CN1431513A - Immune colloidal gold reagent for detecting IgM antibody of toxoplasma and its preparing method - Google Patents
Immune colloidal gold reagent for detecting IgM antibody of toxoplasma and its preparing method Download PDFInfo
- Publication number
- CN1431513A CN1431513A CN 03115144 CN03115144A CN1431513A CN 1431513 A CN1431513 A CN 1431513A CN 03115144 CN03115144 CN 03115144 CN 03115144 A CN03115144 A CN 03115144A CN 1431513 A CN1431513 A CN 1431513A
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- CN
- China
- Prior art keywords
- monoclonal antibody
- preparation
- pad
- reagent
- colloid gold
- Prior art date
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 19
- 241000223996 Toxoplasma Species 0.000 title claims description 9
- 238000000034 method Methods 0.000 title abstract description 11
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 12
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 12
- 241001494479 Pecora Species 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims description 20
- 239000000084 colloidal system Substances 0.000 claims description 19
- 239000010931 gold Substances 0.000 claims description 19
- 229910052737 gold Inorganic materials 0.000 claims description 19
- 230000000274 adsorptive effect Effects 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 230000002829 reductive effect Effects 0.000 claims description 5
- 201000005485 Toxoplasmosis Diseases 0.000 claims description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 claims description 3
- 206010003445 Ascites Diseases 0.000 claims description 3
- 241000283707 Capra Species 0.000 claims description 3
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 3
- 241000699670 Mus sp. Species 0.000 claims description 3
- 210000000683 abdominal cavity Anatomy 0.000 claims description 3
- 238000001042 affinity chromatography Methods 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 210000004408 hybridoma Anatomy 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
- 235000019263 trisodium citrate Nutrition 0.000 claims description 3
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 101710181478 Envelope glycoprotein GP350 Proteins 0.000 abstract 1
- 230000001938 anti-toxoplasmal effect Effects 0.000 abstract 1
- 239000012528 membrane Substances 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 8
- 230000002950 deficient Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000005030 aluminium foil Substances 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000005325 percolation Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- -1 cleaning Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The reagent adheres a sample pad and a combination pad coated with an anti-u chain monoclonal antibody and a colloidal gold conjugate to one end of a PVC back lining in sequence, adheres a nitrocellulose membrane coated with an anti-toxoplasma specific surface membrane antigen and sheep (rabbit) anti-mouse IgG to the middle, and adheres a water absorption pad to the other end in sequence. The reagent has the advantages of strong specificity, high sensitivity, simplicity, rapidness, low cost and capability of on-site detection, the whole process only needs 30 minutes, and operators can finish the operation according to instructions without professional training.
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 detects toxoplasma antibody IgM mainly is to detect this two kinds of equal defectiveness of method or deficiency by enzyme linked immunological absorption (EIA) and immune colloid gold percolation.The defective of enzyme linked immunological absorption (EIA) method is: need special instrument and equipment such as microplate reader to be used; Reagent needs cryopreservation; 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; Be unfavorable for promoting the use of in grass-roots unit.The defective of immune colloid gold percolation is: operating process is relatively complicated, can not realize single stepping; Reagent needs cryopreservation; The detection required time is long, the required expense of detection is higher.
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.
Detect toxoplasma antibody IgM immune colloid gold reagent and comprise 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 arc worm 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, resisting toxoplasmosis specific surfaces membranous 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 resisting toxoplasmosis specific surfaces membranous antigen and the anti-mouse IgG of sheep (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, resisting toxoplasmosis specific surfaces membranous 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 and detect toxoplasma antibody IgM immune colloid gold reagent.
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 toxoplasma 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 toxoplasma 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 toxoplasma antibody IgM immune colloid gold reagent and preparation method, 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 arc worm specific surfaces membranous antigen and the anti-mouse IgG of sheep (or rabbit) on the nitrocellulose filter.
2, detection toxoplasma antibody IgM immune colloid gold reagent according to claim 1 and preparation method, 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, resisting toxoplasmosis specific surfaces membranous 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 03115144 CN1431513A (en) | 2003-01-24 | 2003-01-24 | Immune colloidal gold reagent for detecting IgM antibody of toxoplasma and its preparing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03115144 CN1431513A (en) | 2003-01-24 | 2003-01-24 | Immune colloidal gold reagent for detecting IgM antibody of toxoplasma and its preparing method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1431513A true CN1431513A (en) | 2003-07-23 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03115144 Pending CN1431513A (en) | 2003-01-24 | 2003-01-24 | Immune colloidal gold reagent for detecting IgM antibody of toxoplasma and its preparing method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1431513A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102426241A (en) * | 2011-09-19 | 2012-04-25 | 厦门市湖里区妇幼保健院 | Reagent strip for toxoplasma gondii immune globulin M (IgM) antibody colloidal gold immunochromatography detection, and preparation method thereof |
| CN102435744A (en) * | 2011-09-19 | 2012-05-02 | 厦门大学附属中山医院 | Colloidal gold immunochromatography assay reagent strip for total antibodies to Toxoplasma gondii and preparation method thereof |
| CN104745536A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | Monoclonal antibody hybridoma 3A6 and monoclonal antibody for toxoplasma protein TgVPI |
| CN105759030A (en) * | 2016-03-15 | 2016-07-13 | 中国农业大学 | Colloidal gold test strip for testing toxoplasma gondii antibodies |
-
2003
- 2003-01-24 CN CN 03115144 patent/CN1431513A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102426241A (en) * | 2011-09-19 | 2012-04-25 | 厦门市湖里区妇幼保健院 | Reagent strip for toxoplasma gondii immune globulin M (IgM) antibody colloidal gold immunochromatography detection, and preparation method thereof |
| CN102435744A (en) * | 2011-09-19 | 2012-05-02 | 厦门大学附属中山医院 | Colloidal gold immunochromatography assay reagent strip for total antibodies to Toxoplasma gondii and preparation method thereof |
| CN104745536A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | Monoclonal antibody hybridoma 3A6 and monoclonal antibody for toxoplasma protein TgVPI |
| CN104745536B (en) * | 2015-03-02 | 2018-03-09 | 南方医科大学 | A kind of toxoplasma protein TgVP1 monoclonal antibody hybridomas 3A6 and monoclonal antibody |
| CN105759030A (en) * | 2016-03-15 | 2016-07-13 | 中国农业大学 | Colloidal gold test strip for testing toxoplasma gondii antibodies |
| CN105759030B (en) * | 2016-03-15 | 2018-01-02 | 中国农业大学 | A kind of colloidal gold strip for detecting toxoplasma antibody |
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| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: BODUN BIOTEST TECHNOLOGY(HANGZHOU) CO., LTD Free format text: FORMER OWNER: HUZHOU RUIZE BIOTECHNOLOGY CO., LTD Effective date: 20040325 |
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| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20040325 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 |