis product of a new generation than indirect method.The sensitivity of fast diagnosis reagent and specificity are met or exceeded need the level of the ELISA reagent that specialized laboratory and professional test; The envelope antigen that contains Core and NS3 simultaneously can detect all HCV in theory and infect, if add the sensitivity that NS4 and NS5 can further improve detection again.This test paper is used for the examination or the clinical diagnosis of HCV antibody, have susceptibility height, high specificity, easy and simple to handle, reaction fast, be fit to on-the-spot the detection and advantage such as economical and practical.
Description of drawings:
Fig. 1 HCV antibody fast diagnose test paper structural representation.
The reference numeral explanation:
1: go up the sample pad; 2: the collaurum pad;
The 3:NC film; 4: inhale the sample pad
5: detect (T) line; 6: Quality Control (C) line;
7: plastic support board
Embodiment
The preparation of embodiment 1:HCV antibody fast diagnose test paper
1 main material
1.1 mixing recombinant antigen: be the CORE of HCV, the NS3 fused antigen: U.S. Biotech AtlanticInc (BBI). with Military Medical Science Institute's product, be respectively applied for the bag quilt and the mark of test paper; HCV antigen/antibody combination: BBI company product; Gold chloride: Sigma company product, cellulose nitrate (NC) film: Millipore company product; Bovine serum albumin(BSA) (BSA), polyglycol (PEG) 20000, caseinhydrolysate: Sigma product.Other common agents is analytical reagent.
1.2 HCV antibody country reference material (collaurum class): Nat'l Pharmaceutical ﹠ Biological Products Control Institute's inspection development.Comprise 20 parts of positive serums, be numbered P1-P20,20 parts of negative serums are numbered N1-N20, and 4 parts of sensitivity serum are numbered L1_1, L1_2, and L2_1, L2_2,1 part of precision serum is numbered J.
2 methods
2.1 the colloid gold label gold chloride-trisodium citrate reduction method of HCV mixing recombinant antigen prepares the colloidal gold solution that diameter is 40nm, gets three parts of collaurums after preparation is finished, with 0.2MK2CO3 solution being transferred to pH7.0, pH8.0 and pH9.0 respectively.Then solution is placed on the magnetic stirring apparatus and slowly stir, slowly be added drop-wise to albumen in the colloidal gold solution recombinant antigen by the every adding of 100ml solution 0.5mg, 1mg, 1.5mg, continue to stir 2 hours, be added dropwise to final concentration again and be 0.1% PEG2000 and 1% BSA and seal 30min, it is centrifugal with 12000r/m that mark finishes the back, abandon supernatant, precipitation is pressed original volume and is redissolved to the collaurum dilution (borate buffer solution of different proportionings, pH8.0, contain BSA, caseinhydrolysate, sucrose and surfactant) in.Then the mark colloidal gold solution is pressed 1ml solution shop 22cm
2The ratio application of sample on nonwoven fabrics, at temperature 20-25 ℃, relative humidity is made the collaurum pad, drying for standby dry 2-4 hour of＜30% drying room.
2.2 the bag of HCV mixing recombinant antigen is diluted to 0.5mg/ml, 1mg/ml, 2mg/ml with 0.01M pH7.2 PBS respectively with the HCV recombinant antigen, then with spray film instrument with recombinant antigen on the NC film by the 1ul/cm bag quilt of ruling, on the NC film, wrap simultaneously by HCV antigen/antibody combination, be used for the Quality Control of product, it is, standby after bag is done with NC film dry 8-10 hour at drying room.
2.3 HCV antibody fast diagnose test paper be assembled in dry indoor temperature 20-25 ℃, relative humidity＜30%) gets plastic support board, paste at the middle part that the NC film that wraps quilt is placed on plastic support board, paste at NC film T line one side overlap joint collaurum pad (take collaurum pad 1/3rd), paste sample pad (cost sample pad 1/10th) at collaurum pad opposite side overlap joint; Inhale sample pad (take inhale sample pad 1/10th) at NC film C line one side overlap joint; Paste one deck marking film topmost, will post plastic plate with cutter at last and be cut into 3mm or the wide test strips of 4mm.The test strips that cuts can reinstall in the plastic clip, forms HCV antibody diagnosing reagent card.
2.4 detect principle and method with tested serum or blood plasma balance to the greenhouse, the test strips or the reagent card that prepare are kept flat, during detection, on last sample pad, add the 50-100ul test sample, if contain HCV antigen/antibody combination in the sample, then with sample pad on the collaurum combination of mark HCV antigen, form compound, and be diffused on the NC film further chromatography, when running into the pairing antigen that is coated on T line place on the NC film, compound then again with the bag by the combination of HCV antigen, be trapped in bag and located, when captive colloidal gold composite reaches some, then form a macroscopic T line; If do not contain specific antibody in the serum, then can not form immune complex, also can not form the T line, the C line is as the quality control standard of reagent, and positive and negative sample all can occur when detecting.The appearance situation of Direct observation C, T line in 15,20,30 and 45 minutes with the naked eye, and judgement testing result.
2.5 after the reaction system technology assessment method development preparation of product sample, be quality-control product with HCV antibody country's reference material (collaurum class), detect, determine best product reaction system and process route.
3 results
According to the testing result of sample, the optimum mark pH value of having determined test paper is 8.0; The optimum mark amount of reorganization hybrid antigen is the every 100ml colloidal gold solution of 1mg; Best collaurum dilution is the 50mM borate buffer solution, and pH8.0 contains 0.5%BSA, 1% caseinhydrolysate, 2% sucrose, and 0.1%Tween 20; Best hybrid antigen bag is 1mg/ml by concentration.The optimal decision time of testing result is to judge in 30 minutes.
The performance evaluation of embodiment 2:HCV antibody fast diagnose test paper
1 main material
1.1 HCV antibody diagnosis test paper: the preparation method sees embodiment one;
1.2 HCV antibody country reference material (collaurum class): Nat'l Pharmaceutical ﹠ Biological Products Control Institute's inspection development.
Specify and see embodiment one;
1.3 contain chaff interference serum: comprise respectively 50 parts of common interference object height blood fat, haemolysis, jaundice serum, contain respectively 50 parts of relevant infectious disease HAV, HBV, HIV, TP and HP antibody positive serum, contain each 20 parts of different anti-coagulants heparin, EDTA and sodium citrate blood plasma, collect checking and preservation by our company in the relevant hospital of Efficiency in Buildings in Tianjin Area, above serum or blood plasma detect through two or more ELISA and are HCV negative antibody serum.
1.4 clinical serum is collected in the relevant hospital of Efficiency in Buildings in Tianjin Area by company, totally 1180 parts, is outpatient service collection check serum.
1.5 HCV antibody diagnosing reagent kit (ELISA): Shanghai Kehua Bio-engineering Co., Ltd, Beijing Tso Biological Pharmaceutical Co and Pu Sheng (Tianjin) Science and Technology Ltd..
2 methods
2.1 sample detection method: see embodiment one, in 30 minutes, judge testing result.
Take out HCV antibody country reference material (collaurum class) serum dish 2.2 HCV antibody country reference material (collaurum class) detects, detect with this test paper after equilibrating to room temperature.
2.3 HCV antibody country reference material (collaurum class) part strong positive blood-serum P 9, P13, P14 are got in the sensitivity for analysis assessment, weak positive serum P2, P4 and L2_2, make doubling dilution respectively, compare detection with this test paper and ELISA reagent, until can not detecting positive findings.
2.4 analyzing the specificity assessment detects the chaff interference sample that contains that company preserves with test paper.
2.5 stability test is preserved this test paper at ambient temperature, takes out the part test paper at 1,3,6,9 month, tests with HCV antibody country's reference material (collaurum class); This test paper is placed under 37 ℃, the 50 ℃ conditions,, test with HCV antibody country's reference material (collaurum class) every 7 days taking-up portion test paper.
2.6 the clinical sample test is collected clinical serum by enterprise from relevant hospital, carry out control test with a kind of ELISA reagent and this test paper, if run into the inconsistent sample of testing result, detect with other 2 kinds of ELISA reagent again, if two kinds or with ELISA reagent positive, result of determination is positive, and two kinds or above ELISA reagent are negative, and result of determination is negative.
3 results
3.1 HCV antibody country's reference material (collaurum class) detects national reference material (collaurum class) serum dish is tested, national Specification is passed through in result and expection (table 1) in full accord.
This test paper of table 1 is to the test result of HCV antibody country's reference material (collaurum class)
| Interventions Requested | Quantity | Standard code | Testing result |
| HCV (+) HCV (-) accuracy sample sensitivity sample | 20 20 1 L1_1 L2_1 L1_2 L2_2 | ≥19/20 ≥19/20 + + + +/- +/- | 20/20 20/20 + + + + + |
3.2 the sensitivity for analysis assessment is with behind the national reference material part positive serum doubling dilution, comparison and detection result, the sensitivity for analysis of P2, P4 and L2_2 sample copy test paper surpasses the ELISA reagent sensitivity in 6 duplicate samples, P9, P13, P14, sample analysis sensitivity basically identical, the sensitivity of this test paper of analysis-by-synthesis meets or exceeds the quality of ELISA reagent.
Show that this test paper detects all negative to common interference object height blood fat, haemolysis, jaundice serum 3.3 analyze specificity assessment testing result, all negative to containing relevant infectious disease HAV, HBV, HIV, TP and the detection of HP antibody positive serum, detect all negative to containing different anti-coagulants heparin, EDTA and sodium citrate blood plasma.Explanation does not have nonspecific reaction to above material.
3.4 this test paper of stability analysis is preserved at ambient temperature, tests with HCV country's reference material (collaurum class); 1, detecting in 3,6,9 months all can be by test, there was no significant difference as a result, and test for more time is also underway.Accelerating the failure property test under 37 ℃ of conditions detected for 6 weeks altogether, and accelerating the failure property test under 50 ℃ of conditions detected for 3 weeks altogether, all can be by test, and the test paper performance does not have remarkable decline.According to the test findings that accelerates the failure, the stability of test paper at ambient temperature should be more than 18 months.
3.5 clinical sample test: detected 1180 parts of the clinical samples that enterprise collects altogether, ELISA reagent detects 32 parts of positive, and this detection paper goes out 35 parts of positive, and concrete outcome is as follows:
This test paper of table 2 is to clinical sample HCV detection of antibodies result
| ELISA | This test paper | Sum | |
| + | - | ||
| +-sum | 32 3 35 | 0 1145 1145 | 32 1148 1180 |
Sensitivity=32/32=100%; Specificity=1145/1148=99.7%
Claims (7)
1. hepatitis C virus antibody quick diagnosis test paper, comprise sample pad (1), closely be connected in the collaurum pad (2) that contains underlined HCV hybrid antigen of sample pad one end, with the close-connected NC of the other end of collaurum pad (cellulose nitrate) film (3) with closely be connected in the suction sample pad (4) of the NC film other end, the NC film is coated with detection (T) line (5) and Quality Control (C) line (6) that is separated from each other, last sample pad, the collaurum pad, NC film and suction sample pad paste plastic support board (7) and go up the formation test paper, it is characterized in that, described T line is the HCV hybrid antigen that is coated on the NC film, described collaurum pad contains HCV reorganization hybrid antigen, and described C line is the antibody that is coated on the anti-HCV hybrid antigen on the NC film.
2. hepatitis C virus antibody quick diagnosis test paper according to claim 1, it is characterized in that, mark and bag are cAg (Core), non-structural protein (NS) 3, NS4 and the NS5 antigen of reorganization expression of HCV with HCV hybrid antigen composition, and antigen is genetic engineering single expression or two kinds and above amalgamation and expression albumen; The HCV hybrid antigen can be that Core, NS3 mix, or Core, NS3, NS4 mix, and Core, NS3, NS5 mix or four kinds of whole hybrid antigens.
3. hepatitis C virus antibody quick diagnosis test paper according to claim 1 is characterized in that described upward sample pad is glass fibre membrane or nonwoven fabrics, inhales the sample pad and is made of absorbent filter.
4. according to claim 1 and 2 described hepatitis C virus antibody quick diagnosis test papers, it is characterized in that, the method for coating of antigen is: the solution that hybrid antigen is mixed with 1mg/ml with 0.01M pH 7.2 phosphate buffers (PBS), rule with the parameter of 1ul/cm in NC film bottom with spray film instrument, bag is wrapped by HCV antigen/antibody combination as the C line on NC film top simultaneously by the T line.After the line with the NC film at drying room, temperature 20-25 ℃, humidity is less than 30%, and is dry 8-10 hour, standby.
5. according to claim 1 and 2 described hepatitis C virus antibody quick diagnosis test papers, it is characterized in that, the method of hybrid antigen mark colloid gold particle is: prepare the colloidal gold solution that diameter is 30-50nm with gold chloride-trisodium citrate reduction method, get 100ml collaurum liquid after preparation is finished and be placed in the beaker, use 0.2M K
2CO
3Transfer to pH8.0, press the 100ml colloidal gold solution and add 1mg HCV reorganization hybrid antigen, stirring at room 2 hours adds protective agent, sealing 20min, and centrifugal 30 minutes of 12000r/m abandons supernatant, redissolves to 100ml with the collaurum working fluid, presses 1ml solution shop 22cm
2Ratio be layered on equably on glass fibre membrane or the nonwoven fabrics, put drying room again, temperature 20-25 ℃, humidity is less than 30%, dry 2-4 hour, make the collaurum pad, standby.
6. according to claim 1 and 3 described hepatitis C virus antibody quick diagnosis test papers, it is characterized in that, the assembly method of test paper is: in hothouse, temperature 20-25 ℃, humidity is got the plastic support board plate less than 30%, and paste at the middle part that the NC film that wraps quilt is placed on the plastic support board plate, paste at NC film T line one side overlap joint collaurum pad (take collaurum pad 1/3rd), paste sample pad (cost sample pad 1/10th) at collaurum pad opposite side overlap joint; Inhale sample pad (take inhale sample pad 1/10th) at NC film C line one side overlap joint; Paste one deck marking film topmost, will post plastic plate with cutter at last and be cut into 3mm or the wide test strips of 4mm, the test strips that cuts can reinstall in the plastic clip, forms HCV antibody diagnosing reagent card.
7. hepatitis C virus antibody quick diagnosis test paper according to claim 1, it is characterized in that, detection method is: with tested serum or blood plasma balance to the greenhouse, test strips or reagent card are kept flat, on last sample pad, add the 50-100ul test sample, the sample dissolution collaurum and on the NC film chromatography, the appearance situation of Direct observation C, T line in 30 minutes with the naked eye then, and judge testing result.
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