CN1431505A - Immunity collodal gold reagent for detecting IgM antibody of herpes simplex virus I type and its preparing method - Google Patents
Immunity collodal gold reagent for detecting IgM antibody of herpes simplex virus I type and its preparing method Download PDFInfo
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- CN1431505A CN1431505A CN 03115136 CN03115136A CN1431505A CN 1431505 A CN1431505 A CN 1431505A CN 03115136 CN03115136 CN 03115136 CN 03115136 A CN03115136 A CN 03115136A CN 1431505 A CN1431505 A CN 1431505A
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- monoclonal antibody
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Abstract
The sample pad and the binding pad, which is the conjugate coated with the anti U chain monoclonal antibody and colloidal gold, are adhered to the end with PVC back covered in sequence. The cellulose nitrate film coated with the specificity surface film antigen of herpes simple virus I type and the anti rat IgG of sheep (or rabbit) is adhered to the middle of the reagent. The water uptake pad is adhered to another end of the reagent. The reagent possesses the features of the high specificity and sensitivity, simple and fast. The reagent is applicable to the detection in site within 30 minutes for the full testing procedure without need of professional training for the operation personnel.
Description
Technical field
The present invention relates to a kind of immune colloid gold reagent that detects antibody, also relate to the preparation method of this reagent.
Background technology
The existing method that is used to detect the herpes simplex virus I-type antibody IgM mainly contains enzyme linked immunological absorption, radioimmunoassay, immune colloid gold percolation, western blot test (WBA) etc.The defective of enzyme linked immunological absorption (EIA) method is: need special instrument and equipment such as microplate reader to be used; The detecting operation personnel need pass through professional training; Operating process is relatively complicated, and it is long to detect required time; It is higher to detect required expense, can not realize that single part is detected.The defective of immune colloid gold percolation is: operating process is relatively complicated, can not realize single stepping; Reagent needs cryopreservation; It is higher to detect required expense; It is longer than colloidal gold immunity chromatography to detect required time.The defective of radioimmunoassay is: operating personnel need pass through professional training; Complicated operating process requires high; Reagent needs cryopreservation; Because test relates to radioactive isotope, test is not very safe, so seldom adopt; Testing cost is higher; The detection required time is long.The defective of western blot test (WBA) is: operating personnel need pass through professional training; Complicated operating process requires high; Reagent needs cryopreservation; Still the Western blot detection kit of not having at present commercial HSV serum antibody emerges, and this method only limits to carry out in some specialized laboratory; The testing cost height; The detection required time is long.
Summary of the invention
The objective of the invention is in order to overcome the defective that current techniques exists in promoting the use of, providing a kind of does not need the auxiliary detectable of particular instrument equipment, and can reduce the detection cost effectively, alleviates the burden that needs the testing staff.The preparation method of this reagent is provided simultaneously.
The immune colloid gold reagent that detects the herpes simplex virus I-type antibody IgM comprises sample pad, pad, nitrocellulose filter, adsorptive pads and PVC backing, PVC backing one end adheres to sample pad, pad successively, the middle nitrocellulose filter that adheres to, the other end adheres to adsorptive pads, it is characterized in that the pad bag by the bond of anti-u chain monoclonal antibody-collaurum, bag is by herpes simplex virus I-type specific surfaces membranous antigen and the anti-mouse IgG of sheep (or rabbit) on the nitrocellulose filter.
The preparation method of this reagent may further comprise the steps: (1) preparation monoclonal antibody: get hybridoma cell strain and conventional cultivate, go down to posterity external, with humanized's u chain injection BALB/C mice abdominal cavity, collect ascites, with the affinity chromatography anti-u chain monoclonal antibody of purifying; (2) preparation polyclonal antibody: usefulness humanized's u chain is immune mouse repeatedly, extracts antiserum immune goat or rabbit, gets sheep anti-mouse igg or rabbit anti-mouse igg behind the purifying; (3) preparation collaurum: the colloid gold particle that gold chloride is reduced into 20nm～40nm with reductive agents such as trisodium citrates; (4) the anti-u chain monoclonal antibody colloid gold label thing of preparation: with collaurum and monoclonal antibody in 1: 0.005～0.015 (ml/mg) ratio mixing, make collaurum and antibody form stabilized complex, concentrate by purifying and form anti-u chain monoclonal antibody-colloid gold label thing; (4) will resist u chain monoclonal antibody colloid gold label thing to be coated on the collaurum pad, anti-herpes simplex virus I type specificity surface film antigen and the anti-mouse IgG of sheep (rabbit) will be coated on the detection zone and the control zone of nitrocellulose filter, fully dry.
Good effect of the present invention is: cheap, production procedure is simple, and cost is low, and the expense of detection is than using other detection method to want considerably cheaper; Detection speed is fast, and overall process only needs 30 minutes, can realize that the oneself detects; Can on-the-spotly detect; Specificity is good, highly sensitive, good reproducibility; Easy and simple to handle, fast qualitative, the result is accurately, fast, and is easy and simple to handle, need not flushing process and standard control, can be in batches or single sample in time detect; Be easy to promote the use of, operating personnel need not professional training, and by specification gets final product complete operation.
Embodiment
PVC backing one end sample pad, bag have been adhered to successively by the pad of anti-u chain monoclonal antibody and collaurum bond, the middle bag that adheres to is by the nitrocellulose filter of herpes simplex virus I-type specific surfaces membranous antigen and the anti-mouse IgG of sheep (or rabbit), and the other end adheres to adsorptive pads.
Preparation according to the following steps: (1) preparation monoclonal antibody: get hybridoma cell strain and conventional cultivate, go down to posterity,, collect ascites, with the affinity chromatography anti-u chain monoclonal antibody of purifying with humanized's u chain injection BALB/C mice abdominal cavity external; (2) preparation polyclonal antibody: usefulness humanized's u chain is immune mouse repeatedly, extracts antiserum immune goat or rabbit, gets sheep anti-mouse igg or rabbit anti-mouse igg behind the purifying; (3) preparation collaurum: the particle that 100ml 0.01% chlorauride is reduced into the 40nm size with 0.9ml 1% trisodium citrate; (4) the anti-u chain monoclonal antibody colloid gold label thing of preparation: use 0.1mol/LK
2CO
3The pH value of colloidal gold solution is transferred to about 8.2, colloidal gold solution and monoclonal antibody are mixed in the ratio that adds the 1mg monoclonal antibody in the 100ml colloidal gold solution, make collaurum and antibody form stable colloidal gold composite, again by repeatedly centrifugal, abandon supernatant, cleaning, concentrate formation anti-u chain monoclonal antibody-colloid gold label thing by purifying, refrigerate standby; (5) will resist u chain monoclonal antibody colloid gold label thing to be sprayed on the collaurum pad with Biodot point film machine, anti-herpes simplex virus I type specificity surface film antigen and the anti-mouse IgG of sheep (rabbit) will be sprayed on the nitrocellulose filter, fully dry; (6) nitrocellulose filter, collaurum pad, sample pad, adsorptive pads etc. are bonded on the PVC backing successively; (7) the PVC material that glues is cut into the reagent strip of certain width, promptly makes the immune colloid gold reagent that detects the herpes simplex virus I-type antibody IgM.
Before detection, earlier sample and reagent strip (plate) are placed on placement a period of time (10 minutes) under the room temperature condition, make it restore to room temperature; Take out the detectable bar from aluminium foil bag, by the direction shown in the arrow under the MARK line reagent strip is immersed in the sample solution, liquid level must not surpass the MARK line, takes out after 5 seconds～8 seconds, lies on the operator's console; If agent plate: from aluminium foil bag, take out the detectable plate, lie on the operator's console, in well, drip 3 (about 120ul) sample solutions (serum); Get final product judged result in 3 minutes～15 minutes, the result who judges after 30 minutes is invalid.
The result judges: if there be " the herpes simplex virus I-type antibody IgM " that will detect to exist in the sample, then red stripes appears in the detection line place, red stripes also occurs on the nature controlling line simultaneously, and this moment, the result was positive; If " the herpes simplex virus I-type antibody IgM " that will not detect in the sample exists, then the detection line place does not have the band appearance, but occurs red stripes on the nature controlling line, and this moment, the result was negative.If there is not red stripes to occur on the nature controlling line, then product is invalid.
Claims (2)
1, detects the immune colloid gold reagent and the preparation method of herpes simplex virus I-type antibody IgM, this reagent comprises sample pad, pad, nitrocellulose filter, adsorptive pads and PVC backing, PVC backing one end adheres to sample pad, pad successively, the middle nitrocellulose filter that adheres to, the other end adheres to adsorptive pads, it is characterized in that the pad bag by the bond of anti-u chain monoclonal antibody-collaurum, bag is by herpes simplex virus I-type specific surfaces membranous antigen and the anti-mouse IgG of sheep (or rabbit) on the nitrocellulose filter.
2, the immune colloid gold reagent and the preparation method of detection herpes simplex virus I-type antibody IgM according to claim 1, the preparation method who it is characterized in that this reagent may further comprise the steps: (1) preparation monoclonal antibody: get hybridoma cell strain and cultivate, go down to posterity in external routine, u chain injection BALB/C mice abdominal cavity with the humanized, collect ascites, with the affinity chromatography anti-u chain monoclonal antibody of purifying; (2) preparation polyclonal antibody: usefulness humanized's u chain is immune mouse repeatedly, extracts antiserum immune goat or rabbit, gets sheep anti-mouse igg or rabbit anti-mouse igg behind the purifying; (3) preparation collaurum: the colloid gold particle that gold chloride is reduced into 20nm～40nm with reductive agents such as trisodium citrates; (4) the anti-u chain monoclonal antibody colloid gold label thing of preparation: with collaurum and monoclonal antibody in 1: 0.005～0.015 (ml/mg) ratio mixing, make collaurum and antibody form stabilized complex, concentrate by purifying and form anti-u chain monoclonal antibody-colloid gold label thing; (4) will resist u chain monoclonal antibody colloid gold label thing to be coated on the collaurum pad, anti-herpes simplex virus I type specificity surface film antigen and the anti-mouse IgG of sheep (rabbit) will be coated on the detection zone and the control zone of nitrocellulose filter, fully dry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03115136 CN1431505A (en) | 2003-01-24 | 2003-01-24 | Immunity collodal gold reagent for detecting IgM antibody of herpes simplex virus I type and its preparing method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03115136 CN1431505A (en) | 2003-01-24 | 2003-01-24 | Immunity collodal gold reagent for detecting IgM antibody of herpes simplex virus I type and its preparing method |
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| Publication Number | Publication Date |
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| CN1431505A true CN1431505A (en) | 2003-07-23 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 03115136 Pending CN1431505A (en) | 2003-01-24 | 2003-01-24 | Immunity collodal gold reagent for detecting IgM antibody of herpes simplex virus I type and its preparing method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266620A (en) * | 2018-09-04 | 2019-01-25 | 四川迈克生物新材料技术有限公司 | Anti-human igg monoclonal antibody, its hybridoma cell strain and application |
-
2003
- 2003-01-24 CN CN 03115136 patent/CN1431505A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266620A (en) * | 2018-09-04 | 2019-01-25 | 四川迈克生物新材料技术有限公司 | Anti-human igg monoclonal antibody, its hybridoma cell strain and application |
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Owner name: BODUN BIOTEST TECHNOLOGY(HANGZHOU) CO., LTD Free format text: FORMER OWNER: HUZHOU RUIZE BIOTECHNOLOGY CO., LTD Effective date: 20040326 |
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Effective date of registration: 20040326 Address after: 311215 four road, Xiaoshan economic and Technological Development Zone, Hangzhou, Zhejiang Applicant after: Bioden Inspection Biotech (Hangzhou) Co., Ltd. Address before: 313000 Zhejiang Province, Huzhou City Longxi Road No. 208 Applicant before: Ruize Biotechnology Co., Ltd., Huzhou |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |