CN104151431A - Hybridoma cell strain FQ-Aa6 and anti-heavy-metal-cadmium monoclonal antibody generated by hybridoma cell strain FQ-Aa6 - Google Patents
Hybridoma cell strain FQ-Aa6 and anti-heavy-metal-cadmium monoclonal antibody generated by hybridoma cell strain FQ-Aa6 Download PDFInfo
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- CN104151431A CN104151431A CN201410399821.7A CN201410399821A CN104151431A CN 104151431 A CN104151431 A CN 104151431A CN 201410399821 A CN201410399821 A CN 201410399821A CN 104151431 A CN104151431 A CN 104151431A
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Abstract
The invention relates to a hybridoma cell strain FQ-Aa6 and an anti-heavy-metal-cadmium monoclonal antibody generated by the hybridoma cell strain FQ-Aa6. The hybridoma cell strain FQ-Aa6 is collected in the CCTCC (China Center for Type Culture Collection) with the collection number CCTCC NO.C201458. The monoclonal antibody secreted by the hybridoma cell strain FQ-Aa6 can be used for identifying heavy metal ion cadmium and has high sensitivity and high specificity, and the 50 percent inhibition concentration IC50 of the monoclonal antibody on the heavy metal ion cadmium is 5.2ng.mL<-1>. An enzyme-linked immunosorbent assay method researched by using the monoclonal antibody can be used for realizing detection of heavy metal cadmium ions in water.
Description
Technical field
The present invention relates to hybridoma cell strain FQ-Aa6, and the monoclonal antibody of the preventing from heavy metal cadmium ion of secretion.
Background technology
Along with the fast development of modern industry, when bringing great convenience to human lives, also cause the severe contamination of environment, comprise heavy metal contamination.In environment, the main pollution source of heavy metal cadmium comprises: the waste water and the waste gas that in exploitation, ore dressing and the smelting process of plumbous zinc ore, produce; The production of steel alloy and the course of processing; The factory effluent of cadmium plating, production and the courses of processing such as dyestuff, agricultural chemicals, paint, glass, pottery, photographic material.Residual by food chain enrichment gradually in environment of cadmium, the cadmium of denier just can work the mischief to human body.Within 1993, tumor research mechanism of the world has been defined as cadmium mankind's carcinogens (group I), and cadmium has caused common concern to the pollution of environment.Therefore the testing of heavy metal cadmium in environment is more and more received people's concern.
At present, the detection of heavy metal adopts the chemical apparatus methods such as atomic absorption spectrum, inductively coupled plasma atomic emission spectrometry, ion chromatograph, x ray fluorescence spectrometry, gas chromatography and mass spectromentry coupling method more.Though these traditional detection methods have higher tolerance range and accuracy, detecting instrument costliness, detect effort, time-consuming relatively, and testing often needs metastable laboratory environment, is not suitable with the demand to fast and convenient detection in reality.
The immunologic detection method of heavy metal has advantages of fast, cheap, easy, special, portable and be conducive to carry out field monitoring, overcome the shortcoming of traditional detection method.But the immune prototype of heavy metal ion itself is low, be difficult to prepare the monoclonal antibody for heavy metal ion.Therefore, for overcoming this difficulty, the present invention is by chemosynthesis high-selectivity heavy metal ion trap sequestrant, preparation heavy metal ion inner complex, obtain complete antigen with carrier protein couplet, filter out the hybridoma cell strain that can secrete for the monoclonal antibody specific of heavy metal cadmium ion in ambient water.And utilize the secreted monoclonal antibody of this hybridoma cell strain to set up the enzyme-linked immune detection method for heavy metal cadmium ion in ambient water.
Summary of the invention
The object of this invention is to provide hybridoma cell strain FQ-Aa6, and the monoclonal antibody of the preventing from heavy metal cadmium ion of secretion.
Object of the present invention can be achieved through the following technical solutions:
1) antigen preparation:
Take respectively hemocyanin and bovine serum albumin 10mg is dissolved in 0.5mL, concentration is 10mM, in N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid damping fluid of pH9.0, forms hemocyanin and bovine serum albumin solution;
Taking 10mg metal chelator 1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA) is dissolved in and in 1mL dimethyl sulfoxide (DMSO), forms 23nM metal-chelating agent solution;
Get respectively metal-chelating agent solution that 78.1 μ L concentration are 23nM and stir down gently and be dropwise added drop-wise in 0.5mL hemocyanin and bovine serum albumin solution, regulate pH to 9.0 with 10MKOH, under room temperature, react 24 hours;
Reaction product is purified with the super filter tube of 30Kd, super filter tube soaks with 100mM ethylenediamine tetraacetic acid (EDTA) in advance, after fully rinsing with ultrapure water, in super filter tube, add reaction product ultrafiltration, in super filter tube, first use 10mM, N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid damping fluid of pH9.0 rinses 3 times, use again 10mM, N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid damping fluid of pH7.4 is washed and is removed wherein unreacted small molecules metal chelator for 2 times, products therefrom is respectively hemocyanin-1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA) and bovine serum albumin-1-(4-isothiocyano phenyl)-edta solution of purifying, bovine serum albumin-1-(4-isothiocyano phenyl)-edta solution is divided into two parts, a directly as envelope antigen, another part and cadmium metal ion chelating,
53.7mg purity is that 99.999% cadmium grain is dissolved in 100 μ L top grade concentrated nitric acids, treats fully to dissolve to add ultrapure water to make final volume be 1mL, forms 477.8mM cadmium solution;
Getting cadmium solution that 18.8 μ L concentration are 477.8mM is dropwise added drop-wise under stirring in hemocyanin-1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA) that purifying crosses, getting cadmium solution that 8.9 μ L concentration are 477.8mM is dropwise added drop-wise under stirring in bovine serum albumin-1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA) that purifying crosses, with 0.5M salt acid for adjusting pH to 7.0, under room temperature, react 3 hours.Carry out purifying with the super filter tube of 30Kd and remove unreacted metal ion, the product after purifying is respectively hemocyanin-1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA)-Cd and bovine serum albumin-1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA)-Cd.Wherein hemocyanin-1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA)-Cd is immunogen, and bovine serum albumin-1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA)-Cd is coated source.
2) mouse immune
By the female BAIB/C mouse in immunogen immune 6-8 age in week preparing, the immunogen consumption of each every mouse is 200 μ g (calculating with protein content), altogether immunity five times.Since the 4th time, latter one week of each immunity, caudal vein blood sampling, with tiring and specificity of indirect non-competing ELISA method detection antibody.
3) cytogamy
Screen BAIB/C mouse by variance rate, variance rate exceedes BAIB/C mouse N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid damping fluid diluted blood azurin-1-(4-isothiocyano phenyl)-ethylenediamine tetraacetic acid (EDTA)-Cd direct immunization of 50%, after three days, get spleen, the SP2/O myeloma cell of splenocyte and logarithmic phase prepares hybridoma by hybridoma technology, 37 DEG C, 5% CO2gas incubator was cultivated after 10-14 days, merged hole supernatant screening hybridoma by detection;
4) filtering hybridoma
Merge hole supernatant and first adopt indirect non-competing ELISA method to carry out preliminary screening, behind the fusion hole that obtains tiring higher, confirm to merge hole supernatant (antibody of hybridoma secretion) by indirect competitive ELISA method whether heavy metal cadmium ion is identified.To existing the fusion hole of identity to carry out subclone (gradient dilution method), repeat above-mentioned screening procedure.Through the screening in 3-5 cycle, determine and can identify the hybridoma cell strain of heavy metal cadmium ion and the monoclonal antibody of secretion thereof.
5) preservation of hybridoma cell strain
The hybridoma cell strain that can secrete identification heavy metal cadmium ion monoclonal antibody involved in the present invention is preserved in Chinese Typical Representative culture collection center on March 31st, 2014; Preservation address, Wuhan, China Wuhan University; Deposit number CCTCC NO:C201458; Specific name: hybridoma cell strain FQ-Aa6.
Beneficial effect
(1) good stability of antibody: Cryopreservation of Hybridoma Cells at least keeps 5 years in liquid nitrogen, the stability of antibody also can be relatively good, and antibody-20 of purifying DEG C refrigerator at least can be deposited 2 years.
(2) antibodies specific is high: preventing from heavy metal cadmium monoclonal antibody and Hg that hybridoma cell strain FQ-Aa6 provided by the invention is secreted
2+cross reaction be 1.21%, do not find temporarily cross reaction with all the other metals.
Claims (2)
1. for detection of a monoclonal antibody for heavy metal cadmium, the hybridoma cell strain FQ-Aa6 that is CCTCC NO:C201458 by deposit number produces, and is IgG1 type, and light chain of antibody is kappa chain.
2. the hybridoma cell strain FQ-Aa6 that produces the monoclonal antibody for detection of heavy metal cadmium claimed in claim 1, is preserved in Chinese Typical Representative culture collection center on March 31st, 2014, and deposit number is CCTCC NO:C201458.
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| CN201410399821.7A CN104151431A (en) | 2014-08-13 | 2014-08-13 | Hybridoma cell strain FQ-Aa6 and anti-heavy-metal-cadmium monoclonal antibody generated by hybridoma cell strain FQ-Aa6 |
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| CN201410399821.7A CN104151431A (en) | 2014-08-13 | 2014-08-13 | Hybridoma cell strain FQ-Aa6 and anti-heavy-metal-cadmium monoclonal antibody generated by hybridoma cell strain FQ-Aa6 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016337A (en) * | 2007-03-02 | 2007-08-15 | 南京农业大学 | Preparing method of heavy metal cadmium monoclonal antibody |
| CN101139396A (en) * | 2007-08-08 | 2008-03-12 | 华南理工大学 | Cadmium chelate complex monoclonal antibody and preparation method and usage thereof |
| CN101240023A (en) * | 2008-03-14 | 2008-08-13 | 南京大学 | Preparation of heavy metal cadmium polyclonal antibody and method for measuring enzyme linked immunity absorption |
| CN101445557A (en) * | 2008-12-16 | 2009-06-03 | 中国农业大学 | Cadmium ion antigen and preparation method and application thereof |
| CN103076446A (en) * | 2011-10-25 | 2013-05-01 | 天津科技大学 | Antibody against heavy metal cadmium and application of same in detection of cadmium residue in traditional Chinese medicine |
-
2014
- 2014-08-13 CN CN201410399821.7A patent/CN104151431A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016337A (en) * | 2007-03-02 | 2007-08-15 | 南京农业大学 | Preparing method of heavy metal cadmium monoclonal antibody |
| CN101139396A (en) * | 2007-08-08 | 2008-03-12 | 华南理工大学 | Cadmium chelate complex monoclonal antibody and preparation method and usage thereof |
| CN101240023A (en) * | 2008-03-14 | 2008-08-13 | 南京大学 | Preparation of heavy metal cadmium polyclonal antibody and method for measuring enzyme linked immunity absorption |
| CN101445557A (en) * | 2008-12-16 | 2009-06-03 | 中国农业大学 | Cadmium ion antigen and preparation method and application thereof |
| CN103076446A (en) * | 2011-10-25 | 2013-05-01 | 天津科技大学 | Antibody against heavy metal cadmium and application of same in detection of cadmium residue in traditional Chinese medicine |
Non-Patent Citations (1)
| Title |
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| 朱晓霞: "抗重金属镉、铅单克隆抗体、单链抗体的研制及单链抗体三维结构的模建", 《中国博士学位论文全文数据库 农业科技辑》 * |
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Application publication date: 20141119 |