CN101845096A

CN101845096A - Cu2<+> specific monoclonal antibody and preparation method thereof

Info

Publication number: CN101845096A
Application number: CN200910218168A
Authority: CN
Inventors: 孔涛; 刘国文; 李小兵; 王哲; 张燚; 唐佳; 杨威; 孙佳; 许超; 张鹏
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2009-12-30
Filing date: 2009-12-30
Publication date: 2010-09-29

Abstract

The invention relates to a Cu2<+> specific monoclonal antibody and a preparation method thereof. The preparation method comprises the following steps of: reacting Cu2<+> with a difunctional chelating agent to form a half hapten; reacting Cu2<+>-chelating agent with carrier protein to form a complete antigen; immuning a BALB (Binaural Alternate Loudness Balance)/c mouse by using the complete antigen; and fusing a splenocyte of the mouse with an SP2/0 cell to prepare a hybridoma cell and obtaining a Cu2<+>-resisting monoclonal antibody. The monoclonal antibody has the specificity on the Cu2<+>, high titer and strong specificity. The invention also provides a preparation method of the Cu2<+>-resisting monoclonal antibody. The antibody and a detection technology are used for detecting residual Cu2<+> in agricultural and animal products and have the advantages of high detection speed, high sensitivity, strong selectivity and low detection cost.

Description

Cu 2+Monoclonal antibody specific and preparation method thereof

Technical field:

The invention provides a kind of Cu ²⁺Monoclonal antibody specific also discloses this MONOCLONAL ANTIBODIES SPECIFIC FOR method simultaneously, belongs to technical field of bioengineering.

Background technology:

Copper accumulates in soil He in the farm crop, can pollute the grain seed, and then copper there be in various degree residual in products such as farming animals, cause that copper staining exceeds standard in the agricultural and animal products, have a strong impact on China's agricultural and animal products quality and international competitiveness, but also the serious harm mankind's is healthy.Therefore, the control and the monitoring of environment and agricultural-food heavy metal contamination all attached great importance in countries in the world.Traditional heavy metal copper detection method as: atomic absorption spectrochemical analysis, inductively coupled plasma emmission spectrometric analysis etc. need the analytical instrument of large-scale costliness, can't be used for on-the-spot the detection, be difficult to conform and the spot check of agricultural and animal products and the requirement that the product import and export speed passage through customs.Therefore, need a kind of new heavy metal copper detection method of invention to address the above problem.

Immunological detection method has fast, cheap, easy, sensitive, special advantage, can be portable and carry out field monitoring.Overcome the shortcoming of traditional detection method.By preparing at Cu ²⁺Specific monoclonal antibody is set up the heavy metal copper immunological detection method.Finishing of this method with solution Cu ²⁺Gordian techniquies such as chelating, coupling, MONOCLONAL ANTIBODIES SPECIFIC FOR are for setting up Cu ²⁺The immunology detection technology lays the foundation.This patent not only is a food safety detection, and provides effective technical means and detection method for the entry and exit detection of agricultural products in China etc., the water area monitoring of environmental monitoring department.Sustainable sound development, solution food-safety problem to agricultural products in China have important practical significance and important society, economic worth.

Summary of the invention:

The invention provides a kind of Cu ²⁺Monoclonal antibody specific has the characteristic of antibody titers height, high specificity.

The present invention also further provides Cu ²⁺The preparation method of monoclonal antibody specific is applicable to suitability for industrialized production.

Cu provided by the invention ²⁺Monoclonal antibody specific is characterized in that: described monoclonal antibody is to Cu ²⁺Has specificity.

Described monoclonal antibody specificity is in conjunction with Cu ²⁺Inner complex, described Cu ²⁺Inner complex is Cu ²⁺-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Cu ²⁺-DOTA) or Cu ²⁺-DOTA-bovine serum albumin conjugate or Cu ²⁺-DOTA-ovalbumin conjugate.

The invention provides a kind of Cu ²⁺The preparation method of monoclonal antibody specific, described method comprises the steps:

(1) preparation of complete antigen:

Get the water-soluble mantoquita of 0.5～10mg and be dissolved in 50 μ L water, the Cu of preparation 2.0～80mg/mL ²⁺Solution; Get 0.8～20mg bifunctional chelating agent S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) is dissolved in 50 μ L dimethyl sulfoxide (DMSO), the p-SCN-Bn-DOTA solution of preparation 16～400mg/mL; Get 2.0～20.0mg carrier proteins and be dissolved in 1mL HEPES buffered soln or carbonate buffer solution, the carrier proteins solution of preparation 2～20mg/mL; The Cu of 50 μ L2.0～80mg/mL with preparation ²⁺Solution dropwise joins in the 50 μ Lp-SCN-Bn-DOTA solution, in 15～55 ℃, pH4.5～6.5, oscillating condition down reaction 15min～3h form haptens solution; Again this haptens solution is dropwise joined in the carrier proteins solution of 1mL 2～20mg/mL oscillatory reaction 24h under 4 ℃ or room temperature, pH7.5～10.5 conditions; Remove unreacted lower-molecular substance with the method for ultrafiltration or dialysis, get Cu ²⁺Immunizing antigen and detect antigen;

(2) mouse immune:

Immunizing antigen immune mouse with step (1) preparation;

(3) cytogamy

Select serum titer height and the high mouse of variance rate, the splenocyte and the myeloma cell of immune mouse are merged;

(4) screening of hybridoma cell strain and clone

Two kinds of detection antigens with same concentrations wrap respectively by elisa plate, carry out the screening of hybridoma cell strain; Select positive value height and the high cell strain of variance rate, clone with limiting dilution assay.

Among the above-mentioned preparation method, used water-soluble mantoquita comprises Cu (NO in the step (1) ₃) ₂Or CuCl ₂, CuSO ₄, Cu (CH ₃COO) ₂, CuBr ₂Any one.

Among the above-mentioned preparation method, used carrier albumen is any one in keyhole limpet hemocyanin or bovine serum albumin or ovalbumin or the human serum albumin in the step (1).

Among the above-mentioned preparation method, carrier proteins and p-SCN-Bn-DOTA and Cu in step (1) reaction soln ²⁺Mass ratio be: (8～12): (8～12): 1.

Among the above-mentioned preparation method: the buffered soln of dissolving carrier proteins is the HEPES damping fluid in the step (1), and concentration is that 30～100mM, pH are 7.5～10.5; Or carbonate buffer solution, pH is 9.0～10.5.

Among the above-mentioned preparation method, the damping fluid that is used to dissolve, preserve complete antigen in the step (1) is a HEPES buffered soln, and concentration is that 30～100mM, pH are 6.5～8.0; Or the ammonium citrate damping fluid, concentration is that 10～30mM, pH are 6.5～8.0.

Among the above-mentioned preparation method, when adopting in the step (1) hyperfiltration process to remove unreacted lower-molecular substance, selecting molecular weight cut-off for use is the ultra-filtration centrifuge tube of 10000～30000dal, 6000rpm～8000rpm ultrafiltration 6 times, each 5～30min.

Among the above-mentioned preparation method, immunizing antigen is Cu ²⁺-p-SCN-Bn-DOTA-KLH or Cu ²⁺-p-SCN-Bn-DOTA-BSA or Cu ²⁺-p-SCN-Bn-DOTA-OVA or Cu ²⁺Any one of-p-SCN-Bn-DOTA-HAS.

Among the above-mentioned preparation method, used detection antigen is Cu in the step (4) ²⁺-p-SCN-Bn-DOTA-BSA, p-SCN-Bn-DOTA-BSA use respectively; Or Cu ²⁺-p-SCN-Bn-DOTA-OVA, p-SCN-Bn-DOTA-OVA use respectively; Or Cu ²⁺-p-SCN-Bn-DOTA-HAS, p-SCN-Bn-DOTA-HAS use respectively.

Among the above-mentioned preparation method, merge in the step (3) with the splenocyte of mouse and myeloma cell with 3～12: 1 mixes.

The present invention compared with prior art has following advantage and beneficial effect:

1, utilize antibody among the present invention and detection technique to the Cu in the agricultural and animal products ²⁺The residual detection, detection speed is fast, highly sensitive, selectivity strong, it is low to detect cost.

2, utilize antibody among the present invention and detection technique to the Cu in the agricultural and animal products ²⁺The residual detection only needs a microplate reader to detect, simple portable, and can be used for on-the-spot the detection.

Description of drawings:

Fig. 1 is complete antigen electrophoresis result figure;

1：Cu ²⁺-p-SCN-Bn-DOTA-BSA，2：p-SCN-Bn-DOTA-BSA，3：BSA

Fig. 2 is Cu ²⁺The UV spectrum detected result of-p-SCN-Bn-DOTA-BSA, p-SCN-Bn-DOTA-BSA, BSA;

Fig. 3 is the competitive ELISA result of sequestrant to antigen antibody reaction;

Fig. 4 is the competitive ELISA result of metallic copper to antigen antibody reaction.

Embodiment:

For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood that it is to explaination of the present invention but not to any type of restriction of the present invention.

Embodiment 1

(1) preparation of complete antigen:

Take by weighing 4mg Cu (NO ₃) ₂Be dissolved in 50 μ L ultrapure waters, 10mg p-SCN-Bn-DOTA is dissolved in 50 μ LDMSO, and 20mg carrier proteins KLH or BSA, OVA, HSA are dissolved in 1mL 100mM HEPES damping fluid (pH9.5).With Cu (NO ₃) ₂Solution dropwise joins in the p-SCN-Bn-DOTA solution, constantly stirs reaction 3h down, forms Cu ²⁺-p-SCN-Bn-DOTA haptens.With Cu ²⁺-p-SCN-Bn-DOTA solution dropwise joins in the 0.5mL carrier proteins solution, room temperature, oscillatory reaction 24h.It is in the 30000dal ultrafiltration pipe that antigen is transferred to molecular weight cut-off, the centrifugal 10min of 7500rpm, with 100mM HEPES damping fluid (pH7.5) washing 6 times, again with antigen with 100mMHEPES damping fluid (pH7.5) dilution, packing, Cu ²⁺Complete antigen is in-20 ℃ of preservations.

(2) mouse immune:

Immunizing antigen Cu with step (1) preparation ²⁺-p-SCN-Bn-DOTA-KLH immunity BALB/c mouse in 6～8 age in week.The 1st, 14,21,35d injects to mouse peritoneal, each immunizing antigen consumption be 50 μ g/ only.Initial immunity equivalent Freund's complete adjuvant mixing and emulsifying, the 2nd immunity Freund's incomplete adjuvant mixing and emulsifying.From the 3rd immunity, each immunity back 10d takes a blood sample from mouse tail vein, surveys the mice serum antibody titer with indirect ELISA.

With coating buffer to detecting antigens c u ²⁺-p-SCN-Bn-DOTA-BSA and p-SCN-Bn-DOTA-BSA dilute, with the Cu of same concentrations ²⁺-p-SCN-Bn-DOTA-BSA and p-SCN-Bn-DOTA-BSA wrap respectively by elisa plate 100 μ L/ holes, and 4 ℃ of placements are spent the night or 37 ℃ of effect 2h.With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time.With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h.With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time.The serum to be checked that adding is diluted with phosphate buffered saline buffer, 100 μ L/ holes, 37 ℃ of effect 1h.With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time.The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h.With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time.O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min.The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm surveys the OD value on the microplate reader.With immune serum OD value/normal mouse serum OD value＞2.1, and OD value＞0.2 is judged as the positive.

(3) cytogamy:

Select serum titer height and the high mouse of variance rate, 3d impacts immunity before cytogamy, and the immunizing antigen consumption is 100 μ g/.With the splenocyte of immune mouse and myeloma cell with 3～12: 1 mixes, adding 0.7～1.2mL, 50% polyoxyethylene glycol in the 1min merges, leave standstill 90s, in 5min, add 25mL serum-free RPMI RPMI-1640 then, stop fusion reaction, the centrifugal 10min of 800～1200rpm/min, resuspended with the complete RPMI RPMI-1640 that contains 2%HAT, cell suspension is added the 96 porocyte culture plates that contain feeder cell, and every hole 100 μ L put 37 ℃, 5%CO with culture plate ₂Cultivate in the incubator.Use the HAT nutrient solution after fusion in the 7d, the 7th～14d uses the HT nutrient solution instead, cultivates with complete RPMI RPMI-1640 behind the 14d.

(4) screening of hybridoma cell strain and clone:

Cu with same concentrations ²⁺-p-SCN-Bn-DOTA-BSA and p-SCN-Bn-DOTA-BSA wrap respectively by elisa plate, 100 μ L/ holes, and 4 ℃ of placements are spent the night; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; Add cells and supernatant to be checked, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min; The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm reading on the microplate reader.With hybridoma supernatant OD value/myeloma cell's supernatant serum OD value＞2.1, and OD value＞0.2 is judged as the positive.

(5) evaluation of monoclonal antibody:

Positive colony supernatant by the indirect ELISA primary dcreening operation further determines that with the indirect competitive ELISA method whether secreted antibody is at Cu ²⁺, and do not react with sequestrant.Concrete steps are as follows: antigens c u ²⁺-p-SCN-Bn-DOTA-BSA bag is by elisa plate, and 4 ℃ of placements are spent the night; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h; With the hybridoma supernatant equal-volume mixing that p-SCN-Bn-DOTA solution of series concentration (1,2,5,10,20,50,100mM) and primary dcreening operation are positive, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min; The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm reading on the microplate reader.With hybridoma supernatant OD value/myeloma cell's supernatant serum OD value＞2.1, and OD value＞0.2 is judged as the positive.

(6) preparation of ascites and purifying:

Get 8～10 age in week BALB/c mouse, abdominal injection whiteruss 0.5ml/ only, the hybridoma that is in logarithmic phase that pneumoretroperitoneum inoculation in 7 days is diluted with serum-free RPMI1640 nutrient solution, every injected in mice cell count is 0.5～2 * 10 ⁶Individual, observe the mouse ascites production every day, treat that mouse web portion expands, spiritual variation, dying when motionless, with No. 16 syringe needle aseptic collection ascites.Ascites is centrifugal, and supernatant liquor is anti-Cu ²⁺Monoclonal antibody, packing ,-80 ℃ are frozen standby.With behind the ammonium sulfate precipitation method preliminary purification again through the protein g affinity chromatography column purification.

Embodiment 2

(1) preparation of complete antigen:

Take by weighing 4mg Cu (NO ₃) ₂Be dissolved in 50 μ L ultrapure waters, 10mg p-SCN-Bn-DOTA is dissolved in 50 μ L DMSO, and 20mg carrier proteins KLH or BSA, OVA, HSA are dissolved in 1mL 50mM carbonate buffer solution (pH9.5).With Cu (NO ₃) ₂Solution dropwise joins in the p-SCN-Bn-DOTA solution, constantly stirs reaction 3h down, forms Cu ²⁺-p-SCN-Bn-DOTA haptens.With Cu ²⁺-p-SCN-Bn-DOTA solution dropwise joins in the 0.5mL carrier proteins solution, room temperature, oscillatory reaction 24h.It is in the 30000dal ultrafiltration pipe that antigen is transferred to molecular weight cut-off, the centrifugal 10min of 7500rpm, with 15mM ammonium citrate damping fluid (pH7.5) washing 6 times, again with antigen with 15mM ammonium citrate damping fluid (pH7.5) dilution, packing, Cu ²⁺Complete antigen is in-20 ℃ of preservations.

(2) mouse immune:

(3) cytogamy:

(4) screening of hybridoma cell strain and clone:

(5) evaluation of monoclonal antibody:

(6) preparation of ascites and purifying:

Embodiment 3

(1) preparation of complete antigen:

Take by weighing 4mg Cu (NO ₃) ₂Be dissolved in 50 μ L ultrapure waters, 10mg p-SCN-Bn-DOTA is dissolved in 50 μ L DMSO, and 20mg carrier proteins KLH or BSA, OVA, HSA are dissolved in 1mL 100mM HEPES damping fluid (pH9.5).With Cu (NO ₃) ₂Solution dropwise joins in the p-SCN-Bn-DOTA solution, constantly stirs reaction 3h down, forms Cu ²⁺-p-SCN-Bn-DOTA haptens.With Cu ²⁺-p-SCN-Bn-DOTA solution dropwise joins in the 0.5mL carrier proteins solution, room temperature, oscillatory reaction 24h.It is in the 30000dal ultrafiltration pipe that antigen is transferred to molecular weight cut-off, the centrifugal 10min of 7500rpm, with 100mM HEPES damping fluid (pH7.5) washing 6 times, again with antigen with 100mMHEPES damping fluid (pH7.5) dilution, packing, Cu ²⁺Complete antigen is in-20 ℃ of preservations.

(2) mouse immune:

With coating buffer to detecting antigens c u ²⁺-p-SCN-Bn-DOTA-OVA and p-SCN-Bn-DOTA-OVA dilute, with the Cu of same concentrations ²⁺-p-SCN-Bn-DOTA-OVA and p-SCN-Bn-DOTA-OVA wrap respectively by elisa plate 100 μ L/ holes, and 4 ℃ of placements are spent the night or 37 ℃ of effect 2h.With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time.With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h.With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time.The serum to be checked that adding is diluted with phosphate buffered saline buffer, 100 μ L/ holes, 37 ℃ of effect 1h.With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time.The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h.With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time.O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min.The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm surveys the OD value on the microplate reader.With immune serum OD value/normal mouse serum OD value＞2.1, and OD value＞0.2 is judged as the positive.

(3) cytogamy:

(4) screening of hybridoma cell strain and clone:

Cu with same concentrations ²⁺-p-SCN-Bn-DOTA-OVA and p-SCN-Bn-DOTA-OVA wrap respectively by elisa plate, 100 μ L/ holes, and 4 ℃ of placements are spent the night; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; Add cells and supernatant to be checked, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min; The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm reading on the microplate reader.With hybridoma supernatant OD value/myeloma cell's supernatant serum OD value＞2.1, and OD value＞0.2 is judged as the positive.Select positive value height and the high cell strain of variance rate, clone with limiting dilution assay.

(5) evaluation of monoclonal antibody:

Positive colony supernatant by the indirect ELISA primary dcreening operation further determines that with the indirect competitive ELISA method whether secreted antibody is at Cu ²⁺, and do not react with sequestrant.Concrete steps are as follows: antigens c u ²⁺-p-SCN-Bn-DOTA-OVA bag is by elisa plate, and 4 ℃ of placements are spent the night; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h; With the hybridoma supernatant equal-volume mixing that p-SCN-Bn-DOTA solution of series concentration (1,2,5,10,20,50,100mM) and primary dcreening operation are positive, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min; The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm reading on the microplate reader.With hybridoma supernatant OD value/myeloma cell's supernatant serum OD value＞2.1, and OD value＞0.2 is judged as the positive.

(6) preparation of ascites and purifying:

Embodiment 4

(1) preparation of complete antigen:

Take by weighing 4mg Cu (NO ₃) ₂Be dissolved in 50 μ L ultrapure waters, 10mg p-SCN-Bn-DOTA is dissolved in 50 μ LDMSO, and 20mg carrier proteins KLH or BSA, OVA, HSA are dissolved in 1mL 50mM carbonate buffer solution (pH9.5).With Cu (NO ₃) ₂Solution dropwise joins in the p-SCN-Bn-DOTA solution, constantly stirs reaction 3h down, forms Cu ²⁺-p-SCN-Bn-DOTA haptens.With Cu ²⁺-p-SCN-Bn-DOTA solution dropwise joins in the 0.5mL carrier proteins solution, room temperature, oscillatory reaction 24h.It is in the 30000dal ultrafiltration pipe that antigen is transferred to molecular weight cut-off, the centrifugal 10min of 7500rpm, with 15mM ammonium citrate damping fluid (pH7.5) washing 6 times, again with antigen with 15mM ammonium citrate damping fluid (pH7.5) dilution, packing, Cu ²⁺Complete antigen is in-20 ℃ of preservations.

(2) mouse immune:

(3) cytogamy:

(4) screening of hybridoma cell strain and clone

(5) evaluation of monoclonal antibody:

(6) preparation of ascites and purifying:

Embodiment 5

(1) preparation of complete antigen:

(2) mouse immune:

Immunizing antigen Cu with step (1) preparation ²⁺-p-SCN-Bn-DOTA-BSA immunity BALB/c mouse in 6～8 age in week.The 1st, 14,21,35d injects to mouse peritoneal, each immunizing antigen consumption be 50 μ g/ only.Initial immunity equivalent Freund's complete adjuvant mixing and emulsifying, the 2nd immunity Freund's incomplete adjuvant mixing and emulsifying.From the 3rd immunity, each immunity back 10d takes a blood sample from mouse tail vein, surveys the mice serum antibody titer with indirect ELISA.

(3) cytogamy

(4) screening of hybridoma cell strain and clone

(5) evaluation of monoclonal antibody:

(6) preparation of ascites and purifying:

Embodiment 6

(1) preparation of complete antigen:

(2) mouse immune:

Immunizing antigen Cu with step (1) preparation ²⁺-p-SCN-Bn-DOTA-HSA immunity BALB/c mouse in 6～8 age in week.The 1st, 14,21,35d injects to mouse peritoneal, each immunizing antigen consumption be 50 μ g/ only.Initial immunity equivalent Freund's complete adjuvant mixing and emulsifying, the 2nd immunity Freund's incomplete adjuvant mixing and emulsifying.From the 3rd immunity, each immunity back 10d takes a blood sample from mouse tail vein, surveys the mice serum antibody titer with indirect ELISA.

(3) cytogamy:

(4) screening of hybridoma cell strain and clone:

(5) evaluation of monoclonal antibody:

(6) preparation of ascites and purifying:

Embodiment 7

(1) preparation of complete antigen:

Take by weighing 3.2mg CuCl ₂Be dissolved in 50 μ L ultrapure waters, 10mg p-SCN-Bn-DOTA is dissolved in 50 μ L DMSO, and 20mg carrier proteins KLH or BSA, OVA, HSA are dissolved in 1mL 100mM HEPES damping fluid (pH9.5).With CuCl ₂Solution dropwise joins in the p-SCN-Bn-DOTA solution, constantly stirs reaction 3h down, forms Cu ²⁺-p-SCN-Bn-DOTA haptens.With Cu ²⁺-p-SCN-Bn-DOTA solution dropwise joins in the 0.5mL carrier proteins solution, room temperature, oscillatory reaction 24h.It is in the 30000dal ultrafiltration pipe that antigen is transferred to molecular weight cut-off, the centrifugal 10min of 7500rpm, with 100mM HEPES damping fluid (pH7.5) washing 6 times, again with antigen with 100mMHEPES damping fluid (pH7.5) dilution, packing, Cu ²⁺Complete antigen is in-20 ℃ of preservations.

(2) mouse immune:

(3) cytogamy:

(4) screening of hybridoma cell strain and clone:

Cu with same concentrations ²⁺-p-SCN-Bn-DOTA-BSA and p-SCN-Bn-DOTA-BSA wrap respectively by elisa plate, 100 μ L/ holes, and 4 ℃ of placements are spent the night; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; Add cells and supernatant to be checked, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min; The 2M H that adds 50 μ L/ holes ₂S0 ₄Termination reaction.490nm reading on the microplate reader.With hybridoma supernatant OD value/myeloma cell's supernatant serum OD value＞2.1, and OD value＞0.2 is judged as the positive.Select positive value height and the high cell strain of variance rate, clone with limiting dilution assay.

(5) evaluation of monoclonal antibody:

(6) preparation of ascites and purifying:

Get 8～10 age in week BALB/c mouse, abdominal injection whiteruss 0.5ml/ only, the hybridoma that is in logarithmic phase that pneumoretroperitoneum inoculation in 7 days is diluted with serum-free RPMI1640 nutrient solution, every injected in mice cell count is 0.5～2 * 10 ⁶Individual, observe the mouse ascites production every day, treat that water mouse belly expands, spiritual variation, dying when motionless, with No. 16 syringe needle aseptic collection ascites.Ascites is centrifugal, and supernatant liquor is anti-Cu ²⁺Monoclonal antibody, packing ,-80 ℃ are frozen standby.With behind the ammonium sulfate precipitation method preliminary purification again through the protein g affinity chromatography column purification.

Embodiment 8

(1) preparation of complete antigen:

Take by weighing 4.4mg CuSO ₄5H ₂O is dissolved in 50 μ L ultrapure waters, and 10mg p-SCN-Bn-DOTA is dissolved in 50 μ LDMSO, and 20mg carrier proteins KLH or BSA, OVA, HSA are dissolved in 1mL 100mM HEPES damping fluid (pH9.5).With CuSO ₄5H ₂O solution dropwise joins in the p-SCN-Bn-DOTA solution, constantly stirs reaction 3h down, forms Cu ²⁺-p-SCN-Bn-DOTA haptens.With Cu ²⁺-p-SCN-Bn-DOTA solution dropwise joins in the 0.5mL carrier proteins solution, room temperature, oscillatory reaction 24h.It is in the 30000dal ultrafiltration pipe that antigen is transferred to molecular weight cut-off, the centrifugal 10min of 7500rpm, with 100mM HEPES damping fluid (pH7.5) washing 6 times, again with antigen with 100mM HEPES damping fluid (pH7.5) dilution, packing, Cu ²⁺Complete antigen is in-20 ℃ of preservations.

(2) mouse immune:

(3) cytogamy:

(4) screening of hybridoma cell strain and clone:

Cu with same concentrations ²⁺-p-SCN-Bn-DOTA-BSA and p-SCN-Bn-DOTA-BSA wrap respectively by elisa plate, 100 μ L/ holes, and 4 ℃ of placements are spent the night; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; Add cells and supernatant to be checked, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min; The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm reading on the microplate reader.With hybridoma supernatant OD value/myeloma cell's supernatant serum OD value＞2.1, and OD value＞0.2 is judged as the positive.Select positive value height and the high cell strain of variance rate, clone with limiting dilution assay.

(5) evaluation of monoclonal antibody:

(6) preparation of ascites and purifying:

Embodiment 9

(1) preparation of complete antigen:

Take by weighing 3.4mg Cu (CH ₃COO) ₂Be dissolved in 50 μ L ultrapure waters, 10mg p-SCN-Bn-DOTA is dissolved in 50 μ LDMSO, and 20mg carrier proteins KLH or BSA, OVA, HSA are dissolved in 1mL 100mM HEPES damping fluid (pH9.5).With Cu (CH ₃COO) ₂Solution dropwise joins in the p-SCN-Bn-DOTA solution, constantly stirs reaction 3h down, forms Cu ²⁺-p-SCN-Bn-DOTA haptens.With Cu ²⁺-p-SCN-Bn-DOTA solution dropwise joins in the 0.5mL carrier proteins solution, room temperature, oscillatory reaction 24h.It is in the 30000dal ultrafiltration pipe that antigen is transferred to molecular weight cut-off, the centrifugal 10min of 7500rpm, with 100mM HEPES damping fluid (pH7.5) washing 6 times, again with antigen with 100mM HEPES damping fluid (pH7.5) dilution, packing, Cu ²⁺Complete antigen is in-20 ℃ of preservations.

(2) mouse immune:

(3) cytogamy:

(4) screening of hybridoma cell strain and clone:

(5) evaluation of monoclonal antibody:

(6) preparation of ascites and purifying:

Get 8～10 age in week BALB/c mouse, abdominal injection whiteruss 0.5ml/ only, the hybridoma that is in logarithmic phase that pneumoretroperitoneum inoculation in 7 days is diluted with serum-free RPMI1640 nutrient solution, every injected in mice cell count is 0.5～2 * 10 ⁶Individual, observe the mouse ascites production every day, treat that mouse web portion expands, spiritual variation, dying when motionless, with No. 16 syringe needle aseptic collection ascites.Ascites is centrifugal, and supernatant liquor is anti-Cu2+ monoclonal antibody, packing, and-80 ℃ are frozen standby.With behind the ammonium sulfate precipitation method preliminary purification again through the protein g affinity chromatography column purification.

Embodiment 10

(1) preparation of complete antigen:

Take by weighing 3.7mg CuBr2 and be dissolved in 50 μ L ultrapure waters, 10mg p-SCN-Bn-DOTA is dissolved in 50 μ L DMSO, and 20mg carrier proteins KLH or BSA, OVA, HSA are dissolved in 1mL 100mM HEPES damping fluid (pH9.5).With CuBr ₂Solution dropwise joins in the p-SCN-Bn-DOTA solution, constantly stirs reaction 3h down, forms Cu ²⁺-p-SCN-Bn-DOTA haptens.With Cu ²⁺-p-SCN-Bn-DOTA solution dropwise joins in the 0.5mL carrier proteins solution, room temperature, oscillatory reaction 24h.It is in the 30000dal ultrafiltration pipe that antigen is transferred to molecular weight cut-off, the centrifugal 10min of 7500rpm, with 100mM HEPES damping fluid (pH7.5) washing 6 times, again with antigen with 100mMHEPES damping fluid (pH7.5) dilution, packing, Cu ²⁺Complete antigen is in-20 ℃ of preservations.

(2) mouse immune:

(3) cytogamy:

Select serum titer height and the high mouse of variance rate, 3d impacts immunity before cytogamy, and the immunizing antigen consumption is 100 μ g.With the splenocyte of immune mouse and myeloma cell with 3～10: 1 mixes, adding 0.7～1.2mL, 50% polyoxyethylene glycol in the 1min merges, leave standstill 90s, in 5min, add 25mL serum-free RPMI RPMI-1640 then, stop fusion reaction, the centrifugal 10min of 800～1200rpm/min, resuspended with the complete RPMI RPMI-1640 that contains 2%HAT, cell suspension is added the 96 porocyte culture plates that contain feeder cell, and every hole 100 μ L put 37 ℃, 5%CO with culture plate ₂Cultivate in the incubator.Use the HAT nutrient solution after fusion in the 7d, the 7th～14d uses the HT nutrient solution instead, cultivates with complete RPMI RPMI-1640 behind the 14d.

(4) screening of hybridoma cell strain and clone:

(5) evaluation of monoclonal antibody:

(6) preparation of ascites and purifying:

Test example 1

The complete antigen that the present invention is made carries out following detection:

(1) the SDS-PAGE electrophoresis detection of complete antigen:

The SDS-PAGE electrophoresis result shows that the travelling speed of BSA is faster than p-SCN-Bn-DOTA-BSA mixture, the travelling speed of p-SCN-Bn-DOTA-BSA mixture compares Cu ²⁺-p-SCN-Bn-DOTA-BSA mixture is fast, shows Cu ²⁺The molecular weight of-p-SCN-Bn-DOTA-BSA mixture is greater than the p-SCN-Bn-DOTA-BSA mixture, and the molecular weight of p-SCN-Bn-DOTA-BSA mixture is greater than BSA.According to this result can the preliminary evaluation complete antigen synthetic success.Detected result is seen Fig. 1.

(2) ultraviolet spectrophotometry of complete antigen detects:

Complete antigen is carried out 200～800nm full wavelength scanner, and the result shows that the ultraviolet absorption peak of complete antigen compares carrier proteins and drift about, and peak value is compared with carrier proteins and changed, and further confirms the synthetic success of complete antigen in view of the above.With Cu ²⁺-p-SCN-Bn-DOTA-BSA, p-SCN-Bn-DOTA-BSA are example, and detected result is seen Fig. 2.

(3) Cu of complete antigen ²⁺Content measurement:

Graphite furnace atomic absorption spectrometry detects the Cu in the complete antigen ²⁺Content, the result shows Cu in the complete antigen ²⁺Content reaches 4.0 μ g/mL～20.0 μ g/mL, and this result further shows the synthetic success of complete antigen.With Cu ²⁺-p-SCN-Bn-DOTA-KLH, Cu ²⁺-p-SCN-Bn-DOTA-BSA, p-SCN-Bn-DOTA-BSA are example, and detected result sees Table 1.

Cu in table 1 complete antigen ²⁺Content detection result

Test example 2

The specific evaluation of monoclonal antibody:

Primary dcreening operation positive cells supernatant further determines that with the indirect competitive ELISA method whether secreted antibody is at Cu ²⁺, and do not react with sequestrant.With 4 strain cell conditioned mediums wherein is example, and concrete steps are as follows: antigens c u ²⁺-p-SCN-Bn-DOTA-BSA bag is by elisa plate, and 4 ℃ of placements are spent the night; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h; With the hybridoma supernatant equal-volume mixing that p-SCN-Bn-DOTA solution of series concentration (1,2,5,10,20,50,100mM) and primary dcreening operation are positive, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min; The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm reading on the microplate reader.With hybridoma supernatant OD value/myeloma cell's supernatant serum OD value＞2.1, and OD value＞0.2 is judged as the positive.Sequestrant concentration is less than 5mmol/L as a result, and sequestrant is to not influence of antigen antibody reaction.Detected result is seen Fig. 3.

(2) adopt the indirect competitive ELISA method, the positive cell supernatant is identified further the concrete steps method is as follows: with complete antigen Cu ²⁺-p-SCN-Bn-DOTA-BSA wraps by elisa plate, 100 μ L/ holes, and 4 ℃ of placements are spent the night; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ the effect 1.5h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; With the HEPES damping fluid that contains 2mM p-SCN-Bn-DOTA serial dilution copper standardized solution step by step, the cadmium standardized solution, plumbous standardized solution, the zinc standardized solution, the manganese standardized solution, the mercury standardized solution, the nickel standardized solution, the magnesium standardized solution, calcium standard solution becomes 0.5mmol/L, 0.25mmol/L, 0.125mmol/L, 0.0625mmol/L, 0.03125mmol/L, 0.015625mmol/L, 0.0078125mmol/L, 0.00390625mmol/L series concentration, the solution that dilution is good is put 37～50 ℃, 0.5～1h, again with the copper antibody equal-volume mixing room temperature reaction 1h that is diluted to certain multiple, join in the elisa plate, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; The sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h; With the phosphate buffered saline buffer that contains 0.5% polysorbas20 (PBST) washing, 3min * 3 time; O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min; The 2M H that adds 50 μ L/ holes ₂SO ₄Termination reaction.490nm reading on the microplate reader.Calculate the half-inhibition concentration of metal pair antibody.With the copper standardized solution is that the example detected result is seen Fig. 4.

Test example 3

Adopt the indirect competitive ELISA method, detection curve is carried out desk study, method is as follows:

Bag quilt: with complete antigen Cu ²⁺-p-SCN-Bn-DOTA-BSA wraps by elisa plate, 100 μ L/ holes, and 4 ℃ of placements are spent the night;

Washing: with phosphate buffered saline buffer (PBST) washing that contains 0.5% polysorbas20,3min * 3 time;

Sealing: with 20% rabbit anteserum as confining liquid, 100 μ L/ holes, 37 ℃ act on 1.5h;

Application of sample: with the HEPES damping fluid that contains 2mM p-SCN-Bn-DOTA step by step serial dilution copper standardized solution become 0.5mmol/L, 0.25mmol/L, 0.125mmol/L, 0.0625mmol/L, 0.03125mmol/L, 0.015625mmol/L, 0.0078125mmol/L, 0.00390625mmol/L series concentration, the solution that dilution is good is put 37～50 ℃, 0.5～1h, again with the copper antibody equal-volume mixing room temperature reaction 1h that is diluted to certain multiple, join in the elisa plate, 100 μ L/ holes, 37 ℃ of effect 1h;

Add ELIAS secondary antibody: the sheep anti mouse two that adds the HRP enzyme labelling of using 5000 times of dilutions of PBS is anti-, 100 μ L/ holes, 37 ℃ of effect 1h;

Colour developing: O-Phenylene Diamine (OPD) colour developing, 100 μ L/ holes, 37 ℃ of lucifuge reaction 10min;

Stop: the 2M H that adds 50 μ L//holes ₂SO ₄Termination reaction.490nm reading on the microplate reader.

490nm reading on the microplate reader.The IC of antibody as a result ₅₀=0.399mg/L, result show that the antibody of preparation is to Cu ²⁺Good recognition capability is arranged, can be used for developing Cu ²⁺Quick detection kit and colloidal gold immune chromatography test.

Claims

1. Cu ²⁺Monoclonal antibody specific is characterized in that, described monoclonal antibody is to Cu ²⁺Has specificity.

2. monoclonal antibody according to claim 1 is characterized in that, described monoclonal antibody is that specificity is in conjunction with Cu ²⁺Inner complex, described Cu ²⁺Inner complex is Cu ²⁺-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid or Cu ²⁺-DOTA-bovine serum albumin conjugate or Cu ²⁺-DOTA-ovalbumin conjugate.

3. according to the described Cu of claim 1 ²⁺The preparation method of monoclonal antibody specific comprises the steps:

(1) preparation of complete antigen:

Get the water-soluble mantoquita of 0.5～10mg and be dissolved in 50 μ L water, the Cu of preparation 2.0～80mg/mL ²⁺Solution; Get 0.8～20mg bifunctional chelating agent S-2-(4-isothiocyanatobenzyl)-1,4,7,1 0-tetraazacyclododecane-1,4,7,10-tetraaceticacid (p-SCN-Bn-DOTA) is dissolved in 50 μ L dimethyl sulfoxide (DMSO), the p-SCN-Bn-DOTA solution of preparation 16～400mg/mL; Get 2.0～20.0 mg carrier proteinss and be dissolved in 1mL HEPES buffered soln or carbonate buffer solution, the carrier proteins solution of preparation 2～20mg/mL; The Cu of 50 μ L, 2.0～80 mg/mL with preparation ²⁺Solution dropwise joins in the 50 μ L p-SCN-Bn-DOTA solution, in 15～55 ℃, pH4.5～6.5, oscillating condition down reaction 15min～3h form haptens solution; Again this haptens solution is dropwise joined in the carrier proteins solution of 1mL2～20mg/mL oscillatory reaction 24h under 4 ℃ or room temperature, pH7.5～10.5 conditions; Remove unreacted lower-molecular substance with the method for ultrafiltration or dialysis, get Cu ²⁺Immunizing antigen and detect antigen;

(2) mouse immune:

Immunizing antigen immune mouse with step (1) preparation;

(3) cytogamy

(4) screening of hybridoma cell strain and clone

4. according to the described preparation method of claim 3, it is characterized in that: used water-soluble mantoquita comprises Cu (NO in the step (1) ₃) ₂Or CuCl ₂, CuSO ₄, Cu (CH ₃COO) ₂, CuBr ₂Any one.

5. according to the described preparation method of claim 3, it is characterized in that: used carrier albumen is any one in keyhole limpet hemocyanin or bovine serum albumin or ovalbumin or the human serum albumin in the step (1).

6. according to the described preparation method of claim 3, it is characterized in that: carrier proteins and p-SCN-Bn-DOTA and Cu in step (1) reaction soln ²⁺Mass ratio be: (8～12): (8～12): 1.

7. according to the described preparation method of claim 3, it is characterized in that: when adopting in the step (1) hyperfiltration process to remove unreacted lower-molecular substance, selecting molecular weight cut-off for use is the ultra-filtration centrifuge tube of 10000～30000dal, 6000rpm～8000rpm ultrafiltration 6 times, each 5～30min.

8. according to the described preparation method of claim 3, it is characterized in that: immunizing antigen is Cu ²⁺-p-SCN-Bn-DOTA-KLH or Cu ²⁺-p-SCN-Bn-DOTA-BSA or Cu ²⁺-p-SCN-Bn-DOTA-OVA or Cu ²⁺Any one of-p-SCN-Bn-DOTA-HAS.

9. according to the described preparation method of claim 3, it is characterized in that:

Used detection antigen is Cu in the step (4) ²⁺-p-SCN-Bn-DOTA-BSA, p-SCN-Bn-DOTA-BSA use respectively; Or Cu ²⁺-p-SCN-Bn-DOTA-OVA, p-SCN-Bn-DOTA-OVA use respectively; Or Cu ²⁺-p-SCN-Bn-DOTA-HAS, p-SCN-Bn-DOTA-HAS use respectively.

10. according to the described preparation method of claim 3, it is characterized in that: merge in the step (3) with the splenocyte of mouse and myeloma cell with 3～12: 1 mixes.