CN101948808A - Antibody capable of resisting heavy metal mercury ions and application thereof - Google Patents

Abstract

The invention discloses an antibody capable of resisting heavy metal mercury ion and application thereof. The antibody capable of resisting heavy metal mercury ions is a monoclonal antibody secreted by a hybridoma cell strain H1H8 CCTC No.C 200949. The acquisition of the monoclonal antibody of the metal mercury ion chelate of the invention lays the basis for building the immunological detection method of mercury ion residue. The above monoclonal antibody can be used for researching and building the immunological detection method of heavy metal. The advantages of high speed, low price, agility and strong specificity of the monoclonal antibody are utilized to develop a portable method for on-site detection so as to adapt to the sampling detection of agricultural and animal products and quick detection of import and export custom clearance. Thus, the invention has important realistic significance for improving the efficiency and quality of risk evaluation and ensuring food safety.

Description

Preventing from heavy metal mercury ion antibody and application thereof

Technical field

The present invention relates to a kind of preventing from heavy metal mercury ion antibody and application thereof.

Background technology

In recent years, the heavy metal contamination incident emerges in an endless stream, grows in intensity, and the air that the mankind depend on for existence, soil, vegetables and animal all have been subjected to the pollution of poisonous substances such as heavy metal.The abuse of environmental pollution and feed medicated premix, cause the animal products heavy-metal residual exceed standard in various degree (the auspicious Yue of Pang. problem and countermeasure that China animal products quality safety exists. Heilungkiang grain, 2007, (4): 20-22).The heavy metal ion that accumulates in the crop enters in people's carcass by the biological magnification of food chain, the threat human and livestock health (Lang Minglin, Zhang Yuxiu, Chai Tuanyao. the progress of genetically engineered improvement plant heavy metal resistance and accumulation ability. the biotechnology journal, 2004,20 (2): 157-164).Mercury is the stronger element of cumulative effect, mainly accumulates in animal body.Waterplant in lake, the marsh, fishery products are easily accumulated a large amount of mercury.Later stage, used since the mercurous miticide on the agricultural, mercury is serious day by day to soil, natural stream networks and atmospheric pollution.Mercury pollution is in some place in the world, become general health public hazards (Mei Guangquan. the harm of heavy metal wastewater thereby and improvement. trace element and health research, 2004,21 (4): 54-56).The heavy metal content that detects rapidly and sensitively in the food is the basis that ensures food safety, the analytical procedure of traditional detection heavy-metal residual comprises atomic absorption spectrochemical analysis (Atomic Absorption Spectroscopy, AAS) and inductively coupled plasma emmission spectrum (Inductively Coupled Plasma Atomic Emission Spectroscopy, ICP-AES) etc. be sensitive, (Bontidean I reliably, Lloyd JR, Hobman JL, et al.Bacterial metal-resistance proteins and their use in biosensors for the detection of bioavailable heavy metals.J Inorg Biochemy, 2000,79 (1-4): 225-229.).But the application that the analyst of expensive instrument, specialty and complicated sample pre-treatment have limited these methods.Detection must be carried out in possessing the key lab of large-sized analytic instrument, can't be used for on-the-spot the detection, and be subjected to restriction (Liu Gongliang such as expense height, treatment capacity finite sum detection time is long, Wang Jufang. the immunodetection progress of heavy metal ion. the biotechnology journal, 2006,22 (6): 877-881).Traditional heavy metal detection method is not suitable for the sample that freshness dates such as vegetables, fruit are short, profit is low and the big flux of need detects.

Detection method routinely to heavy metal has had a variety of, some also has been unusual proven technique, but because the wide range that heavy metal ion exists, according to accuracy of detection, comfort level and detection cost, every kind of technology all has their limitation, also need suitable place simultaneously, therefore all be unfavorable for applying aborning.Setting up quicker, more economical immunoassay detection mercury ion is to produce and needs of economic development.

Summary of the invention

The purpose of this invention is to provide a kind of preventing from heavy metal mercury ion antibody and application thereof

Preventing from heavy metal mercury ion antibody provided by the invention is hybridoma cell strain H1H8 CCTCC № .C200949 excretory monoclonal antibody.

Hybridoma cell strain H1H8 CCTCC № .C200949 also belongs to the scope of protection of the invention; be preserved in Chinese typical culture collection center on July 8th, 2009 and (be called for short CCTCC; the address is: Wuhan City, Hubei Province Wuhan University), preservation registration number is CCTCC № .C200949.

Hybridoma cell strain H1H8 CCTCC № .C200949 or its application of excretory monoclonal antibody in the mercury ion immunodetection also belong to protection scope of the present invention.

Hybridoma cell strain H1H8 CCTCC № .C200949 excretory monoclonal antibody provided by the invention is a monoclonal antibody specific.Its preparation process is because of Hg ²⁺Can not cause immune response to such an extent as to ion is too little, so (diethylene triamine pentacetic acid (DTPA), DTPA) (keyhole limpet hemocyanin KLH) couples together with metal ion and carrier proteins with sequestrant.Success is synthetic, identify the mercury compound as antigen after, immune BALB/c mouse has obtained the hybridoma of stably excreting antibody by cytogamy.With Method of Limited Dilution method subclone,, finally obtained the cell strain (H1H8) of the anti-mercury ion antibody of stably excreting by the ELISA screening.Mouse peritoneal injection 1 * 10 ⁷The H1H8 cell strain prepares ascites, and ascites antibody is tired more than 1: 51200.Through identifying that hybridoma is the IgG1 subclass, light chain is that kappa type and secretory antibody stability are better.Hybridoma cell strain H1H8 CCTCC № .C200949 excretory monoclonal antibody provides technical foundation for the foundation of the residual immunological detection method of mercury ion, and to improving the efficient and the quality of risk assessment work, important realistic meaning has ensured food safety.

The acquisition of the monoclonal antibody of mercury metal ion chelate complex of the present invention is for the foundation of the residual immunological detection method of mercury ion lays the foundation.Can utilize said monoclonal antibody, study and set up the immunological detection method of heavy metal.And utilize its fast, the advantage of cheap, sensitivity and high specificity, the on-the-spot portable method that detects is convenient in development, thereby is applicable to the sampling Detection of agricultural and animal products and the quick test that import and export are open to the custom.To improving the efficient and the quality of risk assessment work, important realistic meaning has ensured food safety.

Description of drawings

Fig. 1 is the variance rate after mouse five is exempted from;

The positive hybridoma screening of Fig. 2

Fig. 3 clones back cell conditioned medium antibody titer for the second time for H1H8

Fig. 4 is the evaluation of hybridoma H1H8 karyotype

Fig. 5 is the detection of H1H8 ascitic type monoclonal antibody

Fig. 6 is a H1H8 secretory antibody hypotype

Fig. 7 is the stability of H1H8 secretory antibody

Embodiment

The preparation and the evaluation of the hybridoma of embodiment 1, secretion preventing from heavy metal mercury ion antibody

One, the preparation of the hybridoma of secretion preventing from heavy metal mercury ion antibody

1, antigenic preparation and evaluation

Hg-DTPA-KLH: take by weighing 5.65mg diethylene triamine pentacetic acid (DTPA) (DTPA) and add in the HEPES damping fluid of 1mL pH 9.73 and form A liquid, change reactor over to, adding concentration again is the Hg (NO of 3.46mol/L ₃) ₂10 μ L add the HEPES damping fluid of 0.4mL pH 9.73 and the keyhole limpet hemocyanin (KLH) that 1.7mL concentration is 5.9g/L behind the reaction 30min under the room temperature simultaneously, regulate pH to 9.0, stirring reaction 12h under the room temperature.After the linked reaction, protein complex is carried out separation and purification, remove the Hg that does not participate in reaction with pretreated CentriconYM-30 ultrafiltration pipe ²⁺, sequestrant DTPA and Hg-DTPA mixture.

The synthetic method of Hg-DTPA-OVA is the same, and (OVA (ovalbumin) substitutes keyhole limpet hemocyanin (KLH), and the synthetic method of DTPA-OVA is the same to replace metal ion to get final product with ultrapure water with chicken ovalbumin.

Use BCA kit measurement complex proteins concentration (Robert C.Blake II after the ultrafiltration, Andrey R.Pavlov, Mehraban Khosraviani, et al.Blake.Novel Monoclonal Antibodies with Specificity for Chelated Uranium (VI): Isolation and Binding Properties.Bioconjugate Chem, 2004,15 (5): 1125-1136).With reference to GB GB/T 17141-1997, with the supernatant after the graphite furnace atomic absorption spectrometry detection ultrafiltration, Hg in filtered solution and the HEPES liquid ²⁺Concentration (Khosraviani M, Pavlov AR, Flowers GC, et al.Detection of heavy metals by immunoassay:optimization and validation of a rapid, portable assay for ionic cadmium.Environ Sci Ttechnoy, 1998,32 (1): 137-142).

The result shows, uses BCA kit measurement protein concentration after the ultrafiltration, behind the drawing standard curve, according to formula y=0.0008x+0.0065 (R ²=0.9995) y represents OD ₅₇₀, x represents that protein concentration (mg/L) calculates that complete antigen Hg-DTPA-KLH protein concentration is 4.615g/L in the supernatant, protein concentration is 0.01g/L in the filtered solution.

Detect Hg with graphite furnace atomic absorption spectrometry ²⁺Result: Hg-DTPA-KLH, DTPA-KLH, the solubility of mercury ion is respectively 208mg/Kg in the HEPES solution, 0mg/Kg, 0mg/Kg.If antigen does not synthesize successfully, free Hg ²⁺Or Hg-DTPA can be filtered along with ultrafiltration, can not contain Hg in the supernatant after the ultrafiltration ²⁺, and detected Hg our supernatant after ultrafiltration ²⁺And content is higher, proves that antigen synthesizes successfully.

2, the selection of immunity of mouse and fusion mouse

The immunity of mouse: five of immune BALB/c mouse (purchasing Experimental Animal Center) in Chinese Military Medical Science Institute, when head exempts from, emulsification 1h～2h (Palmarini M behind immunizing antigen and the abundant mixing of isopyknic Freund's complete adjuvant, Murgia C, Fan H.Spliced and prematurely polyadenylated Jaagsiekte sheep retrovirus (JSRV)-specific RNAs from infected or transfected cells.Virology, 2002,294 (1): 180-188), every subcutaneous multi-point injection 200 μ g antigens of BABL/c mouse.Three week back immunity for the second time, the fully emulsified back immunity of same dose antigen and isopyknic Freund's incomplete adjuvant, later on every two all immunity once, method is with immunity for the second time.Immunity for the third time and after each immunity back 7d, tail vein blood, separation of serum is measured tiring and producing the specificity of antibody of serum.

Merge the selection of mouse: select to merge mouse with indirect elisa method, method is as follows:

1. wrap quilt: usefulness HBS (10mmol/L pH7.4) dilutes Hg-DTPA-OVA and DTPA-OVA respectively, and wraps respectively by to 96 hole enzyme plates with identical concentration 5mg/L, 50 μ L/ holes, and 4 ℃ are spent the night or 37 ℃ of hatching 2h; 2. sealing: PBST (1L PBST component: NaCl 8g, KCl 0.2g, NaHPO ₄12H ₂O 3.63g, KH ₂PO ₄0.24g, Tween20 500 μ L, pH7.3) washing is 5 times, pats dry, and adds 3%BSA by 100 μ L/ holes, 37 ℃ of sealing 1h; 3. one is anti-: PBST washing 5 times, add the mice serum after above-mentioned five immunity of identical thinning ratio respectively, and make negative control with the serum of not immune BALB/c mouse (purchasing Experimental Animal Center), 50 μ L/ holes, 37 ℃ of hatching 1h in Chinese Military Medical Science Institute; 4. ELIAS secondary antibody: washing with step 3., after the sheep anti-mouse igg/IgM of horseradish peroxidase-labeled (mixture) dilution in 1: 5000,1h is hatched for 37 ℃ in 50 μ L/ holes; 5. colour developing: wash the same, the liquid TMB that add to develop the color, 15min is hatched for 37 ℃ in 50 μ L/ holes; 6. termination reaction: add 1N hydrochloric acid termination reaction by 50 μ L/ holes; 7. survey OD on the microplate reader ₄₅₀Value returns to zero with blank well.Calculated difference rate Differnce%=(OD by formula ₄₅₀Of Hg-DTPA-OVA-OD ₄₅₀Of DTPA-OVA)/OD ₄₅₀Of DTPA-OVA * 100%.

The result as shown in Figure 1, five exempt from the back detects in the mice serum behind the antibody titer, the result who calculates the variance rate of each mouse shows that No. 1 mouse variance rate is the highest.Several mouse have produced the antibody of anti-DTPA and Hg-DTPA simultaneously, but the antibody horizontal of No. 1 mouse anti Hg-DTPA is the highest and variance rate is maximum, illustrate that No. 1 mouse produces at Hg ²⁺Antibodies specific the strongest, so select No. 1 mouse to be used for merging (Fig. 1).

3, cytogamy, the screening in positive fusion hole and the clone of positive fused cell

Select described No. 1 mouse of step 2 to carry out cytogamy, 3d booster immunization mouse before merging.1d prepares feeder cell (BABL/c Turnover of Mouse Peritoneal Macrophages) before merging, and cell concn is transferred to 1 * 10 ⁵/ mL is added in the 96 porocyte culture plates by the 0.1mL/ hole, and is standby.The SP2/0 myeloma cell strain (available from Tumour Inst., Chinese Medical Academy) of getting No. 1 mouse boosting cell of booster immunization and being in logarithmic phase with 6: 1 cell quantities than mixing, with 50% (quality percentage composition) PEG1450 (purchase of U.S. sigma company) solution mediates fusion, cell after merging is joined in the culture plate that contains feeder cell, 0.1mL/ hole, 37 ℃, 5%CO ₂The incubator cultivation (Li Fengkui, Wang Chunyao chief editor. laboratory animal and animal experiment method are learned. Zhengzhou: the .2007:377 of press of Zhengzhou University).

After treating that cell covers with 1/3 at the bottom of the hole, detect with indirect elisa method and to merge the hole supernatant, method is with the selection of the fusion mouse in the step 2, one anti-be cell conditioned medium, do not do negative control with merging the hole.With Method of Limited Dilution method clone positive cell, the detection method of cell conditioned medium is with positive screening of merging the hole.

The positive results of screening that merges the hole shows, grow macroscopic hybridoma group after 10 days in the 96 porocyte culture plates, treat that cell covers with 1/3 at the bottom of the hole and gets cell conditioned medium and detect antibody titer, the result shows that hybridoma H2H5,3D12, H1H8,2A11 antibody titer are all greater than 2.1 times of negative control, be judged to be positive hybridoma, and the variance rate of H1H8 is than big four strain cell conditioned mediums of variance rate and the Hg-DTPA-OVA reaction OD of 2A11,3D12 ₄₅₀Value all is higher than and DTPA-OVA reaction OD ₄₅₀Value, wherein H1H8 differs greatly (Fig. 2), and the antibody that this two strains emiocytosis is described is at Hg ²⁺The ionic specificity is stronger, selects H1H8 to carry out subclone.

4, the subcloning of positive fused cell

H1H8 raises to some extent through the tiring of antibody of emiocytosis behind twice subcloning, obtains the stable anti-Hg of secretion ²⁺The cell strain of monoclonal antibody illustrates subclone successful (Fig. 3).

H1H8 is preserved in Chinese typical culture collection center on July 8th, 2009, and (be called for short CCTCC, the address is: Wuhan City, Hubei Province Wuhan University), the H1H8 preservation registration number is CCTCC № .C200949.

Two, the evaluation of the hybridoma of secretion preventing from heavy metal mercury ion antibody

1, the evaluation of hybridoma karyotype

Reference literature (the Si Tuzhenqiang chief editor. cell cultures. Xi'an: the .2007:242 of world book press) described method is carried out the evaluation of hybridoma karyotype, concrete grammar is: the vegetative period hybridoma cell strain H1H8 final concentration of taking the logarithm is that the colchicine of 0.05mg/L～0.1mg/L is handled 4h～6h, behind the collecting cell with hypotonic KCL solution-treated 30min, stationary liquid is fixed, drip on the slide glass of cell suspension to 4 ℃ precooling, natural drying at room temperature, Giemsa dyeing, through observation by light microscope, select the finely dispersed cell counting of the complete karyomit(e) of cellular form.5 cells of every strain counting, the record chromosome number is also calculated mean value.

The hybridoma chromosome karyotype analysis is the result show, 68 of the chromosome number average out to of SP2/0 cell, 40 of splenocyte chromosome number average out to, and the chromosome number of hybridoma is all at (Fig. 4) more than 100, the chromosome number that is higher than two parent's cells illustrates the hybridization product that this two strain of hybridoma is SP2/0 cell and splenocyte.

2, ascitic type MONOCLONAL ANTIBODIES SPECIFIC FOR

Get the female BABL/c mouse in 8 ages age～10 week in week, abdominal injection paraffin oil 0.5mL/, all backs, 1 week～2 are abdominal injection 1 * 10 respectively ⁶Individual hybridoma HIH8, healthy state of close observation animal and ascites sign behind inoculating cell 7d～10d are extracted ascites.After room temperature is placed 30min, 12000r/min behind 4 ℃ of placement 1.5h～2h, centrifugal 2min gets supernatant-20 a ℃ preservation and is equipped with inspection.Detect mouse ascites behind the gradient dilution ascitic type antibody and tire, detection method is with the selection of the described fusion of the step 2 in step 1 mouse, one anti-be ascites after the dilution.Obtain to detect behind the cell strain H1H8 ascitic type antibody and draw the H1H8 mouse ascites and tire and to reach 1: 51200 (Fig. 5).

3, the evaluation of anti-mercury ion antibody subtype

Take out the hybridoma HIH8 cell conditioned medium of cultivating, after dilution in 1: 50, according to the operation of mouse monoclonal antibody hypotype identification kit ISOStripTM Monoclonal Antibody Isotyping Kit specification sheets, to get diluent 0.15mL and add in the test kit tubule, room temperature is placed 1min.After treating that it dissolves naturally, mixing gently.Then reagent strip is inserted into tubule bottom, approximately behind the 5min, when band foremost when two occur in the middle of to "+", promptly visible antibody subclass and the light chain type corresponding indication band position secreted, thereby definite antibody subtype with hybridoma cell strain.

The result shows, when band foremost appear at two to "+" in the middle of the time, antibody subclass and light chain indication band be respectively at G1, the k position, promptly the secreted antibody of cell strain HIH8 is the IgG1 subclass, light chain is kappa type (Fig. 6).

4, the mensuration of hybridoma secretory antibody stability

With hybridoma cell strain frozen after, after a while again the recovery and cultured continuously after survey antibody titer.Detection method is used the Hg-DTPA-OVA coated elisa plate with positive screening of merging the hole in the step 3 of step 1, two anti-goat anti-mouse iggs with horseradish peroxidase-labeled.

Showed cell strain H1H8 secretes anti-metal Hg after testing ²⁺The level of monoclonal antibody is not seen obvious decline (Fig. 7), shows that the stability of antibody is better, and provable subclone is more successful simultaneously.Tiring has fluctuation slightly, may be owing to get cell conditioned medium and examine fully due to the error between different and each detection of the timed interval, belongs to normal.

Three, discuss

Because Hg ²⁺Can with the irreversible reaction of biomolecules generation intensive in the animal body, cause animal to poison, therefore, can utilize specific bifunctional chelating agent DTPA chelating Hg ²⁺Formation can be by the inner complex of animal immune system recognition, but being molecular weight, these heavy metal complexs are lower than the haptens of 1kD, immunogenicity is low, be not enough to cause immune response (Yu HN, Jones RM, Blake DA.An immunosensor for autonomous in-line detection of heavy metals:validation.Int J Environ Anal Chem, 2005,85 (12-13): 817-830).DTPA is except the energy chelated metal ions, can also with carrier protein couplet, the envelope antigen and immunizing antigen (the Darwish IA that can successfully prepare heavy metal Hg, Blake DA.One-step competitive immunoassay for cadmium ions:development and validation for enviromental water samples.Anal Chem, 2001,73 (8): 1889-1895; Darwish IA, Blake DA.Development and validation of a one-step immunoassay for determination of cadmium in human serum.Anal Chem, 2002,74 (1): 52-58).There are multiple proteins such as KLH, BSA, the OVA etc. can be as carrier proteins, but from bringing out the angle of immunne response, the immunogenicity of KLH is better than BSA, OVA etc., so this research comes immune mouse in the hope of producing stronger immunne response with KLH as carrier proteins.

Heavy metal Hg ²⁺The key of Monoclonal Antibody is the immunogenic preparation of heavy metal, thus after immunogen identified again immune mouse be the assurance that success obtains the heavy metal monoclonal antibody.This research has been measured the heavy metal complex protein concentration in the supernatant after the ultrafiltration with the BCA method earlier, then with graphite furnace atomic absorption spectrometry detection Hg wherein ²⁺Content, this be after heavy metal Hg ²⁺The successful preparation of monoclonal antibody reliable assurance is provided.

This research is according to the used mouse of variance rate (Difference%) screening fusion, and Difference% is big more, illustrates to produce at Hg ²⁺Antibodies specific strong more, it is just big more to merge the probability that screening obtains positive hybridoma cell.

This research has successfully obtained preventing from heavy metal Hg ²⁺Or the monoclonal antibody of Hg-DTPA, just identify at present in every performance of antibody purification and antagonist.The preparation of finishing to other preventing from heavy metal antibody of this work provides feasible method, also be that the foundation of heavy metal immunologic detection method and application are (as ELISA method, immune colloid gold quick diagnosis method (Chen Fengmei, Li Juan, Qu Yuanjun, Deng. the progress of immune colloid gold and application. Chinese veterinary drug magazine, 2004,38 (8): 33-35) etc.) lay the foundation.

The antibody that the present invention obtains, can be used for immunology detection technology for detection heavy metal, have that detection speed is fast, expense is cheap, the simple portable of instrument, the highly sensitive and selective advantage such as strong, can be used for on-the-spot heavy metal being carried out sensitivity, accurate, real-time, fast check analysis. Immuno analytical method causes that people more and more pay close attention to, and this also is the inexorable trend of immuno analytical method development, also is that the low suitability for industrialized production of cost is desired.

Claims (3)

1. hybridoma cell strain H1H8 CCTCC № .C200949.

2. hybridoma cell strain H1H8 CCTCC № .C200949 excretory monoclonal antibody.

3. hybridoma cell strain H1H8 CCTCC № .C200949 or its application of excretory monoclonal antibody in the mercury ion immunodetection.

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