CN110408598A - One plant of hybridoma cell strain for secreting preventing from heavy metal lead ion monoclonal antibody and its application - Google Patents

One plant of hybridoma cell strain for secreting preventing from heavy metal lead ion monoclonal antibody and its application Download PDF

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Publication number
CN110408598A
CN110408598A CN201811506000.3A CN201811506000A CN110408598A CN 110408598 A CN110408598 A CN 110408598A CN 201811506000 A CN201811506000 A CN 201811506000A CN 110408598 A CN110408598 A CN 110408598A
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liquid
heavy metal
solution
lead ion
monoclonal antibody
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金仁耀
郭建军
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Zhejiang Gongshang University
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Zhejiang Gongshang University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Abstract

The hybridoma cell strain for secreting preventing from heavy metal lead ion monoclonal antibody the present invention provides one plant and its application；The hybridoma cell strain can secrete preventing from heavy metal lead ion monoclonal antibody, and the present invention also provides the preparation methods of the preparation method of the hybridoma cell strain and the preventing from heavy metal lead ion monoclonal antibody；The monoclonal antibody specificity that the present invention is prepared is stronger, has preferably sensitivity, IC to lead ion20Value is 0.45 ngmL‑1, can be used in quickly and accurately detecting heavy metal lead ion.The anti-lead ion monoclonal antibody can be used for the residue detection of lead ion in environmental sample.

Description

One plant secretion preventing from heavy metal lead ion monoclonal antibody hybridoma cell strain and its Using
Technical field
The invention belongs to immunochemistry detection technique fields, and in particular to one plant of secretion preventing from heavy metal lead ion monoclonal is anti- The hybridoma cell strain of body and its application.
Background technique
Heavy metal pollution refers mainly to pollution of the pollutants such as lead, cadmium, mercury, nickel, chromium, arsenic, zinc, copper to environment.Heavy metal point Cloth is extensive, is difficult to degrade, and can enter human body by big gas and water, food chain, have an effect, make with vivo protein and various enzymes They lose activity, and are enriched in certain organs, if it exceeds the limit that human body is resistant to, it is acute or slow to will cause human body Property poisoning, have carcinogenic, teratogenesis and mutagenesis, to human body have very big harm.Therefore, reinforce heavy metal environment, Important means of the residue detection as guarantee heavy metal safety in agricultural product and food, and the research of new detecting technique under the new situation Seem especially urgent with exploitation.
Traditional heavy metal detection method mainly uses atomic absorption spectroscopy (Atomic Absorption Spectroscopy, AAS), inductive coupling plasma emission spectrum (InductiveLy CoupLedpLasma Atomic Emission Spectroscopy, ICP-AES), anodic stripping voltammetry (Anodic Stripping VoLtammetry, ASV), chromatography and various combination detection methods.Although these methods can carry out the heavy metal ion in various environmental samples Effectively analysis, but large-scale instrument is needed mostly, analysis method is at high cost, and sample is needed by resolution, and analysis time is long, is unsuitable for The field quick detection of heavy metal, it is difficult to adapt to the spot check of environment and market product, manufacturing enterprise checks oneself and product disengaging The requirement that mouth speeds passage through customs.
Immunology detection technology is fast with detection speed, analysis capacity is big, low-cost, the simple portable, user of instrument Member's technical requirements are not high, are easy universal and promote, the advantages that high sensitivity and selectivity are strong, are especially suitable for scene screening and a large amount of The quick analysis of sample, it has also become the 21 century most competitive detection and analysis technology with challenge.
The key of heavy metal ion immunology detection is the preparation of preventing from heavy metal specific antibody, and specific antibody system Standby key is therefore how the synthesis of high-quality heavy metal immunogene prepares high-quality heavy metal artificial antigen, how to prepare again Preventing from heavy metal specific antibody, the immunoassay detection heavy metal ion established more rapidly, more economical become what needs solved Problem.
Summary of the invention
To overcome the shortcomings of existing technologies, the present invention provides the hybridization of one plant of secretion preventing from heavy metal lead ion monoclonal antibody Tumor cell strain and its application.
The hybridoma cell strain of one plant of secretion preventing from heavy metal lead ion monoclonal antibody, has been preserved in China Microbiological bacterium Kind preservation administration committee common micro-organisms center, deposit number are CGMCC No.16692.
Preventing from heavy metal lead ion monoclonal antibody, it is by the hybridoma cell strain point that deposit number is CGMCC No.16692 Secrete generation.
Hybridoma cell strain CGMCC No.16692 or the monoclonal antibody of its secretion are in lead ion immune detection Using.
The invention adopts the following technical scheme:
A kind of preparation method of hybridoma cell strain that secreting preventing from heavy metal lead ion monoclonal antibody, this method is for making Standby above-described hybridoma cell strain, comprising the following steps:
(1) synthesis and identification of heterocyclic bifunctional chelating agent lead ion artificial antigen:
5~8mg 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae are weighed, 7- triacetic acid (is abbreviated as o-NH2- Bn-NOTA), it is dissolved in 2mL 0.01molL-1, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution, be made chelating agent it is molten Liquid, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 150~200 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
500~800 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this Reaction solution is D liquid；
It weighs 20~30mg bovine serum albumin(BSA) BSA or chicken egg white OVA is dissolved in 3mL HEPES, at room temperature magnetic force It stirs and evenly mixs, this reaction solution is E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten Liquid (20mg is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, 15~20min, is redissolved with the HEPES solution of the 0.01M of 5~10mL, pH7.4 every time, rear to dispense, and -20 Heavy metal lead artificial antigen Pb-L is made in DEG C cryo-conservation1- BSA or Pb-L1- OVA, L1Indicate 2-S- (2- aminobenzene) -1, 4,7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid,
Synthetic route are as follows:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
(2) mouse is immune:
Female BAl BIc/C mice of 6~8 week old of health is selected to be immunized.By the heavy metal lead artificial antigen of preparation Pb-L1After-BSA and isometric Freund's complete adjuvant are sufficiently mixed emulsification using syringe pressurization, pass through abdomen and oxter multiple spot Injection.Booster immunization is carried out at interval of 21d (day) afterwards, blood sampling measurement potency, potency use indirect ELISA after 3 booster immunizations Method measurement.It carries out last end when potency is no longer significantly raised to exempt from, end carries out cell fusion after exempting from 3d.First in immunologic process Secondary immune using Freund's complete adjuvant, booster immunization uses incomplete Freund's adjuvant, and end is exempted from without using adjuvant, direct immunization original annotation It penetrates immune.
(3) cell fusion and cell strain screen:
Mouse boosting cell and murine myeloma cell SP2/0 are carried out according to the ratio of 5~10:1 by polyethylene glycol method Fusion, HAT selective medium culture, indirect ELISA detects positive hole, and further utilizes indirect competition to positive hole ELISA measures the inhibitory effect of positive cell hole, and then the positive hole good to inhibitory effect carries out limiting dilution assay and carry out 3~4 Time cloning, screening obtains hybridoma cell strain 2C8 after finally being verified with indirect competitive ELISA method.
The preparation method of preventing from heavy metal lead ion monoclonal antibody, comprising the following steps:
It is mono- that the 2C8 screened is injected with method in BALB/C mice intraperitoneal injection norphytane 0.3ml/, after 7~10d (day) Clonal cell line cell, ascites is extracted in sterile working after mouse peritoneal obviously swells, and is centrifuged off after precipitation of grease to get small Mouse ascites fluid McAb；
Ascites after purification, obtains monoclonal antibody purification.
Further, by heterocyclic bifunctional chelating agent L1It is substituted for 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 three azacyclo-s respectively Nonane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as m-NH2- Bn-NOTA) or 2-S- (4- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononane -1, 4,7- triacetic acids (are abbreviated as p-NH2- Bn-NOTA) or 2-S- [4- (3- aminopropyl)-benzene]-Isosorbide-5-Nitrae, 7 7-triazacyclononanes- Isosorbide-5-Nitrae, 7- triacetic acid, using the above identical preparation method can be made different lead ion artificial antigen, hybridoma cell strain, Preventing from heavy metal lead ion monoclonal antibody.
Further, bovine serum albumin(BSA) BSA is replaced with into keyhole limpet hemocyanin KLH or other carrier proteins, used The above identical preparation method, can prepare different lead ion artificial antigens, hybridoma cell strain, monoclonal antibody.
The beneficial effects of the present invention are:
(1) present invention uses heterocyclic bifunctional chelating agent o-NH2- Bn-NOTA prepares lead ion artificial antigen, the chelating Agent can preferably combine heavy metal ion in structure, while can be preferably by the structure of heavy metal lead ion on space structure It shows, the lead ion artificial antigen being prepared has stronger specificity.
(2) the hybridoma cell strain 2C8 that the present invention is prepared can secrete heavy metal lead ion monoclonal antibody, the list Clonal antibody specificity is stronger, has preferably sensitivity, IC to lead ion chelating agent complexes20Value is 0.45ngmL-1, It can be used in quickly detecting heavy metal lead ion.The anti-lead ion monoclonal antibody can be used for the residual of lead ion in environmental sample Stay detection.
Detailed description of the invention
Fig. 1 is that (wherein, Protein represents bovine serum albumin(BSA) to lead ion artificial antigen synthesis schematic diagram in embodiment 1,2 BSA or chicken egg white OVA).
Fig. 2 is that (wherein, Protein represents bovine serum albumin(BSA) to lead ion artificial antigen synthesis schematic diagram in embodiment 3,4 BSA or chicken egg white OVA).
Fig. 3 is that (wherein, Protein represents bovine serum albumin(BSA) to lead ion artificial antigen synthesis schematic diagram in embodiment 5,6 BSA or chicken egg white OVA).
Fig. 4 is that (wherein, Protein represents bovine serum albumin(BSA) to lead ion artificial antigen synthesis schematic diagram in embodiment 7,8 BSA or chicken egg white OVA).
Fig. 5 is lead ion artificial antigen UV scanning figure prepared by embodiment 1,2.
Fig. 6 is lead ion artificial antigen electrophoretogram prepared by embodiment 1,2.
Specific embodiment
Further details of the technical solution of the present invention below, it is noted that specific embodiment is Detailed description of the invention is not construed as limitation of the invention.
Heterocyclic bifunctional chelating agent used in the present invention indicates that heterocyclic bifunctional chelating agent is distinguished in embodiment with L Are as follows: 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as o-NH2- Bn-NOTA) use L1It indicates, 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as m-NH2- Bn-NOTA) use L2It indicates, 2- S- (4- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-NH2- Bn-NOTA) use L3It indicates, 2-S- [4- (3- aminopropyl)-benzene]-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-CH2CH2CH2NH2-Bn- NOTA L) is used4It indicates, these chelating agents are commercially available by commercial sources.
1. the preparation and authentication of heterocyclic bifunctional chelating agent lead ion artificial antigen
Embodiment 1
(1) synthesis of lead ion artificial antigen:
Weigh 6.5mg L1, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES of pH it is molten L is made in liquid1Chelating agent solution, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 175 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde water solution, room temperature be protected from light overnight, this reaction Liquid is D liquid；
It weighs 20mg bovine serum albumin(BSA) BSA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution redissolution of pH 7.4, after Heavy metal lead artificial antigen Pb-L is made in packing, -20 DEG C of cryo-conservations1- BSA, L1Expression 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 three Azepine cyclononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as o-NH2-Bn-NOTA)。
(2) identification of lead ion artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: BSA, Pb-L1- BSA and Pb-L1Being configured to concentration is 1~5mgmL-1Solution in range, The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution Whether line succeeds to identify to synthesize.
Ultraviolet scanning atlas is as shown in figure 5, Pb-L1Compared with BSA solution, maximum absorption wavelength changes-BSA solution, Illustrate to be coupled successfully.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager Photographic analysis, as shown in Figure 6.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 33:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, coupling efficiency is high.In conjunction with than It is higher, illustrate that the quantity that heavy metal ion is coupled on a protein molecular is more, coupling efficiency is also higher, in conjunction with than for 33:1, It then indicates to combine 33 heavy metal ion on a protein molecular.
Embodiment 2
(1) synthesis of lead ion artificial antigen:
Weigh 6.5mg L1, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES of pH it is molten L is made in liquid1Chelating agent solution, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 175 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution For D liquid；
It weighs 20mg chicken egg white OVA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution redissolution of pH 7.4, after Heavy metal lead artificial antigen Pb-L is made in packing, -20 DEG C of cryo-conservations1- OVA, L1Expression 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 three Azepine cyclononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as o-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: OVA, Pb-L1- OVA and Pb-L1Being configured to concentration is 1~5mgmL-1Solution in range, The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution Whether line succeeds to identify to synthesize.
Ultraviolet scanning atlas is as shown in figure 5, Pb-L1Compared with OVA solution, maximum absorption wavelength changes-OVA solution, Illustrate to be coupled successfully.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager Photographic analysis, as shown in Figure 6.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 29:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio.In conjunction with than for 29:1, then table Show and combines 29 heavy metal ion on a protein molecular.
Embodiment 3
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L2, L in alternative embodiment 11；According to 1 phase of embodiment Lead ion artificial antigen Pb-L is made in same step2- BSA, lead ion artificial antigen synthetic route is as shown in Fig. 2, specific synthesis Steps are as follows.
(1) synthesis of lead ion artificial antigen:
Weigh 6.5mg L2, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES of pH it is molten L is made in liquid2Chelating agent solution, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 175 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution For D liquid；
It weighs 20mg bovine serum albumin(BSA) BSA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3-5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH7.4 redissolves, rear to divide Heavy metal lead artificial antigen Pb-L is made in dress, -20 DEG C of cryo-conservations2- BSA, L2Indicate 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 three nitrogen Ononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as m-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: BSA, Pb-L2- BSA and Pb-L2Being configured to concentration is 1~5mgmL-1Solution in range, The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Pb-L2Compared with BSA solution, maximum absorption wavelength changes-BSA solution, illustrates to be coupled Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 28:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, coupling efficiency is higher.In conjunction with Than higher, illustrate that the quantity that heavy metal ion is coupled on a protein molecular is more, coupling efficiency is also higher, in conjunction with than being 28: 1, then it represents that 28 heavy metal ion are combined on a protein molecular.
Embodiment 4
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L2, L in alternative embodiment 21；According to 2 phase of embodiment Lead ion artificial antigen Pb-L is made in same step2- OVA, lead ion artificial antigen synthetic route is as shown in Fig. 2, specific synthesis Steps are as follows.
(1) synthesis of lead ion artificial antigen:
Weigh 6.5mg L2, it is dissolved in 2mL 0.01molL-1, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution, L is made2Chelating agent solution, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 175 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution For D liquid；
It weighs 20mg chicken egg white OVA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3-5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH7.4 redissolves, rear to divide Heavy metal lead artificial antigen Pb-L is made in dress, -20 DEG C of cryo-conservations2- OVA, L2Indicate 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 three nitrogen Ononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as m-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: OVA, Pb-L2- OVA and Pb-L2Being configured to concentration is 1~5mgmL-1Solution, measurement Absorbance in 200~400nm wave-length coverage, and ultraviolet scanning atlas is established, come by comparing the absorption curve of every kind of solution Identify whether synthesis succeeds.
In ultraviolet scanning atlas, Pb-L2Compared with OVA solution, maximum absorption wavelength changes-OVA solution, illustrates to be coupled Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 24:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than then being indicated for 24:1 24 heavy metal ion are combined on one protein molecular.
Embodiment 5
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L3, L in alternative embodiment 11；According to 1 phase of embodiment Lead ion artificial antigen Pb-L is made in same step3- BSA, lead ion artificial antigen synthetic route is as shown in figure 3, specific synthesis Steps are as follows.
(1) synthesis of lead ion artificial antigen:
Weigh 6.5mg L3, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES of pH it is molten L is made in liquid3Chelating agent solution, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 175 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution For D liquid；
It weighs 20mg bovine serum albumin(BSA) BSA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH7.4 redissolves, rear to divide Heavy metal lead artificial antigen Pb-L is made in dress, -20 DEG C of cryo-conservations3- BSA, L3Indicate 2-S- (4- aminobenzene)-Isosorbide-5-Nitrae, 7 three nitrogen Ononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: BSA, Pb-L3- BSA and Pb-L3Being configured to concentration is 1~5mgmL-1Solution in range, The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Pb-L3Compared with BSA solution, maximum absorption wavelength changes-BSA solution, illustrates to be coupled Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 22:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than for 22:1, then table Show and combines 22 heavy metal ion on a protein molecular.
Embodiment 6
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L3, L in alternative embodiment 11；According to 2 phase of embodiment Lead ion artificial antigen Pb-L is made in same step3- OVA, lead ion artificial antigen synthetic route is as shown in figure 3, specific synthesis Steps are as follows.
(1) synthesis of lead ion artificial antigen:
Weigh 6.5mg L3, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES of pH it is molten L is made in liquid3Chelating agent solution, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 175 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution For D liquid；
It weighs 20mg chicken egg white OVA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution redissolution of pH 7.4, after Heavy metal lead artificial antigen Pb-L is made in packing, -20 DEG C of cryo-conservations3- OVA, L3Expression 2-S- (4- aminobenzene)-Isosorbide-5-Nitrae, 7 three Azepine cyclononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: OVA, Pb-L3- OVA and Pb-L3Being configured to concentration is 1~5mgmL-1Solution in range, The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Pb-L3Compared with OVA solution, maximum absorption wavelength changes-OVA solution, illustrates to be coupled Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 18:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than for 18:1, then table Show and combines 18 heavy metal ion on a protein molecular.
Embodiment 7
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L4, L in alternative embodiment 11；According to 1 phase of embodiment Lead ion artificial antigen Pb-L is made in same step4- BSA, lead ion artificial antigen synthetic route is as shown in figure 4, specific synthesis Steps are as follows.
(1) synthesis of lead ion artificial antigen:
Weigh 6.5mg L4, it is dissolved in 2mL 0.01molL-1, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution, L is made4Chelating agent solution, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 175 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution For D liquid；
It weighs 20mg bovine serum albumin(BSA) BSA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten Liquid (20mg is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH7.4 redissolves, rear to divide Heavy metal lead artificial antigen Pb-L is made in dress, -20 DEG C of cryo-conservations4- BSA, L4It indicates 2-S- [4- (3- aminopropyl)-benzene]- Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-CH2CH2CH2NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: BSA, Pb-L4- BSA and Pb-L4Being configured to concentration is 1~5mgmL-1Solution in range, The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Pb-L4Compared with BSA solution, maximum absorption wavelength changes-BSA solution, illustrates to be coupled Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 25:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than for 25:1, then table Show and combines 25 heavy metal ion on a protein molecular.
Embodiment 8
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L4, L in alternative embodiment 11；According to 2 phase of embodiment Lead ion artificial antigen Pb-L is made in same step4- OVA, lead ion artificial antigen synthetic route is as shown in figure 4, specific synthesis Steps are as follows.
(1) synthesis of lead ion artificial antigen:
Weigh 6.5mg L4, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES of pH it is molten L is made in liquid4Chelating agent solution, this solution are A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Plumbi nitras Solution, this solution are B liquid；
The B liquid of 175 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution For D liquid；
It weighs 20mg chicken egg white OVA and is dissolved in 4mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ l sodium borohydrides are added dropwise is molten Liquid (20mg is dissolved in 200 μ l ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1 Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution redissolution of pH 7.4, after Heavy metal lead artificial antigen Pb-L is made in packing, -20 DEG C of cryo-conservations4- OVA, L4Indicate 2-S- [4- (3- aminopropyl)- Benzene]-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (abbreviation p-CH2CH2CH2NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: OVA, Pb-L4- OVA and Pb-L4Being configured to concentration is 1~5mgmL-1Solution in range, The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Pb-L4Compared with OVA solution, maximum absorption wavelength changes-OVA solution, illustrates to be coupled Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 20:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than for 20:1, then table Show and combines 20 heavy metal ion on a protein molecular.
Embodiment 9
The measurement of antiserum titre:
Artificial antigen prepared by embodiment 1,3,5 and 7 is respectively immunized BALB/C mice, initial immunity is using not Family name's Freund's complete adjuvant emulsifies artificial antigen, and after emulsification, injection is measured as 250 μ g/ mouse, after at interval of 21d booster immunization, altogether plus 3 times strong immune, booster immunization is emulsified using Freund's incomplete adjuvant, and it is 150 μ g/ mouse that metering, which is immunized, small after booster immunization 14d Mouse docking blood sampling carries out the titration of polyvalent antibody, and ELISA method measures antiserum titre after serum confining liquid doubling dilution, Using the mice serum before being immunized as negative control, with positive serum OD450nmValue and negative serum OD450nmIt is worth ratio and is greater than 2.1 Place dilution is antiserum titre, and the results are shown in Table 1.It finally carries out end to exempt from, end is exempted from using the direct abdominal cavity note of artificial antigen The mode penetrated carries out, and it is 300 μ g/ mouse that metering, which is immunized,.
Titration uses indirect ELISA method, and steps are as follows for specific experiment:
A. it is coated with: being respectively packet with the artificial antigen in embodiment 2 or embodiment 4 or embodiment 6 or embodiment 8 It is former, pH 9.6,0.05molL-1CBS is coating buffer, and primary concentration of envelope is 10 μ gmL-1, package amount is 100 μ L/ Hole, is coated with 2h in 37 DEG C, and latter PBST washing lotion board-washing 4 times；
B. it closes: 250 hole μ L/ of confining liquid, after 37 DEG C of incubation 30min, PBST washing lotion board-washing 4 times is added；
C. plus antiserum: the antiserum 10000rmin that mouse blood sampling is obtained-1After being centrifuged 5min, draws 10 μ L and be added To 2mL confining liquid, (initial dilution multiple is 200 times) uses 11 gradients of confining liquid doubling dilution and 1 negative control afterwards, 100 holes μ L/, 4 repetitions of each gradient, 37 DEG C of incubation 1h are rear to use PBST washing lotion board-washing 4 times；
Plus ELIAS secondary antibody d.: PBST board-washing 4 times after reaction, after 10000 times of sheep anti mouse secondary antibody dilutions of HRP label plus Enter ELISA Plate, 100 holes μ L/, 37 DEG C of incubation 1h, latter PBST washing lotion board-washing 4 times；
E. plus substrate reactions liquid: tmb substrate buffer, 100 holes μ L/, 37 DEG C of incubation 15min are added；
F. it terminates reading: 2molL is added-1OD is read with microplate reader in 50 hole μ L/ of sulfuric acid450nmValue.
Combination ratio and antiserum titre measurement result in 1 embodiment 1-8 of table
Immunizing antigen In conjunction with than Detect antigen In conjunction with than Antiserum titre
Pb-L1- BSA embodiment 1 33:1 Pb-L1- OVA embodiment 2 29:1 310000
Pb-L2- BSA embodiment 3 28:1 Pb-L2- OVA embodiment 4 24:1 256000
Pb-L3- BSA embodiment 5 22:1 Pb-L3- OVA embodiment 6 18:1 190000
Pb-L4- BSA embodiment 7 25:1 Pb-L4- OVA embodiment 8 20:1 220000
Show antiserum titre sequence with antiserum titre measurement result by the combination ratio of table 1 are as follows: embodiment 1 > reality 3 > embodiment of example, 7 > embodiment 5 is applied, in conjunction with the sequence that compares so as to see who is superior are as follows: 7 > embodiment of embodiment 1 > embodiment, 3 > embodiment 5, wherein The antiserum titre highest of embodiment 1, the antiserum titre of embodiment 3 are only below embodiment 1.This illustrates to prepare in embodiment 1 Lead ion artificial antigen Pb-L1- BSA can preferably show heavy-metal antigen determinant, and antigentic specificity is stronger, together When embodiment 1 and embodiment 2 prepared by artificial antigen its combine relatively high, height is combined than being easier to cause strong immune answers Reaction is answered, is conducive to prepare preventing from heavy metal lead ion monoclonal antibody.
Specific preparation method, the measuring method of the use of embodiment 1,3,5,7 are identical, still, the bifunctional chelating agent of use Difference is respectively as follows: o-NH2- Bn-NOTA, m-NH2- Bn-NOTA, p-NH2- Bn-NOTA, p-CH2CH2CH2NH2-Bn-NOTA.This Illustrate-NH in chelating agent2Ortho position in phenyl ring, chelating agent o-NH2After-Bn-NOTA combines heavy metal ion, in space structure On preferably the structure of heavy metal lead ion can be shown, and from the experimental results, the artificial of ortho position structure preparation resists Former combination is than also highest, as antigenic determinant, is more advantageous to preparation affinity and sensitivity is higher, specific stronger anti- Heavy metal monoclonal antibody.
Embodiment 10
The screening of hybridoma cell strain and the preparation purifying of monoclonal antibody
(1) mouse immune:
Select 6~8 week old, weight 18~20g BALB/C female mice.By the immunogene of preparation, (immunogene is used herein : lead ion artificial antigen Pb-L1- BSA) being pressurizeed with isometric Freund's complete adjuvant using syringe is sufficiently mixed emulsification Afterwards, by abdomen and oxter multi-point injection, dosage be 50~100 μ g/ only, after at interval of 21d carry out booster immunization, 3 reinforcements Blood sampling measurement potency, potency are measured using indirect ELISA method after immune, and ELISA method is surveyed after serum confining liquid doubling dilution Antiserum titre is determined, using the mice serum before being immunized as negative control, with positive serum OD450nmIt is worth big with negative serum ratio In 2.1 place dilutions be antiserum titre.It carries out end when potency is no longer significantly raised to exempt from, progress cell melts after 3d is exempted from end It closes.It is immunized for the first time in immunologic process and uses Freund's complete adjuvant, booster immunization uses incomplete Freund's adjuvant, and end exempts from not using Adjuvant, direct immunization original injecting immune.
Titration uses indirect ELISA method, and steps are as follows for specific experiment:
A. it is coated with: with Pb-L1- OVA is coating antigen, pH 9.6,0.05molL-1CBS is coating buffer, and coating antigen is dense Degree is 10 μ gmL-1, package amount is 100 holes μ L/, after being coated with 2h in 37 DEG C, PBST washing lotion board-washing 4 times；
B. it closes: 250 hole μ L/ of confining liquid is added, after 37 DEG C of incubation 30min, PBST board-washing 4 times, every hole adds 300 μ of PBST L, latter PBST washing lotion board-washing 4 times；
C. plus antiserum: the antiserum 10000rmin that mouse blood sampling is obtained-1After being centrifuged 5min, draws 10 μ L and be added To 2mL confining liquid (initial dilution multiple is 200 times), 11 gradients of confining liquid doubling dilution and 1 negative control are used afterwards, 100 holes μ L/, 4 repetitions of each gradient, 37 DEG C of incubation 1h, latter PBST washing lotion board-washing 4 times；
Plus ELIAS secondary antibody d.: board-washing 4 times after reaction are added after 1:10000 times of sheep anti mouse secondary antibody dilution of HRP label ELISA Plate, 100 holes μ L/, 37 DEG C of incubation 1h, latter PBST washing lotion board-washing 4 times；
E. plus substrate reactions liquid: tmb substrate buffer, 100 holes μ L/, 37 DEG C of incubation 15min are added；
F. it terminates reading: 2molL is added-1OD is read with microplate reader in 50 hole μ L/ of sulfuric acid450nmValue.
(2) cell fusion and culture:
After 3d is exempted from end, cell fusion is carried out according to conventional polyethylene glycol (PEG1500) method, the specific steps are as follows:
A. the eyeball of mouse bloodletting after end is exempted from, centrifugation sucts clear spare after serum collection, and neck execution is drawn to be placed on 70% wine 3~5min in essence takes mouse spleen under aseptic condition, is placed in sterile grinding bowl and mills after being shredded with aseptic operation, uses RPMI- After 1640 basal mediums blow outstanding cell, crosses 200 mesh cell screen clothes and obtain splenocyte suspension, and carry out cell count；
B. SP2/0 cell (myeloma cell) is collected, it is desirable that cell growth state is good, and cell activity is greater than 90%, sucks New RPMI-1640 basal medium is added after cell conditioned medium and blows and beats cell and suspends, carries out cell count afterwards；
C. according to cell counts, after splenocyte and SP2/0 cell are mixed according to the ratio of 5~10:1,1800r min-1Supernatant is sucked after centrifugation 5min, remaining cell is added 0.5mL PEG and is gently mixed mixing in 1min inner edge edged, after adding RPMI-1640 basal medium 45mL, rear 1500rmin are added from slow to fast after standing 1min-1Supernatant is removed after centrifugation 5min, 96 porocyte culture plates are plated on after selectivity HAT culture medium is added, every 250 μ L of hole is placed in 37 DEG C, 5%CO2Incubator in Culture.
D. it after cultivating 3~5d (day), is changed liquid 1 time with HAT culture medium, 10d is changed to HT culture medium culture.
(3) cell screening and cell strain are established:
When cell to be fused grows into covering 10%~30% hole floor space of culture hole, supernatant is taken to be screened with indirect ELISA Antibody positive wells, envelope antigen is Pb-L when screening1- OVA cross-linking agent, and negative control is made with OVA, BSA.The positive filtered out Reacting hole is further with the detection sensitivity of competitive ELISA analysis antibody.According to the standard curve of foundation, IC is calculated50Value and IC20Value, (abscissa is the natural logrithm of lead ion standard concentration to selection standard slope of curve range, and ordinate is inhibiting rate The competition test curve of foundation) between 8~13, IC50Value is lower than 200ngml-1, IC20Value is lower than 20ngml-1Cell Strain is further cloned and is screened, and is continuously cloned with limiting dilution assay 3~4 times, final to obtain a strain of hybridoma strain 2C8, deposit number are CGMCC No.16692.Comparatively, IC20Numerical value is lower, IC50The suppression of the lower cell strain of numerical value Effect processed is preferable, is conducive to prepare the preferable hybridoma cell strain of characteristic.
Hybridoma cell strain 2C8 is preserved in China Committee for Culture Collection of Microorganisms on November 02nd, 2018 Common micro-organisms center (abbreviation CGMCC, address: Institute of Microorganism, Academia Sinica, BeiChen West Road, Chaoyang District, BeiJing City 1 Institute 3, postcode 100101).
On the one hand the cell strain is moved into cell cryopreservation tube after expanding culture by the hybridoma cell strain 2C8 finally obtained And be put into liquid nitrogen and save for a long time, the cell strain is on the other hand used for ascites preparation, the purifying and application of monoclonal antibody.
(3) preparation, purifying and identification of monoclonal antibody
Monoclonal antibody is prepared using method is induced in animal body.
6~8 week old health BALB/C female mices are selected, norphytane 0.3mL/ is injected intraperitoneally only in BALB/C mice, 7~ After 10d with method injection screen 2C8 monoclonal cell strain cell (0.4mL/ only, every ml cell strain quantity is 2.5 × 106~1 ×107Between), ascites is extracted after mouse peritoneal obviously swells after 5~7d, is centrifuged off after precipitation of grease to get mouse abdomen Water McAb.
After ascites uses caprylic acid-ammonium preliminary purification, crossing affinity chromatographic column, (filler is the production of TOSOH company TOYOPEARL AF-rProtein A HC-650F) it is further purified, Purified monoclonal is measured respectively with ultraviolet specrophotometer The ultraviolet 260nm of antibody and the optical density of 280nm, calculating MAb concentration with Lowry-kalokar formula is 2.45mg·mL-1, remaining purifying -70 DEG C of monoclonal antibody save backup.
Specific step is as follows for ascites purifying:
1) by ascites 60mmolL-1The acetate buffer solution of pH 4.0 is diluted by 1:4 volume ratio, uses 0.1mol L-1NaOH adjusts pH value to 4.5；
2) according to 33 μ lmL under magnetic agitation-1Octanoic acid is added dropwise in the ratio of ascites, after stir 30min at room temperature, it is quiet in 4 DEG C Set 2h, rear 10000rmin-1It is centrifuged 30min；
3) after supernatant is filtered with qualitative filter paper, 0.1molL is added according to 1:10 (PBS: filtered supernatant) ratio- 1PBS uses 1molL-1NaOH adjusts pH to 7.4, is placed in 4 DEG C of 30min；
4) under magnetic stirring according to 0.277gmL-1Ammonium sulfate is added portionwise in (ammonium sulfate: mixed liquor) ratio, after adding It is stirred to react 30min, 4 DEG C of standings 3h, 12000rmin-1It is centrifuged 30min, abandons supernatant；
5) a small amount of 0.01molL of precipitating-1PBS dissolution is placed in bag filter, with pH 7.4,0.01molL-1's PBS dialysis, centre is changed liquid 4~6 times；
6) monoclonal antibody solution of preliminary purification crosses Protein A (TOYOPEARL AF-rProtein A HC- 650F) affinity chromatographic column is purified, and is saved backup after being after purification freeze-dried antibody in -70 DEG C.
Embodiment 11
The foundation of monoclonal antibody indirect competitive ELISA method
The measurement of monoclonal antibody working concentration is carried out using orthogonal test, and steps are as follows for specific experiment:
1) it is coated with: with pH 9.6,0.05molL-1CBS is coating buffer, Pb-L1- OVA is coating antigen, and package amount is pressed According to 10 μ gmL-1Starting doubling dilution, coating volume is 100 holes μ L/, after 37 DEG C of incubation 2h, PBST washing lotion board-washing 4 times；
2) it closes: 250 hole μ L/ of confining liquid, after 37 DEG C of incubation 30min, PBST washing lotion board-washing 4 times is added；
3) it is loaded: monoclonal antibody after purification is added, according to 10 μ gmL-1Coating hole is added after starting doubling dilution In, 100 holes μ L/, after 37 DEG C of reaction 1h, PBST washing lotion board-washing 4 times；
4) add ELIAS secondary antibody: board-washing 4 times after reaction, the sheep anti mouse secondary antibody of HRP label is diluted with 10000 times of confining liquid After be added ELISA Plate, 100 holes μ L/, after 37 DEG C of reaction 1h, PBST washing lotion board-washing 4 times；
5) add substrate reactions liquid: tmb substrate buffer, 100 holes μ L/, 37 DEG C of incubation 15min are added；
6) it terminates reading: 2molL is added-150 hole μ L/ of sulfuric acid, microplate reader read OD450nmValue.
7) OD is chosen450nmThe antigen-antibody concentration combination being worth in the section 1.0-1.2 is working concentration, and it is dense to choose 4 work Degree combination, concentration combination respectively (the 0.625 μ gmL of antigen, antibody-1, 5 μ gmL-1)、(1.25μg·mL-1, 2. μ g·mL-1)、(2.5μg·mL-1, 1.25 μ gmL-1) and (5 μ gmL-1, 0.625 μ gmL-1), using indirect competitive ELISA The selected 4 working concentrations combination of method carries out sensitivity analysis screening, the results are shown in Table 2, by the slope of standard curve of foundation, IC50The parameters such as value and standard curve r value carry out overall merit, determine that optimal working concentration is 2.5 μ g of antigen coat concentration mL-1, antibody concentration is 1.25 μ gmL-1。
2 working concentration of table analyzes result
Specific step is as follows for indirect competitive ELISA:
A. it is coated with: with pH 9.6,0.05molL-1CBS is coating buffer, Pb-L1- OVA is coating antigen, is coated with volume For 100 holes μ L/, peridium concentration is the best effort concentration of aforementioned screening, after 37 DEG C of incubation 2h, PBST washing lotion board-washing 4 times；
B. it closes: 250 hole μ L/ of confining liquid, after 37 DEG C of incubation 30min, PBST washing lotion board-washing 4 times is added；
C. sample pre-treatments: 5mmolL-12-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid That is L1It is mixed in equal volume with various concentration gradient heavy metal lead ion standard specimen, 37 DEG C of incubation 1h；
D. it is loaded: pre- mixed 50 hole μ L/ of sample is added, twice of best effort concentration primary antibody (i.e. above embodiments is added The anti-lead ion monoclonal antibody of 10 preparations) 50 holes μ L/, after concussion mixes after 37 DEG C of reaction 1h, PBST washing lotion board-washing 4 times；
Plus ELIAS secondary antibody e.: after reaction, board-washing 4 times, the sheep anti mouse secondary antibody of HRP label is dilute with 10000 times of confining liquid It is added ELISA Plate after releasing, 100 holes μ L/, after 37 DEG C of reaction 1h, PBST washing lotion board-washing 4 times；
F. plus substrate reactions liquid: tmb substrate buffer, 100 holes μ L/, 37 DEG C of incubation 15min are added；
G. it terminates reading: 2molL is added-150 hole μ L/ of sulfuric acid, microplate reader read OD450nmValue.
Antibody specificity test experience step:
The cross reacting rate of heavy metal zinc, iron, cadmium and lead is detected with the indirect competitive ELISA method of foundation.Measuring method ginseng According to indirect competitive ELISA step, difference is to replace lead ion to be detected with heavy metal ion to be measured, according to the standard of foundation IC is calculated in curve50Value, cross reacting rate calculation formula are CR (%)=IC50(lead ion)/IC50(heavy metal to be measured) × 100%.Specific detection the results are shown in Table 3.
The cross reaction of table 3 monoclonal antibody and heavy metal ion
Using indirect ELISA measurement monoclonal antibody purification to the sensitivity for analysis IC of lead ion chelating agent complexes20For 0.45ng·mL-1, antibody specificity test experience the result shows that, prepared monoclonal antibody and intersecting for other heavy metals are anti- Should rate be below 4.3%, this preventing from heavy metal lead ion monoclonal antibody for being prepared of explanation has very high detection to lead ion Sensitivity and well specificity, can be used in quickly detecting heavy metal lead ion.
Solution is prepared:
Carbonate buffer solution (CBS): Na is weighed2CO31.59g NaHCO32.93g is added pure water to 990mL, adjusts pH extremely 9.6, then it is settled to 1000mL with pure water, 4 DEG C of storages are spare.
Phosphate buffer (PBS): 8.5g NaCl, 2.2g Na2HPO4·12H2O, 0.2g NaH2PO4·2H2O is dissolved in In 900mL pure water, pH to 7.4 is adjusted, 1000mL is settled to.
PBST washing lotion: 500mL 0.01molL is taken-10.25mL polysorbas20 is added in PBS, mixes spare.
Confining liquid: 1g skimmed milk power is dissolved in 50mL pH 7.4,0.01molL-1PBS。
For tmb substrate buffer by A liquid, B liquid and C liquid composition, specific formula and use volume ratio are as follows:
Developing solution A:2.35g citric acid, 9.2g Na2HPO4·12H2O adds pure water to be settled to 490mL, adjusts pH value 5.0 ~5.4, then it is settled to 500mL with pure water, 4 DEG C of storages are spare.
Developing solution B: taking 30% hydrogen peroxide 1.25mL, is settled to 50mL with pure water, 4 DEG C of storages are spare.
Developing solution C: taking 200mg tetramethyl benzidine (TMB) to be dissolved in 100ml dehydrated alcohol, and 4 DEG C of storages are standby after dissolution With.
Tmb substrate buffer uses preceding interim adapted, and adapted ratio is 42 μ L, C liquid 0.5mL of A liquid 9.5mL, B liquid, mixes After use.
Sheep anti mouse HRP marks ELIAS secondary antibody to be purchased from Sigma company.
Embodiment 12
The experiment of TIANZHU XINGNAO Capsul in water sample sample
Recovery experiment is added to actual sample, respectively with tap water, Lake Water is experimental subjects, heavy metal lead ion Addition concentration be respectively 10ngmL-1And 50ngmL-1, sample pre-treatments step are as follows:
Lake water sample: after Lake Water is first filtered with qualitative filter paper, lead ion standard solution is added, tap water directly adds lead Ion standard solution, lead ion ultimate density is 10ngmL after addition-1And 50ngmL-1, after addition mixes, draw respectively Tap water, lake water sample and 5mmolL after 100 μ L mixing-1L1The 50 above-mentioned premixs of μ L are added in isometric premix, rear every hole Tap water, lake water sample after conjunction is into reacting hole, 3 repeating holes, adds the primary antibody of 50 twice of best effort concentration of μ L (i.e. the anti-lead ion monoclonal antibody of the preparation of above embodiments 10), mixing is placed on 37 DEG C of incubations.Subsequent experimental procedure with it is upper It is consistent to state indirect competitive ELISA reaction step in embodiment 11.In tap water, lake water sample TIANZHU XINGNAO Capsul experimental result is shown in Table 4.
4 water sample sample TIANZHU XINGNAO Capsul result of table
Lake water sample TIANZHU XINGNAO Capsul is respectively 103.1% and 96.2%, and the coefficient of variation is respectively 6.8%, 7.4%；From Water TIANZHU XINGNAO Capsul is respectively 97.1% and 98.3%, and the coefficient of variation is respectively 7.2%, 7.7%.
By TIANZHU XINGNAO Capsul experiment it can be concluded that the accuracy of testing result, above TIANZHU XINGNAO Capsul value can illustrate to adopt The testing result accuracy measured with the method is higher, and the testing result coefficient of variation is low, illustrates that testing result stability is good. The preventing from heavy metal lead ion monoclonal antibody that this explanation is prepared can be used in accurate, quick detection heavy metal lead ion.
In above-described embodiment 10-12, by artificial antigen Pb-L1- BSA replaces with Pb-L2- BSA, coating antigen Pb-L1- OVA is replaced It is changed to Pb-L2- OVA, or by artificial antigen Pb-L1- BSA replaces with Pb-L3- BSA, coating antigen Pb-L1- OVA replaces with Pb- L3- OVA, or by artificial antigen Pb-L1- BSA replaces with Pb-L4- BSA, coating antigen Pb-L1- OVA replaces with Pb-L4- OVA is adopted Different hybridoma cell strain, preventing from heavy metal lead ion monoclonal antibody can be made with the above identical preparation method and make Lead ion is detected with indirect competitive ELISA method.
In above-described embodiment 1-12, by bovine serum albumin(BSA) BSA therein be substituted for keyhole limpet hemocyanin KLH or other Carrier protein can also prepare lead ion artificial antigen, hybridoma cell strain, monoclonal using the above identical preparation method Antibody and lead ion is detected using indirect ELISA.

Claims (5)

1. the hybridoma cell strain of one plant of secretion preventing from heavy metal lead ion monoclonal antibody, is preserved in Chinese microorganism strain preservation Administration committee's common micro-organisms center, deposit number are CGMCC No.16692.
2. preventing from heavy metal lead ion monoclonal antibody, which is characterized in that the hybridization that it is CGMCC No.16692 by deposit number Tumor cell strain secretion generates.
3. hybridoma cell strain CGMCC No.16692 or the preventing from heavy metal lead ion monoclonal antibody of its secretion are in lead ion Application in immune detection.
4. a kind of preparation method for the hybridoma cell strain for secreting preventing from heavy metal lead ion monoclonal antibody, which is characterized in that should Method is used to prepare hybridoma cell strain described in claim 1, comprising the following steps:
(1) synthesis of lead ion artificial antigen:
5-8mg 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae are weighed, 7- triacetic acid is dissolved in 2mL 0.01mol·L-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution of pH, be made chelating agent solution, this solution be A liquid；
It weighs 124.2mg plumbi nitras to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Lead nitrate solution, This solution is B liquid；
The B liquid of 150~200 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
500~800 μ L, 20mmolL are added into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution be D Liquid；
It weighs 20~30mg bovine serum albumin(BSA) BSA or chicken egg white OVA is dissolved in 3mL HEPES, at room temperature magnetic agitation It mixes, this reaction solution is E liquid；
D liquid is added in E liquid, is protected from light at room temperature for 24 hours, 150~200 μ L sodium borohydride solutions are added afterwards, keep away at room temperature Light reaction 1h；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1Centrifugation Washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH 7.4 redissolves, rear to divide Heavy metal lead artificial antigen Pb-L is made in dress, -20 DEG C of cryo-conservations1- BSA or Pb-L1- OVA, L1Indicate 2-S- (2- amino Benzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid；
Synthetic route are as follows:
(2) mouse is immune:
Selection BALB/C mice is immunized, by the heavy metal lead artificial antigen Pb-L of preparation1- BSA is helped completely with isometric Freund After agent mixing and emulsifying, by abdomen and oxter multi-point injection, after at interval of certain time carry out booster immunization, 3 booster immunizations Blood sampling measurement potency afterwards, carries out last end when potency is no longer significantly raised and exempts from, end carries out cell fusion after exempting from 3 days；
(3) cell fusion and cell strain screen:
Mouse boosting cell and murine myeloma cell SP2/0 are merged according to the ratio of 5~10:1 by polyethylene glycol method, Be added culture medium culture, indirect ELISA screening antibodies positive hole, the hybridoma limiting dilution assay good to inhibitory effect into 3~4 time cloning of row obtains hybridoma cell strain 2C8 after screening.
5. the preparation method of preventing from heavy metal lead ion monoclonal antibody, which is characterized in that this method is used to prepare claim 2 institute The monoclonal antibody stated, comprising the following steps:
In BALB/C mice intraperitoneal injection norphytane 0.3ml/, after 7~10 days, the 2C8 monoclonal cell strain screened is injected Cell, ascites is extracted in sterile working after mouse peritoneal obviously swells, and is centrifuged off after precipitation of grease to get mouse ascites McAb；
Ascites after purification, obtains monoclonal antibody purification.
CN201811506000.3A 2018-12-10 2018-12-10 One plant of hybridoma cell strain for secreting preventing from heavy metal lead ion monoclonal antibody and its application Pending CN110408598A (en)

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Publication number Priority date Publication date Assignee Title
CN112379088A (en) * 2020-12-04 2021-02-19 福建农林大学 Detection test paper for detecting lead residue based on gold nanoflower technology

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US20100254987A1 (en) * 2009-02-27 2010-10-07 Massachusetts Institute Of Technology Engineered proteins with high affinity for dota chelates
CN101914153A (en) * 2010-07-05 2010-12-15 吉林大学 Pb<2+> antigen and corresponding monoclonal antibody and preparation method thereof
CN108264553A (en) * 2018-02-09 2018-07-10 浙江工商大学 A kind of application of the preparation method and NOTA of heavy metal lead artificial antigen in heavy metal lead artificial antigen reagent is prepared

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US20030049800A1 (en) * 2000-12-15 2003-03-13 Saravis Calvin A. Compositions and methods for producing antibodies to low molecular weight analytes
US20100254987A1 (en) * 2009-02-27 2010-10-07 Massachusetts Institute Of Technology Engineered proteins with high affinity for dota chelates
CN101914153A (en) * 2010-07-05 2010-12-15 吉林大学 Pb<2+> antigen and corresponding monoclonal antibody and preparation method thereof
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112379088A (en) * 2020-12-04 2021-02-19 福建农林大学 Detection test paper for detecting lead residue based on gold nanoflower technology
CN112379088B (en) * 2020-12-04 2022-05-31 福建农林大学 Detection test paper for detecting lead residue based on gold nanoflower technology

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