CN1300581C - SARS virus antibody detecting method, rapid diagnosis kit and preparation method - Google Patents
SARS virus antibody detecting method, rapid diagnosis kit and preparation method Download PDFInfo
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- CN1300581C CN1300581C CNB031344194A CN03134419A CN1300581C CN 1300581 C CN1300581 C CN 1300581C CN B031344194 A CNB031344194 A CN B031344194A CN 03134419 A CN03134419 A CN 03134419A CN 1300581 C CN1300581 C CN 1300581C
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The present invention relates to a detection method, a rapid diagnostic reagent kit and a preparation method for SARS virus antibodies. The detection method comprises the steps: (1) loading an antihuman Ig antibody marked by colloidal gold on a carrier; coating a detection line formed from SARS coronavirus variant antigens and a control line formed from anti-rat IgG antibodies on a detection carrier connected with the carrier of the antibody marked by colloidal gold; (2) dripping human serum onto the carrier of the antibody marked by colloidal gold. If SARS coronavirus variant antibodies are contained in blood serum, then the detection line and the control line formed on the detection carrier are positive, else the two lines are negative. The diagnostic reagent kit comprises a detection board marked by SARS coronavirus variant antibody gold; the detection board comprises a water absorption layer at the sample adding end, a detection layer and a water absorption layer at the water absorption end, wherein gold marked antihuman Ig antibody layer is arranged between the detection layer and the water absorption layer at the water absorption end. The detection line formed from SARS coronavirus variant antigens and the control line formed from anti-rat IgG antibodies are coated on the detection layer. The method of the present invention is a one-step detection method, and has the advantages of high sensitivity, strong specificity, high speed, simplicity, convenience and no need of special facility.
Description
Technical field
The present invention relates to a kind of method that is used to diagnose, detect sars coronavirus antibody, and the sars coronavirus antibody gold label quick diagnosis reagent kit and its preparation method that are used for this method.
Background technology
SARS (Severe Acute Respiratory Syndrome) (SARS) is a kind of human sudden infectious disease that betides world many countries in the recent period, is the human a kind of precipitate disaster that is faced.The control of SARS has become current most important work, and vast medical personnel is fighting bravely the front of resisting SARS, and the countries in the world scientist is studying the prevention and treatment method of SARS, comprises diagnostic method, medicine and prevention vaccine.May 1 published two parts of SARS virus genome series of studies papers by U.S.'s " science " magazine, and this is to pass through peer review SARS virus genome research result in the first batch.Two scientific research groups of the U.S. and Canadian Studies composition of personnel confirm that in result of study SARS virus is a kind of brand-new coronavirus.China Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C cooperates with Beijing Institute of Gene Science, Chinese Academy of Sciences, has also finished April 15 whole genome sequence of sars coronavirus is measured, and genome sequence is classified 29,727 nucleotide as.The mensuration of sars coronavirus genome sequence is for anti-SARS drug screening, vaccine development with set up fast diagnosis reagent and be significant.
To accomplish discovery early, early treatment, early isolate SARS at present.So require both to have detected patient's SARS antigen, detect patient's SARS antibody again.Antibody test also will detect the special IgM of its virus except IgG, early stage or acute stage can detect because IgM is in morbidity, have in addition can detect in the latter stage of hiding.
SARS current diagnosis method mainly contains three kinds of (1) viral nucleic acid detection-pcr amplification methods, early detection, (2) antibody test: ELISA and immunofluorescence technique.(3) SARS virus infection cell culture model.It is antigen that above immunoassay technology all adopts the SARS virus infection cell, and there is the preparation difficulty in the viral antigen of cultivation, and certain danger is arranged, and can not produce in a large number, and poor specificity can occur.
Summary of the invention
First purpose of the present invention is for a kind of sars coronavirus variant detection of antibodies method is provided, and this method is the single stage method detection method, highly sensitive, high specificity; And speed is fast, easy and simple to handle, need not Special Equipment, is applicable to multiple occasions such as clinical detection, epidemiology survey and place quarantine.
Second purpose of the present invention is for a kind of sars coronavirus variant antibody gold label quick diagnosis reagent kit is provided, antibody test provides a kind of easy and direct single stage method detection box to this kit at the sars coronavirus variant, utilize this kit to detect highly sensitive, the high specificity of sars coronavirus variant antibody, and speed is fast, easy and simple to handle, need not specialized facilities.
The 3rd purpose of the present invention is for a kind of preparation method of sars coronavirus variant antibody gold label quick diagnosis reagent kit is provided, and this method is easy, good stability, good reproducibility.
First purpose of the present invention can realize by following measure:
A kind of sars coronavirus variant antibody detection method comprises the steps: that (1) comprises that with the anti-people Ig antibody of colloid gold label IgG and IgM are stated from glass fibre or the nonwoven portion cloth carrier; And with detection carrier that the colloid gold label antibody carrier links to each other on the detection line that formed by sars coronavirus variant antigen of bag and the control line that forms by anti-mouse IgG antibody; (2) get human serum and drip on the carrier of colloid gold label antibody, sars coronavirus variant antibody then forms detection line on the detection carrier, the control line two-wire is positive as containing in the serum, otherwise negative.
Second purpose of the present invention can realize by following measure:
Establish sars coronavirus variant antibody gold label check-out console in a kind of sars coronavirus variant antibody gold label quick diagnosis reagent kit.
Described sars coronavirus variant antibody gold label check-out console is to be provided with application of sample end water accepting layer, detection layers and suction side water accepting layer on liner plate, is provided with the anti-people Ig antibody layer of gold mark between detection layers and application of sample end water accepting layer; Be coated with detection line that forms by sars coronavirus variant antigen and the control line that forms by anti-mouse IgG antibody on the detection layers.
The 3rd purpose of the present invention can realize by following measure:
A kind of preparation method of sars coronavirus variant antibody gold label quick diagnosis reagent kit mainly comprises the golden labeling antibody layer of preparation and the control line of detection layers and the bag quilt of detection line, combination S ARS coronavirus variant antibody gold label test strip and check-out console on liner plate then.
The preparation method of described golden labeling antibody layer comprises the steps: the preparation of i collaurum, in concentration is that 1.3-2%, the concentration that adds its volume in the gold chloride of 0.004-0.012% is the trisodium citrate of 1-3%, boiled 10-20 minute, and can obtain the colloidal gold solution of 15-50 nanometer; (1gG, IgM) antibody are transferred pH8.0-8.4 with the 0.2mol solution of potassium carbonate to the anti-people Ig of ii colloid gold label in colloidal gold solution, add anti-people Ig antibody by 4-12mg/100ml, after the stirring, press 0.1-0.6g/100ml again and add animal blood serum albumen in solution, 4 ℃ left standstill 2-4 hour; Iii through 2000 rev/mins of centrifugal 10-15 minutes, removes above-mentioned mixed solution to sediment, gets supernatant; Iv with supernatant through 10000 rev/mins of centrifugal 60-80 minutes centrifugal sediment; V with sediment by 4-10ml/100ml be dissolved in 0.02M, the Ph7.4Tris-HCI damping fluid gets golden labeling antibody solution; The animal blood serum albumen and the 0.01-0.06% Sodium azide that contain 0.2-0.6% in this damping fluid; Till vi immerses golden labeling antibody solution glass fibre or nonwoven fabrics to liquid and begins to ooze out and form golden labeling antibody layer.
Described colloid gold label antibody be anti-people Ig (1gG, gM).
Described detection layers is to be provided with genetic engineering sars coronavirus variant antigen detection line that constitutes and the control line that has sheep, rabbit anti-mouse igg antibody to form on cellulose membrane.Its preparation method is to be the fixing agent that adds 1-3% in the antigen of 0.8-2.4mg/ml or the antibody-solutions in concentration, utilize Membrane jetter that antigen or antibody are sprayed on the cellulose membrane again, 37 ℃ of dryings behind the spray film, the PBS that contains 10% calf serum again with 0.01mlpH7.0, sealed 30 minutes down at 37 ℃, 0.01ml the PBS rinsing of pH7.0,37 ℃ of dryings.
The present invention has following advantage compared to existing technology:
The method of 1, detection of the present invention, diagnosing SARS corona virus variant antibody is a single stage method, has higher specificity and sensitivity, and easy and simple to handle, need not specialized facilities.
Have higher specificity and sensitivity when 2, diagnostic kit of the present invention detects sars coronavirus variant antibody, testing process operation news speed, quick and easy is applicable to multiple occasions such as clinical examination, epidemiology survey and place quarantine,
3, easy, the good stability of the preparation method of diagnostic kit of the present invention, good reproducibility.
Description of drawings
Fig. 1 is the surface structure synoptic diagram of check-out console of the present invention
1-check-out console 2-well 3-viewport
Fig. 2 is the inner structure synoptic diagram of check-out console of the present invention
4-application of sample end water accepting layer 5-gold labeling antibody layer 6-control line 7-detection line
8-detection layers 9-suction side water accepting layer 10-liner plate
Concrete embodiment
The present invention also will be described in further detail in conjunction with the embodiments
Embodiment one:
A kind of sars coronavirus variant antibody detection method comprises the steps: that (1) comprises that with the anti-people Ig antibody of colloid gold label IgG and IgM are stated from glass fibre or the nonwoven portion cloth carrier; And with detection carrier that the colloid gold label antibody carrier links to each other on the detection line that formed by genetic engineering sars coronavirus variant antigen of bag and the control line that forms by anti-mouse IgG antibody; (2) get human serum and drip on the carrier of colloid gold label antibody, as serum contain sars coronavirus variant antibody then detect on the carrier form detection line, the control line two-wire is positive, and is otherwise negative.
With reference to Fig. 1, establish sars coronavirus variant antibody gold label check-out console 1 in a kind of sars coronavirus variant antibody gold label quick diagnosis reagent kit.
With reference to Fig. 2, described sars coronavirus variant antibody gold label check-out console 1 is to be provided with application of sample end water accepting layer 4, detection layers 8 and suction side water accepting layer 9 on liner plate 10, is provided with anti-people's 12 antibody layers 5 of gold mark between detection layers and suction side water accepting layer 9; On detection layers 8, be coated with detection line 7 and control line 6.Wherein application of sample end water accepting layer 4 and suction side water accepting layer 9 are made by multilayer filter paper: detection layers 8 is cellulose membrane, and this cellulose membrane can be nitrocellulose filter, cellulose acetate membrane; Gold labeling antibody layer is that glass fibre or nonwoven fabrics leaching colloid gold label antibody are made.
A kind of preparation method of sars coronavirus variant antibody gold label quick diagnosis reagent kit mainly comprises preparation golden labeling antibody layer (5) and the control line (6) of detection layers (8) and the bag quilt of detection line (7), goes up combination S ARS coronavirus variant antibody gold label test strip and check-out console (1) at liner plate (10) then.Paste application of sample end absorbent paper layer 4 and suction side water accepting layer 9 respectively at the two ends of plastic base plate 10; Section is pasted the cellulose rete 8 of coated antibody therein, at the handing-over position of application of sample end absorbent paper layer 4 in cellulose rete 8, folder pastes the glass fibre that contains golden labeling antibody 5, and 4/5 part of glass fibre is in the middle of application of sample end absorbent paper layer 4, and 1/5 part is on cellulose rete 8.By 4 mm wides, 8 centimeter length specification slittings, be assembled in the plastic casing more then.Well 2 is over against the application of sample end absorbent paper layer 4 of test-strips on the lid, and viewport 3 is over against the detection layers 8 of cellulose membrane.
The preparation method of described golden labeling antibody layer 5 comprises the steps: the preparation of i collaurum, getting the 100mg gold chloride is dissolved in the 1000ml tri-distilled water, adding 15ml, concentration are 1% stubborn lemon acid trisodium, boil 15 minutes, obtain the colloidal gold solution of 15-50 nanometer after the cooling; The anti-people Ig of ii colloid gold label antibody is got the 100ml colloidal gold solution, transfers pH8.4 with the 0.2mol solution of potassium carbonate, adds the anti-human IgG monoclonal antibody of 6mg, stirs 20 minutes, adds the 240mg bovine serum albumin(BSA) again, continues to stir 5 minutes, and 4 ℃ left standstill 2-4 hour; Iii with above-mentioned mixed solution through 2000 rev/mins centrifugal 10 minutes, remove sediment, supernatant; Iv with supernatant through 10000 rev/mins of centrifugal 60 minutes centrifugal sediments; V is dissolved in 0.02M Ph7.4Tris-HCl damping fluid with sediment by 4m10ml and gets golden labeling antibody solution; Contain 0.25% bovine serum albumin(BSA) and 0.02% Sodium azide in this damping fluid; Till vi immerses golden labeling antibody solution glass fibre or nonwoven fabrics to liquid and begins to ooze out, 37 ℃ following 2 hours dry and form golden labeling antibody layer 5.
Described colloid gold label antibody is anti-human IgG monoclonal antibody.
Described detection layers 8 is to be provided with genetic engineering sars coronavirus variant antigen detection line that constitutes and the control line 6 that has sheep, rabbit anti-mouse igg antibody to form on cellulose membrane.Its preparation method is to get genetic engineering sars coronavirus variant antigen, and accent concentration is 1mg/ml, adds 2% formaldehyde, sprays detection line 7 in the cellulose membrane stage casing with Membrane jetter; Getting sheep anti-mouse igg antibody accent concentration again is 2mg/ml, the formaldehyde of adding 2% uses Membrane jetter in the cellulose membrane stage casing, apart from detection line 0.5cm place, spray control line 6, by 20ul/10cm spray film amount, 37 ℃ of dryings of spray film, 2 hours are set, the PBS that contains 10% calf serum again with 0.01mpH7.0, sealed 30 minutes the PBS rinsing of 0.01mlpH7.0,37 ℃ of dryings down at 37 ℃.
Embodiment two:
Sars coronavirus variant antibody detection method and golden label fast diagnosis reagent box thereof are identical with example one.
The antibody that the preparation method of this kit removes the preparation colloid gold label is that anti-people IgM monoclonal anti is external, and other conditions and example are together.
Claims (5)
1, a kind of sars coronavirus variant antibody gold label quick diagnosis reagent kit is characterized in that establishing in this kit sars coronavirus variant antibody gold label check-out console (1); Described sars coronavirus variant antibody gold label check-out console (1) is to be provided with application of sample end water accepting layer (4), detection layers (8) and suction side water accepting layer (9) on liner plate (10), is provided with gold mark anti-people Ig antibody layer (5) between detection layers (8) and application of sample end water accepting layer (4); On detection layers (8), be coated with detection line (7) that forms by sars coronavirus variant antigen and the control line (6) that forms by anti-mouse IgG antibody.
2, the preparation method of the described sars coronavirus variant of a kind of claim 1 antibody gold label quick diagnosis reagent kit, it is characterized in that mainly comprising preparation golden labeling antibody layer (5) and the control line (6) of detection layers (8) and the bag quilt of detection line (7), go up combination S ARS coronavirus variant antibody gold label test strip and check-out console (1) at liner plate (10) then; The preparation method of described golden labeling antibody layer (5) comprises the steps: the preparation of i collaurum, in concentration is that 1.3-2%, the concentration that adds its volume in the gold chloride of 0.004-0.012% is the trisodium citrate of 1-3%, boiled 10-20 minute, and can obtain the colloidal gold solution of 15-50 nanometer; The anti-people Ig of ii colloid gold label, i.e. IgG, IgM antibody adds anti-people Ig antibody by 4-12mg/100ml in colloidal gold solution, press 0.1-0.6g/100ml adding animal blood serum albumen after stirring again in solution; Iii through the centrifugal sediment that goes, gets supernatant with above-mentioned mixed liquor; Iv is with the centrifugal sediment that gets of supernatant; V with sediment by 4-10ml/100ml be dissolved in 0.02M, the Ph7.4Tris-HCl damping fluid obtains golden labeling antibody solution; The animal blood serum albumen and the 0.01-0.06% Sodium azide that contain 0.2-0.6% in this damping fluid; Till vi immerses golden labeling antibody solution glass fibre or nonwoven fabrics to liquid and begins to ooze out and form golden labeling antibody layer (5).
3, the preparation method of sars coronavirus variant antibody gold label quick diagnosis reagent kit as claimed in claim 2 is characterized in that described colloid gold label antibody is anti-people Ig, i.e. IgG, gM.
4, the preparation method of sars coronavirus variant antibody gold label quick diagnosis reagent kit as claimed in claim 2 is characterized in that described detection layers (8) is to be provided with the detection line (7) of sars coronavirus variant antigen formation and the control line (6) that is formed by sheep, rabbit anti-mouse igg antibody on cellulose membrane.
5, the preparation method of sars coronavirus variant antibody gold label quick diagnosis reagent kit as claimed in claim 4 is characterized in that described sars coronavirus variant antigen is to adopt genetic engineering to be prepared from.
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| CNB031344194A CN1300581C (en) | 2003-07-22 | 2003-07-22 | SARS virus antibody detecting method, rapid diagnosis kit and preparation method |
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| CNB031344194A CN1300581C (en) | 2003-07-22 | 2003-07-22 | SARS virus antibody detecting method, rapid diagnosis kit and preparation method |
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| CN1300581C true CN1300581C (en) | 2007-02-14 |
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Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2023502928A (en) * | 2019-11-15 | 2023-01-26 | アボット・ラピッド・ダイアグノスティクス・インターナショナル・アンリミテッド・カンパニー | Device for digital readout of lateral flow assays |
| CN111413500A (en) * | 2020-02-12 | 2020-07-14 | 谱尼测试集团北京检验认证科学研究院有限公司 | Novel coronavirus 2019-nCoV antigen colloidal gold detection technology |
| CN111187354B (en) * | 2020-02-20 | 2020-11-27 | 北京新创生物工程有限公司 | Novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit |
| CN111190012A (en) * | 2020-02-22 | 2020-05-22 | 南京申基医药科技有限公司 | Rare earth up-conversion fluorescent nano test strip for novel coronavirus detection and preparation method thereof |
| CN111443196A (en) * | 2020-02-27 | 2020-07-24 | 深圳市绿诗源生物技术有限公司 | New coronavirus antibody detection card and manufacturing method thereof |
| CN113341136A (en) * | 2020-03-01 | 2021-09-03 | 上海雄图生物科技有限公司 | Detection method for rapidly screening novel coronavirus |
| CN111378018B (en) * | 2020-03-28 | 2021-01-19 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Test strip for detecting novel coronavirus antibody and preparation method and application thereof |
| CN111398603B (en) * | 2020-03-28 | 2021-01-15 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Test strip for detecting novel coronavirus antibody, preparation method and application thereof |
| CN111781349B (en) * | 2020-04-03 | 2022-04-22 | 中国科学院苏州纳米技术与纳米仿生研究所 | Diagnostic kit capable of predicting prognosis of COVID-19 patient |
| CN111693713A (en) * | 2020-04-09 | 2020-09-22 | 泰州市人民医院 | Detection card for detecting novel coronavirus IgM by colloidal gold method and preparation method thereof |
| CN111551708A (en) * | 2020-05-13 | 2020-08-18 | 常州领航量子生物医疗科技有限公司 | Specific antibody detection marker, preparation method, application and kit thereof |
| CN113740538A (en) * | 2020-05-27 | 2021-12-03 | 复旦大学 | Method and product for detecting SARS-CoV-2 novel coronavirus IgM/IgG antibody |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0481020A1 (en) * | 1989-07-06 | 1992-04-22 | Secr Health Brit | Silver enhanced gold-labelled immuno assay method. |
| CN2442272Y (en) * | 2000-09-18 | 2001-08-08 | 王冰 | Test biological chip for TORCH causative agent |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0481020A1 (en) * | 1989-07-06 | 1992-04-22 | Secr Health Brit | Silver enhanced gold-labelled immuno assay method. |
| CN2442272Y (en) * | 2000-09-18 | 2001-08-08 | 王冰 | Test biological chip for TORCH causative agent |
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