CN110408600A - A hybridoma cell line secreting anti-heavy metal copper ion monoclonal antibody and its application - Google Patents

Abstract

The hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody the present invention provides one plant and its application；The hybridoma cell strain can secrete preventing from heavy metal copper ion monoclonal antibody, and the present invention also provides the preparation methods of the preparation method of hybridoma cell strain and preventing from heavy metal copper ion monoclonal antibody；The monoclonal antibody specificity that the present invention is prepared is stronger, has preferably sensitivity, IC to copper ion₂₀Value is 0.14ngmL^‑1, can be used in quickly detecting heavy metal copper ion.The anti-copper ion monoclonal antibody can be used for the residue detection of copper ion in environmental sample.

Description

A hybridoma cell line secreting anti-heavy metal copper ion monoclonal antibody and its application

technical field

The invention belongs to the technical field of immunochemical detection, in particular to a hybridoma cell strain secreting anti-heavy metal copper ion monoclonal antibodies and its application.

Background technique

Heavy metal pollution mainly refers to the environmental pollution caused by pollutants such as copper, cadmium, mercury, nickel, chromium, arsenic, zinc, and copper. Heavy metals are widely distributed and difficult to degrade. They can enter the human body through the atmosphere, water and food chain, and interact with proteins and various enzymes in the body, making them inactive and enriched in some organs. , it will cause acute or chronic poisoning of human body, and has carcinogenic, teratogenic and mutagenic effects, and has great harm to human body. Therefore, strengthening the detection of heavy metal residues in the environment, agricultural products and food has become an important means to ensure the safety of heavy metals, and the research and development of new detection technologies under the new situation is particularly urgent.

Traditional heavy metal detection methods mainly use Atomic Absorption Spectroscopy (AAS), Inductively Coupled Plasma Emission Spectroscopy (InductiveLy CoupLedpLasma AtomicEmission Spectroscopy, ICP-AES), Anodic Stripping Voltammetry (ASV), chromatography and Various combined detection methods. Although these methods can effectively analyze heavy metal ions in various environmental samples, most of them require large-scale instruments, the cost of analysis methods is high, the samples need to be digested, the analysis time is long, and they are not suitable for on-site rapid detection of heavy metals, and are difficult to adapt to the environment and market. Requirements for spot inspection of products, self-inspection of production enterprises and rapid customs clearance for product import and export.

Immunological detection technology has the advantages of fast detection speed, large analysis capacity, low cost, simple and portable instrument, low technical requirements for users, easy popularization and promotion, high sensitivity and strong selectivity, etc. It is especially suitable for on-site screening and analysis of a large number of samples. Rapid analysis has become the most competitive and challenging detection and analysis technology in the 21st century.

The key to the immunological detection of heavy metal ions is the preparation of anti-heavy metal specific antibodies, and the key to the preparation of specific antibodies lies in the synthesis of high-quality heavy metal immunogens. Therefore, how to prepare high-quality artificial antigens for heavy metals, how to prepare anti-heavy metal specific antibodies, and establish Faster and more economical immunoassays for the detection of heavy metal ions have become a problem to be solved.

SUMMARY OF THE INVENTION

In order to overcome the defects of the prior art, the present invention provides a hybridoma cell line secreting anti-heavy metal copper ion monoclonal antibody and its application.

A hybridoma cell line that secretes monoclonal antibodies against heavy metal copper ions has been deposited in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, and the deposit number is CGMCC No.16388.

The anti-heavy metal copper ion monoclonal antibody is secreted and produced by the hybridoma cell line whose deposit number is CGMCC No.16388.

Application of hybridoma cell line CGMCC No.16388 or its secreted monoclonal antibody in copper ion immunodetection.

The present invention adopts following technical scheme:

A preparation method of a hybridoma cell line secreting anti-heavy metal copper ion monoclonal antibody, the method is used for preparing the above-mentioned hybridoma cell line, comprising the following steps:

(1) Synthesis and identification of heterocyclic bifunctional chelating agent copper ion artificial antigen:

Weigh 5-8 mg of 2-S-(3-aminobenzene)-1,4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as o-NH ₂ -Bn-NOTA), dissolve In 2mL of 0.01mol·L ^-1 , pH7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution, a chelating agent solution was prepared, and this solution was A solution;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 150-200 μL of solution B to solution A, and react in the dark for 3-5 hours at room temperature, this reaction solution is solution C;

Add 500-800 μL, 20 mmol·L ^-1 glutaraldehyde solution dropwise to solution C, react overnight at room temperature in the dark, and this reaction solution is solution D;

Weigh 20-30 mg of bovine serum albumin BSA or chicken ovalbumin OVA, dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution (20 mg dissolved in 200 μL ultrapure water) dropwise, and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed with an 8KD dialysis bag for 3 to 5 times, and then centrifuged with a 30KD ultrafiltration centrifuge tube at 7000 to 9000 r·min ^-1 for 3 to 5 times, for 15 to 20 minutes each time, with 5 to 10 mL of 0.01M, The HEPES solution with pH 7.4 was reconstituted, then packaged and stored at -20°C to prepare the heavy metal copper artificial antigen Cu-L ₂ -BSA or Cu-L ₂ -OVA, where L ₂ represents 2-S-(3-amino Benzene)-1,4,7triazacyclononane-1,4,7-triacetic acid

The synthetic route is:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

(2) Immunization of mice:

Healthy 6-8 week old female BALB/C mice were selected for immunization. The prepared heavy metal copper artificial antigen Cu-L ₂ -BSA and an equal volume of Freund's complete adjuvant were fully mixed and emulsified by syringe pressure, and then injected through the abdomen and axilla at multiple points. Afterwards, booster immunization was performed every 21 days (days), and blood was collected to measure the titer after 3 booster immunizations, and the titer was determined by indirect ELISA method. When the titer was no longer significantly increased, the final immune was performed, and cell fusion was performed 3 days after the final immune. In the immunization process, Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for booster immunization.

(3) Cell fusion and cell line screening:

The mouse spleen cells were fused with mouse myeloma cells SP2/0 in a ratio of 5 to 10:1 by the polyethylene glycol method, cultured in HAT selective medium, and the positive wells were detected by indirect ELISA, and the positive wells were further utilized. Indirect competition ELISA was used to measure the inhibitory effect of positive cell wells, and then the positive wells with good inhibitory effect were cloned 3-4 times by limiting dilution method. Finally, the hybridoma cell line 6F9 was obtained after verification by indirect competitive ELISA method.

The preparation method of anti-heavy metal copper ion monoclonal antibody comprises the following steps:

BALB/C mice were intraperitoneally injected with 0.3ml/mice of pristane, and the screened 6F9 monoclonal cells were injected in the same way after 7-10 days (days). After centrifugation to remove the grease precipitation, the mouse ascites McAb was obtained;

After purification of the ascites fluid, purified monoclonal antibodies were obtained.

Further, the heterocyclic bifunctional chelating agent L ₂ was respectively replaced with 2-S-(2-aminobenzene)-1,4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as o-NH ₂ -Bn-NOTA) or 2-S-(4-aminobenzene)-1,4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as p-NH ₂ -Bn -NOTA) or 2-S-[4-(3-aminopropyl)-benzene]-1,4,7 triazacyclononane-1,4,7-triacetic acid, the same preparation method above can be used Different copper ion artificial antigens, hybridoma cell lines and anti-heavy metal copper ion monoclonal antibodies were prepared.

Further, by replacing bovine serum albumin BSA with keyhole limpet hemocyanin KLH or other carrier proteins, different copper ion artificial antigens, hybridoma cell lines and monoclonal antibodies can be prepared by using the same preparation method above.

The beneficial effects of the present invention are:

(1) The present invention adopts the heterocyclic bifunctional chelating agent m-NH ₂ -Bn-NOTA to prepare copper ion artificial antigen. The chelating agent can better bind heavy metal ions in structure and can better The structure of the heavy metal copper ion is revealed, and the prepared copper ion artificial antigen has strong specificity.

(2) The hybridoma cell line 6F9 prepared by the present invention can secrete heavy metal copper ion monoclonal antibody, the monoclonal antibody has strong specificity and good sensitivity to copper ion chelator complexes, and the IC ₂₀ value is 0.14ng ·mL ^-1 , which can be used for rapid detection of heavy metal copper ions. The anti-copper ion monoclonal antibody can be used for the residual detection of copper ion in environmental samples.

Description of drawings

Figure 1 is a schematic diagram of the synthesis of copper ion artificial antigens in Examples 1 and 2 (wherein Protein represents bovine serum albumin BSA or chicken ovalbumin OVA).

Figure 2 is a schematic diagram of the synthesis of artificial antigens of copper ions in Examples 3 and 4 (wherein Protein represents bovine serum albumin BSA or chicken ovalbumin OVA).

Figure 3 is a schematic diagram of the synthesis of artificial antigens of copper ions in Examples 5 and 6 (wherein Protein represents bovine serum albumin BSA or chicken ovalbumin OVA).

Figure 4 is a schematic diagram of the synthesis of artificial antigens of copper ions in Examples 7 and 8 (wherein, Protein represents bovine serum albumin BSA or chicken ovalbumin OVA).

Detailed ways

The technical solutions of the present invention are further described in detail below. It should be noted that the specific embodiments are only detailed descriptions of the present invention and should not be regarded as limitations of the present invention.

The heterocyclic bifunctional chelating agent used in the present invention is represented by L. In the embodiment, the heterocyclic bifunctional chelating agent is respectively: 2-S-(2-aminobenzene)-1,4,7 triazacyclononane -1,4,7-Triacetic acid (abbreviated as o-NH ₂ -Bn-NOTA) is represented by L ₁ , 2-S-(3-aminobenzene)-1,4,7 triazacyclononane-1 , 4,7-triacetic acid (abbreviated as m- _NH2 -Bn-NOTA) is represented by L2, ₂ -S-(4-aminobenzene)-1,4,7triazacyclononane-1,4 , 7-triacetic acid (abbreviated as p- _NH2 -Bn-NOTA) is represented by L3, ₂ -S-[4-(3-aminopropyl)-benzene]-1,4,7 triazacyclononane Alkane-1,4,7-triacetic acid (abbreviated as p-CH ₂ CH 2 CH ₂ CH ₂ NH ₂ -Bn-NOTA) is represented by L ₄ , and these chelating agents are commercially available.

1. Preparation and Identification of Heterocyclic Bifunctional Chelating Agent Copper Ion Artificial Antigen

Example 1

(1) Synthesis of copper ion artificial antigen:

Weigh 6 mg L ₁ , dissolve it in 2 mL of 0.01 mol·L ^-1 , pH 7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution to obtain L ₁ chelating agent solution, which is solution A;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 180 μL of solution B to solution A, and react in the dark for 3 to 5 hours at room temperature. This reaction solution is solution C;

Add 700 μL, 20 mmol·L ^-1 of glutaraldehyde aqueous solution dropwise to solution C, react overnight at room temperature in the dark, and this reaction solution is solution D;

Weigh 20 mg of bovine serum albumin BSA, dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution dropwise (20 mg sodium borohydride dissolved in 200 μL ultrapure water), and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed with an 8KD dialysis bag for 3 to 5 times, and then centrifuged with a 30KD ultrafiltration centrifuge tube at 7000 to 9000 r·min ^-1 for 3 to 5 times, 15 to 20 minutes each time, with 5 to 10 mL of 0.01 mol· L ^-1 , reconstituted in HEPES solution with pH 7.4, then packaged and stored at -20 ℃ to prepare heavy metal copper artificial antigen Cu-L ₁ -BSA, L ₁ represents 2-S-(2-aminobenzene)-1 , 4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as o- _NH2 -Bn-NOTA).

(2) Identification of copper ion artificial antigens:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

Ultraviolet scanning scheme: BSA, Cu-L ₁ -BSA and Cu-L ₁ were prepared into solutions with a concentration in the range of 1-5 mg·mL ^-1 , the absorbance in the wavelength range of 200-400 nm was measured, and an ultraviolet scanning spectrum was established. Comparing the absorbance curves of each solution to identify whether the synthesis was successful or not.

In the UV scanning spectrum, compared with the BSA solution, the maximum absorption wavelength of the Cu _{-L 1 -BSA} solution changed, indicating that the coupling was successful.

SDS-PAGE electrophoresis scheme: choose 5% stacking gel, choose 10% separating gel, load 10 μL per well, stacking gel voltage 75V, separating gel voltage 100V, Coomassie brilliant blue staining for 1h, destaining 4 times , and the gel imager photographed and analyzed.

SDS-PAGE showed that the electrophoretic band of the conjugate had a hysteresis phenomenon compared with that of the single protein, and the molecular weight of the conjugate was larger than that of the single protein, indicating that the coupling was successful.

The binding ratio was calculated to be 24:1 by measuring heavy metal content and conjugate protein concentration. The binding ratio is 24:1, which means that 24 heavy metal ions are bound to one protein molecule.

Example 2

(1) Synthesis of copper ion artificial antigen:

Weigh 7 mg L ₁ , dissolve it in 2 mL of 0.01 mol·L ^-1 , pH 7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution to obtain L ₁ chelating agent solution, which is solution A;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 195 μL of solution B to solution A, and react in the dark for 3 to 5 hours at room temperature. This reaction solution is solution C;

Add 720 μL, 20 mmol·L ^-1 of glutaraldehyde solution dropwise to solution C, and react overnight in the dark at room temperature. This reaction solution is solution D;

Weigh 20 mg of chicken ovalbumin OVA and dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution dropwise (20 mg sodium borohydride dissolved in 200 μL ultrapure water), and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed with an 8KD dialysis bag for 3 to 5 times, and then centrifuged with a 30KD ultrafiltration centrifuge tube at 7000 to 9000 r·min ^-1 for 3 to 5 times, 15 to 20 minutes each time, with 5 to 10 mL of 0.01 mol· L ^-1 , reconstituted in HEPES solution with pH 7.4, then packaged and stored at -20 ℃ to prepare heavy metal copper artificial antigen Cu-L ₁ -OVA, L ₁ represents 2-S-(2-aminobenzene)-1 , 4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as o- _NH2 -Bn-NOTA).

(2) Identification of artificial antigens:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

Ultraviolet scanning scheme: OVA, Cu-L ₁ -OVA and Cu-L ₁ were prepared into solutions with a concentration in the range of 1-5 mg·mL ^-1 , the absorbance in the wavelength range of 200-400 nm was measured, and an ultraviolet scanning spectrum was established. Comparing the absorbance curves of each solution to identify whether the synthesis was successful or not.

In the UV scanning spectrum, the maximum absorption wavelength of Cu-L ₁ -OVA solution was changed compared with that of OVA solution, indicating that the coupling was successful.

SDS-PAGE electrophoresis scheme: choose 5% stacking gel, choose 10% separating gel, load 10 μL per well, stacking gel voltage 75V, separating gel voltage 100V, Coomassie brilliant blue staining for 1h, destaining 4 times , and the gel imager photographed and analyzed.

SDS-PAGE showed that the electrophoretic band of the conjugate had a hysteresis phenomenon compared with that of the single protein, and the molecular weight of the conjugate was larger than that of the single protein, indicating that the coupling was successful.

The binding ratio was calculated to be 21:1 by measuring heavy metal content and conjugate protein concentration. The binding ratio is 21:1, which means that 21 heavy metal ions are bound to one protein molecule.

Example 3

The heterocyclic bifunctional chelating agent selected in this example is L ₂ , which replaces L ₁ in Example 1; the artificial copper ion antigen Cu-L ₂ -BSA was prepared according to the same steps as in Example 1, and the copper ion artificial antigen was The synthetic route is shown in Figure 2, and the specific synthetic steps are as follows.

(1) Synthesis of copper ion artificial antigen:

Weigh 6 mg L ₂ , dissolve it in 2 mL of 0.01 mol·L ^-1 , pH 7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution to obtain L ₂ chelating agent solution, which is solution A;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 180 μL of solution B to solution A, and react in the dark for 3 to 5 hours at room temperature. This reaction solution is solution C;

Add 700 μL, 20 mmol·L ^-1 of glutaraldehyde solution dropwise to solution C, react overnight at room temperature in the dark, and this reaction solution is solution D;

Weigh 20 mg of bovine serum albumin BSA, dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution dropwise (20 mg sodium borohydride dissolved in 200 μL ultrapure water), and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed 3-5 times with an 8KD dialysis bag, and then centrifuged and washed 3-5 times with a 30KD ultrafiltration centrifuge tube at 7000-9000 r·min ^-1 for 15-20 min each time, with 5-10 mL of 0.01 mol· L ^-1 , reconstituted in HEPES solution with pH 7.4, then packaged and stored at -20℃ to prepare heavy metal copper artificial antigen Cu-L ₂ -BSA, L ₂ represents 2-S-(3-aminobenzene)- 1,4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as m- _NH2 -Bn-NOTA).

(2) Identification of artificial antigens:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

Ultraviolet scanning scheme: BSA, Cu-L ₂ -BSA and Cu-L ₂ were prepared into a solution with a concentration in the range of 1-5 mg·mL ^-1 , the absorbance in the wavelength range of 200-400 nm was measured, and an ultraviolet scanning spectrum was established. Comparing the absorbance curves of each solution to identify whether the synthesis was successful or not.

In the UV scanning spectrum, the maximum absorption wavelength of Cu-L ₂ -BSA solution was changed compared with that of BSA solution, indicating that the coupling was successful.

SDS-PAGE electrophoresis scheme: choose 5% stacking gel, choose 10% separating gel, load 10 μL per well, stacking gel voltage 75V, separating gel voltage 100V, Coomassie brilliant blue staining for 1h, destaining 4 times , and the gel imager photographed and analyzed.

SDS-PAGE showed that the electrophoretic band of the conjugate had a hysteresis phenomenon compared with that of the single protein, and the molecular weight of the conjugate was larger than that of the single protein, indicating that the coupling was successful.

The binding ratio was calculated to be 46:1 by measuring the content of heavy metals and the protein concentration of the conjugate, and the coupling efficiency was high. The higher the binding ratio, the greater the number of heavy metal ions coupled to a protein molecule, and the higher the coupling efficiency. When the binding ratio is 46:1, it means that a protein molecule binds 46 heavy metal ions.

Example 4

The heterocyclic bifunctional chelating agent selected in this example is L ₂ , which replaces L ₁ in Example 2; the artificial copper ion antigen Cu-L ₂ -OVA was prepared according to the same steps as in Example 2, and the copper ion artificial antigen was The synthetic route is shown in Figure 2, and the specific synthetic steps are as follows.

(1) Synthesis of copper ion artificial antigen:

Weigh 7 mg L ₂ , dissolve it in 2 mL of 0.01 mol·L ^-1 , pH7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution to obtain L ₂ chelating agent solution, which is solution A;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 195 μL of solution B to solution A, and react in the dark for 3 to 5 hours at room temperature. This reaction solution is solution C;

Add 720 μL, 20 mmol·L ^-1 of glutaraldehyde solution dropwise to solution C, and react overnight in the dark at room temperature. This reaction solution is solution D;

Weigh 20 mg of chicken ovalbumin OVA and dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution dropwise (20 mg sodium borohydride dissolved in 200 μL ultrapure water), and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed 3-5 times with an 8KD dialysis bag, and then centrifuged and washed 3-5 times with a 30KD ultrafiltration centrifuge tube at 7000-9000 r·min ^-1 for 15-20 min each time, with 5-10 mL of 0.01 mol· L ^-1 , reconstituted in HEPES solution with pH 7.4, then packaged and stored at -20 ℃ to prepare heavy metal copper artificial antigen Cu-L ₂ -OVA, L ₂ represents 2-S-(3-aminobenzene)- 1,4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as m- _NH2 -Bn-NOTA).

(2) Identification of artificial antigens:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

Ultraviolet scanning scheme: OVA, Cu-L ₂ -OVA and Cu-L ₂ were prepared into a solution with a concentration of 1-5 mg·mL ^-1 , the absorbance in the wavelength range of 200-400 nm was measured, and an ultraviolet scanning spectrum was established. The absorption curve of the solution was used to identify whether the synthesis was successful.

In the UV scanning spectrum, the maximum absorption wavelength of Cu-L ₂ -OVA solution was changed compared with that of OVA solution, indicating that the coupling was successful.

SDS-PAGE electrophoresis scheme: choose 5% stacking gel, choose 10% separating gel, load 10 μL per well, stacking gel voltage 75V, separating gel voltage 100V, Coomassie brilliant blue staining for 1h, destaining 4 times , and the gel imager photographed and analyzed.

SDS-PAGE showed that the electrophoretic band of the conjugate had a hysteresis phenomenon compared with that of the single protein, and the molecular weight of the conjugate was larger than that of the single protein, indicating that the coupling was successful.

The binding ratio of 40:1 was calculated by measuring the heavy metal content and the conjugated protein concentration, and a binding ratio of 40:1 means that 40 heavy metal ions are bound to one protein molecule.

Example 5

The heterocyclic bifunctional chelating agent selected in this example is L ₃ , which replaces L ₁ in Example 1; the artificial copper ion antigen Cu-L ₃ -BSA was prepared according to the same steps as in Example 1, and the copper ion artificial antigen was The synthetic route is shown in Figure 3, and the specific synthetic steps are as follows.

(1) Synthesis of copper ion artificial antigen:

Weigh 6 mg L ₃ , dissolve it in 2 mL of 0.01 mol·L ^-1 , pH 7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution to obtain L ₃ chelating agent solution, which is solution A;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 180 μL of solution B to solution A, and react in the dark for 3 to 5 hours at room temperature. This reaction solution is solution C;

Add 700 μL, 20 mmol·L ^-1 of glutaraldehyde solution dropwise to solution C, react overnight at room temperature in the dark, and this reaction solution is solution D;

Weigh 20 mg of bovine serum albumin BSA, dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution dropwise (20 mg sodium borohydride dissolved in 200 μL ultrapure water), and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed with an 8KD dialysis bag for 3 to 5 times, and then centrifuged with a 30KD ultrafiltration centrifuge tube at 7000 to 9000 r·min ^-1 for 3 to 5 times, 15 to 20 minutes each time, with 5 to 10 mL of 0.01 mol· L ^-1 , reconstituted in HEPES solution with pH 7.4, then packaged and stored at -20 ℃ to prepare heavy metal copper artificial antigen Cu-L ₃ -BSA, L ₃ represents 2-S-(4-aminobenzene)- 1,4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as p- _NH2 -Bn-NOTA).

(2) Identification of artificial antigens:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

Ultraviolet scanning scheme: BSA, Cu-L ₃ -BSA and Cu-L ₃ were prepared into a solution with a concentration in the range of 1-5 mg·mL ^-1 , the absorbance in the wavelength range of 200-400 nm was measured, and an ultraviolet scanning spectrum was established. Comparing the absorbance curves of each solution to identify whether the synthesis was successful or not.

In the UV scanning spectrum, the maximum absorption wavelength of Cu-L ₃ -BSA solution was changed compared with that of BSA solution, indicating that the coupling was successful.

SDS-PAGE electrophoresis scheme: choose 5% stacking gel, choose 10% separating gel, load 10 μL per well, stacking gel voltage 75V, separating gel voltage 100V, Coomassie brilliant blue staining for 1h, destaining 4 times , and the gel imager photographed and analyzed.

SDS-PAGE showed that the electrophoretic band of the conjugate had a hysteresis phenomenon compared with that of the single protein, and the molecular weight of the conjugate was larger than that of the single protein, indicating that the coupling was successful.

By measuring the heavy metal content and the conjugated protein concentration, the binding ratio was calculated to be 39:1, and the binding ratio was 39:1, which indicated that 39 heavy metal ions were bound to one protein molecule.

Example 6

The heterocyclic bifunctional chelating agent selected in this example is L ₃ , which replaces L _{1 in Example 1} ; the artificial copper ion antigen Cu-L ₃ -OVA was prepared according to the same steps as in Example 2, and the copper ion artificial antigen was The synthetic route is shown in Figure 3, and the specific synthetic steps are as follows.

(1) Synthesis of copper ion artificial antigen:

Weigh 7 mg L ₃ , dissolve it in 2 mL of 0.01 mol·L ^-1 , pH 7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution to obtain L ₃ chelating agent solution, which is solution A;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 195 μL of solution B to solution A, and react in the dark for 3 to 5 hours at room temperature. This reaction solution is solution C;

Add 720 μL, 20 mmol·L ^-1 of glutaraldehyde solution dropwise to solution C, and react overnight in the dark at room temperature. This reaction solution is solution D;

Weigh 20 mg of chicken ovalbumin OVA and dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution dropwise (20 mg sodium borohydride dissolved in 200 μL ultrapure water), and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed with an 8KD dialysis bag for 3 to 5 times, and then centrifuged with a 30KD ultrafiltration centrifuge tube at 7000 to 9000 r·min ^-1 for 3 to 5 times, 15 to 20 minutes each time, with 5 to 10 mL of 0.01 mol· L ^-1 , reconstituted in HEPES solution with pH 7.4, then packaged and stored at -20 ℃ to prepare heavy metal copper artificial antigen Cu-L ₃ -OVA, L ₃ represents 2-S-(4-aminobenzene)-1 , 4,7 triazacyclononane-1,4,7-triacetic acid (abbreviated as p- _NH2 -Bn-NOTA).

(2) Identification of artificial antigens:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

UV scanning scheme: OVA, Cu-L ₃ -OVA and Cu-L ₃ were prepared into solutions with a concentration in the range of 1 to 5 mg·mL ^-1 , the absorbance in the wavelength range of 200 to 400 nm was measured, and the UV scanning spectrum was established. Comparing the absorbance curves of each solution to identify whether the synthesis was successful or not.

In the UV scanning spectrum, the maximum absorption wavelength of Cu-L ₃ -OVA solution was changed compared with that of OVA solution, indicating that the coupling was successful.

SDS-PAGE electrophoresis scheme: choose 5% stacking gel, choose 10% separating gel, load 10 μL per well, stacking gel voltage 75V, separating gel voltage 100V, Coomassie brilliant blue staining for 1h, destaining 4 times , and the gel imager photographed and analyzed.

SDS-PAGE showed that the electrophoretic band of the conjugate had a hysteresis phenomenon compared with that of the single protein, and the molecular weight of the conjugate was larger than that of the single protein, indicating that the coupling was successful.

By measuring the heavy metal content and the conjugate protein concentration, the binding ratio was calculated to be 36:1, and the binding ratio was 36:1, which indicated that 36 heavy metal ions were bound to one protein molecule.

Example 7

The heterocyclic bifunctional chelating agent selected in this example is L ₄ , which replaces L ₁ in Example 1; the artificial copper ion antigen Cu-L ₄ -BSA was prepared according to the same steps as in Example 1, and the copper ion artificial antigen was The synthetic route is shown in Figure 4, and the specific synthetic steps are as follows.

(1) Synthesis of copper ion artificial antigen:

Weigh 6 mg L ₄ , dissolve it in 2 mL of 0.01 mol·L ^-1 , pH7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution to obtain L ₄ chelating agent solution, which is solution A;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 180 μL of solution B to solution A, and react in the dark for 3 to 5 hours at room temperature. This reaction solution is solution C;

Add 700 μL, 20 mmol·L ^-1 of glutaraldehyde solution dropwise to solution C, react overnight at room temperature in the dark, and this reaction solution is solution D;

Weigh 20 mg of bovine serum albumin BSA, dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution (20 mg dissolved in 200 μL ultrapure water) dropwise, and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed with an 8KD dialysis bag for 3 to 5 times, and then centrifuged with a 30KD ultrafiltration centrifuge tube at 7000 to 9000 r·min ^-1 for 3 to 5 times, 15 to 20 minutes each time, with 5 to 10 mL of 0.01 mol· L ^-1 , reconstituted in HEPES solution with pH 7.4, then packaged and stored at -20 ℃ to prepare heavy metal copper artificial antigen Cu-L ₄ -BSA, L ₄ represents 2-S-[4-(3-amino propyl)-benzene] _-1,4,7 triazacyclononane _- 1,4,7-triacetic acid (abbreviated as p- _{CH2CH2CH2NH2 -} Bn-NOTA).

(2) Identification of artificial antigens:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

Ultraviolet scanning scheme: BSA, Cu-L ₄ -BSA and Cu-L ₄ were prepared into solutions with a concentration in the range of 1 to 5 mg·mL ^-1 , the absorbance in the wavelength range of 200 to 400 nm was measured, and an ultraviolet scanning spectrum was established. Comparing the absorbance curves of each solution to identify whether the synthesis was successful or not.

In the UV scanning spectrum, the maximum absorption wavelength of Cu-L ₄ -BSA solution was changed compared with that of BSA solution, indicating that the coupling was successful.

SDS-PAGE electrophoresis scheme: choose 5% stacking gel, choose 10% separating gel, load 10 μL per well, stacking gel voltage 75V, separating gel voltage 100V, Coomassie brilliant blue staining for 1h, destaining 4 times , and the gel imager photographed and analyzed.

SDS-PAGE showed that the electrophoretic band of the conjugate had a hysteresis phenomenon compared with that of the single protein, and the molecular weight of the conjugate was larger than that of the single protein, indicating that the coupling was successful.

By measuring the heavy metal content and the conjugated protein concentration, the binding ratio was calculated to be 42:1, and the binding ratio was 42:1, indicating that 42 heavy metal ions were bound to one protein molecule.

Example 8

The heterocyclic bifunctional chelating agent selected in this example is L ₄ , which replaces L _{1 in Example 1} ; the artificial copper ion antigen Cu-L ₄ -OVA was prepared according to the same steps as in Example 2, and the copper ion artificial antigen was The synthetic route is shown in Figure 4, and the specific synthetic steps are as follows.

(1) Synthesis of copper ion artificial antigen:

Weigh 7 mg L ₄ , dissolve it in 2 mL of 0.01 mol·L ^-1 , pH 7.4 4-hydroxyethylpiperazineethanesulfonic acid HEPES solution to obtain L ₄ chelating agent solution, which is solution A;

Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B;

Add 195 μL of solution B to solution A, and react in the dark for 3 to 5 hours at room temperature. This reaction solution is solution C;

Add 720 μL, 20 mmol·L ^-1 of glutaraldehyde solution dropwise to solution C, and react overnight in the dark at room temperature. This reaction solution is solution D;

Weigh 20 mg of chicken ovalbumin OVA and dissolve it in 4 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution;

Add solution D to solution E dropwise, react in the dark for 24 hours at room temperature, then add 150-200 μl sodium borohydride solution (20 mg dissolved in 200 μl ultrapure water) dropwise, and react in the dark for 1 hour at room temperature;

The reaction solution was first dialyzed with an 8KD dialysis bag for 3 to 5 times, and then centrifuged with a 30KD ultrafiltration centrifuge tube at 7000 to 9000 r·min ^-1 for 3 to 5 times, 15 to 20 minutes each time, with 5 to 10 mL of 0.01 mol· L ^-1 , reconstituted in HEPES solution with pH 7.4, then packaged and stored at -20 ℃ to prepare heavy metal copper artificial antigen Cu-L ₄ -OVA, L ₄ represents 2-S-[4-(3-aminopropane) base)-benzene] _-1,4,7 triazacyclononane _- 1,4,7-triacetic acid (abbreviated as p- _{CH2CH2CH2NH2 -} Bn-NOTA).

(2) Identification of artificial antigens:

The coupling effect was identified by UV scanning and SDS-PAGE. The concentrations of heavy metal ions and proteins were determined by ICP-MS and Bradford methods, respectively, and the coupling-binding ratio of artificial antigens was calculated.

Ultraviolet scanning scheme: OVA, Cu-L ₄ -OVA and Cu-L ₄ were prepared into solutions with a concentration in the range of 1-5 mg·mL ^-1 , the absorbance in the wavelength range of 200-400 nm was measured, and an ultraviolet scanning spectrum was established. Comparing the absorbance curves of each solution to identify whether the synthesis was successful or not.

In the UV scanning spectrum, the maximum absorption wavelength of Cu-L ₄ -OVA solution was changed compared with that of OVA solution, indicating that the coupling was successful.

SDS-PAGE electrophoresis program: choose 5% stacking gel, choose 10% separating gel, load 10 μL per well, stacking gel voltage 75V, separating gel voltage 100V, Coomassie brilliant blue staining for 1h, destaining 4 times , and the gel imager photographed and analyzed.

SDS-PAGE showed that the electrophoretic band of the conjugate had a hysteresis phenomenon compared with that of the single protein, and the molecular weight of the conjugate was larger than that of the single protein, indicating that the coupling was successful.

By measuring the heavy metal content and the conjugated protein concentration, the binding ratio was calculated to be 38:1, and the binding ratio was 38:1, indicating that 38 heavy metal ions were bound to one protein molecule.

Example 9

Determination of antiserum titer:

The artificial antigens prepared in Examples 1, 3, 5 and 7 were respectively immunized to BALB/C mice, and the artificial antigen was emulsified with Freund's complete adjuvant for the initial immunization. The immunization was boosted at intervals of 21 days, and the booster immunization was boosted 3 times in total. The booster immunization was emulsified with incomplete adjuvant, and the immunization dose was 150 μg/mouse. The titer of antiserum was determined by ELISA after doubling the dilution. The mouse serum before immunization was used as the negative control, and the dilution at which the ratio of the OD _450nm value of the positive serum to the OD _450nm value of the negative serum was greater than 2.1 was the antiserum titer. The results are as follows: shown in Table 1. Finally, the final immunization was performed by direct intraperitoneal injection of artificial antigen, and the immunization dose was 300 μg/mouse.

The titer was determined by an indirect ELISA method, and the specific experimental steps were as follows:

a. Coating: Take the artificial antigen in Example 2 or Example 4 or Example 6 or Example 8 as the coating original, pH 9.6, 0.05mol·L ^-1 CBS as the coating buffer, and the coating original The concentration was 10 μg·mL ^-1 , the coating amount was 100 μL/well, and the plate was coated for 2 hours at 37°C, and then the plate was washed 4 times with PBST washing solution;

b. Blocking: Add 250 μL/well of blocking solution, incubate at 37°C for 30 min, and wash the plate 4 times with PBST washing solution;

c. Add antiserum: After centrifuging the antiserum obtained by blood collection of mice at 10000r·min ^-1 for 5 minutes, draw 10 μL into 2mL blocking solution (the initial dilution ratio is 200 times), and then use the blocking solution to dilute 11 times. Gradient and 1 negative control, 100 μL/well, 4 replicates for each gradient, incubated at 37°C for 1 h, and then washed 4 times with PBST wash solution;

d. Add enzyme-labeled secondary antibody: after the reaction, wash the plate 4 times with PBST, and add the HRP-labeled goat anti-mouse secondary antibody 10,000 times to the enzyme-labeled plate, 100 μL/well, incubate at 37 °C for 1 h, and then wash the plate with PBST washing solution 4. Second-rate;

e. Add substrate reaction solution: add TMB substrate buffer, 100μL/well, incubate at 37°C for 15min;

f. Stop reading: add 50 μL/well of 2 mol·L ^-1 sulfuric acid, and read the OD _450nm value with a microplate reader.

Binding ratio and antiserum titer determination results in Table 1 Examples 1-8

immune antigen binding ratio detection antigen binding ratio Antiserum titer Cu-L₁-BSA Example 1 24:1 Cu-L₁-OVA Example 2 21:1 256000 Cu-L₂-BSA Example 3 46:1 Cu-L₂-OVA Example 4 40:1 650000 Cu-L₃-BSA Example 5 39:1 Cu-L₃-OVA Example 6 36:1 550000 Cu-L₄-BSA Example 7 42:1 Cu-L₄-OVA Example 8 38:1 600000

The results of the determination of the binding ratio and antiserum titer in Table 1 show that the order of antiserum titer is: Example 3> Example 7> Example 5> Example 1, and the order of binding ratio is: Example 3> Implementation Example 7>Example 5>Example 1, wherein, the antiserum titer of Example 3 is the highest, and the antiserum titer of Example 7 is only lower than that of Example 3. This shows that the copper ion artificial antigen Cu-L ₂ -BSA prepared in Example 3 can better express the heavy metal antigenic determinant, and the antigen specificity is stronger. At the same time, the artificial antigen prepared in Example 3 and Example 7 has The binding ratio is high, and the high binding ratio is more likely to trigger a strong immune response, which is beneficial to the preparation of anti-heavy metal copper ion monoclonal antibodies.

The specific preparation methods and measurement methods adopted in Examples 1, 3, 5, and 7 are the same, but the bifunctional chelating agents adopted are different, namely: o-NH ₂ -Bn-NOTA, m-NH ₂ -Bn-NOTA, p _{- NH2 -} Bn-NOTA, p- _{CH2CH2CH2NH2 -} Bn-NOTA. This shows that -NH ₂ in the chelating agent is in the meta position of the benzene ring. After the chelating agent m-NH ₂ -Bn-NOTA binds heavy metal ions, it can better display the structure of heavy metal copper ions in terms of spatial structure. The results showed that the artificial antigen prepared by the meta structure also had the highest binding ratio. As an antigenic determinant, it was more conducive to the preparation of anti-heavy metal monoclonal antibodies with higher affinity, sensitivity and specificity.

Example 10

Screening of hybridoma cell lines and preparation and purification of monoclonal antibodies

(1) Mice immunization:

BALB/C female mice aged 6-8 weeks and weighing 18-20 g were selected. The prepared immunogen (the immunogen used here is: copper ion artificial antigen Cu-L ₂ -BSA) and an equal volume of Freund's complete adjuvant are fully mixed and emulsified by syringe pressure, and injected through the abdomen and axilla at multiple points. , the dose is 50-100 μg/a, and then booster immunization is carried out every 21d. After 3 booster immunizations, blood is collected to measure the titer, and the titer is measured by indirect ELISA method. , the mouse serum before immunization was used as the negative control, and the dilution at which the ratio of the positive serum OD _450nm value to the negative serum was greater than 2.1 was used as the antiserum titer. When the titer was no longer significantly increased, the final immunity was performed, and cell fusion was performed 3 days after the final immunity. In the immunization process, Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for booster immunization.

The titer was determined by an indirect ELISA method, and the specific experimental steps were as follows:

a. Coating: Take Cu-L ₂ -OVA as the coating source, pH 9.6, 0.05mol·L ^-1 CBS as the coating buffer, the coating concentration is 10μg·mL ^-1 , and the coating amount is 100μL/ Well, after coating for 2 hours at 37°C, the plate was washed 4 times with PBST wash solution;

b. Blocking: add 250 μL/well of blocking solution, incubate at 37°C for 30 min, wash the plate 4 times with PBST, add 300 μL of PBST to each well, and then wash the plate 4 times with PBST washing solution;

c. Add antiserum: After centrifuging the antiserum obtained by blood collection of mice at 10000r·min ^-1 for 5 minutes, pipette 10μL into 2mL of blocking solution (the initial dilution factor is 200 times), and then use the blocking solution to dilute 11 gradients. and 1 negative control, 100 μL/well, 4 replicates for each gradient, incubate at 37°C for 1 h, and wash the plate 4 times with PBST wash;

d. Add enzyme-labeled secondary antibody: After the reaction, wash the plate 4 times. After the HRP-labeled goat anti-mouse secondary antibody is diluted 10,000 times, add it to the enzyme-labeled plate, 100 μL/well, incubate at 37 °C for 1 h, and then wash the plate 4 times with PBST washing solution. ;

e. Add substrate reaction solution: add TMB substrate buffer, 100μL/well, incubate at 37°C for 15min;

f. Stop reading: add 50 μL/well of 2 mol·L ^-1 sulfuric acid, and read the OD _450nm value with a microplate reader.

(2) Cell fusion and culture:

After 3 days of final immunity, cell fusion was performed according to the conventional polyethylene glycol (PEG1500) method, and the specific steps were as follows:

a. The eyeballs of the mice after the final immunization were bled, the serum was collected, and the supernatant was centrifuged for use. After being sacrificed by neck pulling, they were placed in 70% alcohol for 3-5 minutes. The spleen of the mice was taken under aseptic conditions and cut into pieces with sterile surgical scissors. Then put it into a sterile mortar for grinding, and suspend the cells with RPMI-1640 basal medium, then pass through a 200-mesh cell screen to obtain a spleen cell suspension, and count the cells;

b. Collect SP2/0 cells (myeloma cells), the cell growth state is required to be good, and the cell viability is greater than 90%. After aspirating the cell supernatant, add a new RPMI-1640 basal medium and pipetting the cells to suspend, and then count the cells. ;

c. According to the cell count results, mix splenocytes and SP2/0 cells in a ratio of 5-10:1, centrifuge at 1800 r·min ^-1 for 5 min, remove the supernatant, and add 0.5 mL of PEG to the remaining cells within 1 min. Gently stir and mix well. After adding, let stand for 1 min and then add 45 mL of RPMI-1640 basal medium from slow to fast. After centrifugation at 1500 r·min ^-1 for 5 min, remove the supernatant, add selective HAT medium and plate at 96 Well cell culture plates, 250 μL per well, were placed in an incubator at 37 °C, 5% CO ₂ .

d. After culturing for 3 to 5 days (days), the medium was changed once with HAT medium, and the 10th day was changed to HT medium for culture.

(3) Cell screening and establishment of cell lines:

When the fused cells grow to cover 10% to 30% of the bottom area of the culture well, take the supernatant and screen the antibody _- positive wells by indirect ELISA. as a negative control. The selected positive reaction wells were further analyzed for the detection sensitivity of the antibody by competitive ELISA.

According to the established standard curve, calculate the IC ₅₀ value and IC ₂₀ value, and select the slope range of the standard curve (the abscissa is the natural logarithm of the copper ion standard concentration, and the ordinate is the competition standard curve established by the inhibition rate) between 8 and 13. In between, the cell lines with IC ₅₀ value lower than 200ng·ml ^-1 and IC ₂₀ value lower than 20ng·ml ^-1 were further cloned and screened, and cloned for 3 to 4 times by limiting dilution method, and finally a hybridoma was obtained. Cell line 6F9, its deposit number is CGMCC No.16388. Relatively speaking, a cell line with a lower IC ₂₀ value and a lower IC ₅₀ value has a better inhibitory effect, which is beneficial to the preparation of a hybridoma cell line with better characteristics.

The hybridoma cell line 6F9 has been deposited on September 25, 2018 in the General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC for short, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing , 100101).

After the finally obtained hybridoma cell line 6F9 is expanded and cultured, on the one hand, the cell line is transferred into a cell cryopreservation tube and placed in liquid nitrogen for long-term storage, and on the other hand, the cell line is used for ascites preparation and monoclonal antibody purification. and application.

(3) Preparation, purification and identification of monoclonal antibodies

Monoclonal antibodies were prepared by in vivo induction in animals.

Healthy BALB/C female mice aged 6-8 weeks were selected, BALB/C mice were injected intraperitoneally with 0.3 mL/mice of pristane, and 7-10 days later, the screened 6F9 monoclonal cells (0.4 mL/mice were injected in the same way). The number of cell lines per ml was between 2.5×10 ⁶ and 1×10 ⁷ ). After 5 to 7 days, the ascites was extracted after the abdominal cavity of the mice was obviously distended.

After the ascites was initially purified by the octanoic acid-ammonium sulfate method, it was further purified by an affinity chromatography column (the filler was TOYOPEARL AF-rProtein A HC-650F produced by TOSOH). and the optical density at 280 nm, the concentration of monoclonal antibody was calculated by Lowry-kalokar formula to be 2.43 mg·mL ^-1 , and the remaining purified monoclonal antibodies were stored at -70°C for future use.

The specific steps of ascites purification are as follows:

1) Dilute the ascites with 60 mmol·L ^-1 acetate buffer solution of pH 4.0 at a volume ratio of 1:4, and adjust the pH value to 4.5 with 0.1 mol · L ^-1 NaOH;

2) Caprylic acid was added dropwise at a ratio of 33 μl·mL ^-1 ascites under magnetic stirring, followed by stirring at room temperature for 30 minutes, standing at 4°C for 2 hours, and then centrifuging at 10000 r·min ^-1 for 30 minutes;

3) After the supernatant was filtered with qualitative filter paper, 0.1 mol·L ^-1 PBS was added at a ratio ^of 1:10 (PBS: filtered supernatant), the pH was adjusted to 7.4 with 1 mol·L ^-1 NaOH, and placed at 4°C for 30 min ;

4) Under magnetic stirring, ammonium sulfate was added in batches at a ratio of 0.277 g·mL ^-1 (ammonium sulfate: mixed solution), and after the addition, the reaction was stirred for 30 minutes, left standing at 4°C for 3 hours, centrifuged at 12000 r·min ^-1 for 30 minutes, and discarded. clear;

5) The precipitate is dissolved with a small amount of 0.01mol·L ^-1 PBS and placed in a dialysis bag, dialyzed with pH 7.4, 0.01mol·L ^-1 PBS, and the medium is exchanged 4 to 6 times;

6) The preliminary purified monoclonal antibody solution was purified by protein A (TOYOPEARL AF-rProtein AHC-650F) affinity chromatography column. After purification, the antibody was freeze-dried and stored at -70°C for later use.

Example 11

Establishment of an indirect competitive ELISA method for monoclonal antibodies

The determination of the working concentration of monoclonal antibody is carried out by orthogonal test, and the specific experimental steps are as follows:

1) Coating: take pH 9.6, 0.05mol·L ^-1 CBS as the coating buffer, Cu-L ₂ -OVA as the coating source, the coating amount is diluted by 10 μg·mL ^-1 at the beginning, and the coating volume 100 μL/well, after incubation at 37°C for 2 h, the plate was washed 4 times with PBST wash solution;

2) Blocking: Add 250 μL/well of blocking solution, incubate at 37°C for 30 min, and wash the plate 4 times with PBST washing solution;

3) Sample loading: Add the purified monoclonal antibody, and then add it to the coated wells after initial doubling dilution of 10 μg·mL ^-1 , 100 μL/well, react at 37°C for 1 hour, and wash the plate 4 times with PBST washing solution;

4) Add enzyme-labeled secondary antibody: after the reaction, wash the plate 4 times. HRP-labeled goat anti-mouse secondary antibody is diluted 10,000 times with blocking solution and then added to the enzyme-labeled plate, 100 μL/well, react at 37 °C for 1 h, and wash with PBST washing solution plate 4 times;

5) Add substrate reaction solution: add TMB substrate buffer, 100 μL/well, and incubate at 37°C for 15 min;

6) Stop reading: add 50 μL/well of 2 mol·L ^-1 sulfuric acid, and read the OD _450nm value with a microplate reader.

7) Select the antigen-antibody concentration combination with the OD _450nm value in the range of 1.0-1.2 as the working concentration, and select 4

Working concentration combination, the concentration combination of antigen and antibody is (0.625μg·mL ^-1 , 5μg·mL ^-1 ),

(1.25μg·mL ^-1 , 2.μg·mL ^-1 ), (2.5μg·mL ^-1 , 1.25μg·mL ^-1 ) and (5μg·mL ^-1 ,

0.625μg·mL ^-1 ), using the combination of 4 working concentrations selected by the indirect competitive ELISA method for sensitive

Degree analysis and screening, the results are shown in Table 2, through the established standard curve slope, IC ₅₀ value and standard curve r value

The optimal working concentration was determined to be the antigen coating concentration of 1.25μg·mL ^-1 , and other parameters were comprehensively evaluated.

The antibody concentration was 2.5 μg·mL ^-1 .

Table 2 Working concentration analysis results

The analytical sensitivity IC ₂₀ of the purified monoclonal antibody to the copper chelator complex by indirect ELISA was 0.14ng·mL ^-1 , which indicated that the prepared anti-heavy metal copper ion monoclonal antibody had high detection sensitivity and It has good specificity and can be used for rapid detection of heavy metal copper ions.

The specific steps of indirect competition ELISA are as follows:

a. Coating: with pH 9.6, 0.05mol·L ^-1 CBS as coating buffer, Cu-L ₂ -OVA as coating source, the coating volume is 100 μL/well, and the coating concentration is the best screened above Working concentration, after incubation at 37°C for 2h, the plate was washed 4 times with PBST wash solution;

b. Blocking: Add 250 μL/well of blocking solution, incubate at 37°C for 30 min, and wash the plate 4 times with PBST washing solution;

c. Sample pretreatment: 5mmol·L ^-1 2-S-(2-aminobenzene)-1,4,7 triazacyclononane-1,4,7-triacetic acid ie L ₁ and heavy metals with different concentration gradients The copper ion standard sample was mixed in equal volume and incubated at 37°C for 1 h;

d. Add sample: add 50 μL/well of pre-mixed sample, add 50 μL/well of primary antibody twice the optimal working concentration (that is, the anti-copper ion monoclonal antibody prepared in Example 10 above), shake and mix well and react at 37°C After 1 h, the plate was washed 4 times with PBST washing solution;

e. Add enzyme-labeled secondary antibody: After the reaction, wash the plate 4 times, and add the HRP-labeled goat anti-mouse secondary antibody 10,000-fold with blocking solution and add it to the enzyme-labeled plate, 100 μL/well, react at 37°C for 1 h, wash with PBST Wash the plate 4 times;

f. Add substrate reaction solution: add TMB substrate buffer, 100μL/well, incubate at 37°C for 15min;

g. Stop reading: add 50 μL/well of 2 mol·L ^-1 sulfuric acid, and read the OD _450nm value with a microplate reader.

Solution preparation:

Carbonate buffer solution (CBS): Weigh Na ₂ CO ₃ 1.59 g, NaHCO ₃ 2.93 g, add pure water to 990 mL, adjust pH to 9.6, and then make up to 1000 mL with pure water, store at 4°C for later use.

Phosphate buffered saline (PBS): 8.5g NaCl, 2.2g Na ₂ HPO ₄ ·12H ₂ O, 0.2g NaH ₂ PO ₄ ·2H ₂ O, dissolved in 900 mL of pure water, adjusted to pH 7.4, and dilute to 1000 mL.

PBST washing solution: take 500 mL of 0.01 mol·L ^-1 PBS, add 0.25 mL of Tween 20, and mix well for later use.

Blocking solution: 1 g of skim milk powder was dissolved in 50 mL of pH 7.4, 0.01 mol·L ^-1 PBS.

TMB substrate buffer is composed of solution A, solution B and solution C. The specific formula and volume ratio are as follows:

Color developing solution A: 2.35g citric acid, 9.2g Na ₂ HPO ₄ ·12H ₂ O, add pure water to make up to 490mL, adjust pH to 5.0-5.4, then make up to 500mL with pure water, store at 4°C for later use.

Color developing solution B: Take 1.25 mL of 30% hydrogen peroxide, dilute to 50 mL with pure water, and store at 4°C for later use.

Color developing solution C: Dissolve 200 mg of tetramethylbenzidine (TMB) in 100 ml of absolute ethanol, and store at 4° C. for later use after dissolving.

The TMB substrate buffer is temporarily prepared before use, and the mixing ratio is 9.5 mL of A solution, 42 μL of B solution, and 0.5 mL of C solution, and used after mixing.

Goat anti-mouse HRP-labeled secondary antibody was purchased from Sigma Company.

Example 12

Experiment of adding recovery rate in water sample

Addition and recovery experiments were carried out on the actual samples. Tap water and lake water were used as experimental objects. The concentration of heavy metal copper ions was 10ng·mL ^-1 and 50ng·mL ^-1 respectively. The sample pretreatment steps were as follows:

Lake water sample: The lake water is filtered with qualitative filter paper, then the copper ion standard solution is ^added , and the ^tap water is directly added with the copper ion standard solution. Pipette 100 μL of the mixed tap water and lake water samples and premix them with an equal volume of 5 mmol·L ^-1 L ₂ respectively, and then add 50 μL of the above premixed tap water and lake water samples to each well. Then add 50 μL of primary antibody twice the optimal working concentration (ie, the anti-copper ion monoclonal antibody prepared in Example 10 above), mix well, and incubate at 37°C. Subsequent experimental steps are consistent with the indirect competition ELISA reaction steps in Example 11 above. Table 4 shows the experimental results of adding recovery rates to tap water and lake water samples.

Table 4 Water sample addition recovery results

The accuracy of the detection results can be obtained from the addition recovery experiment. The above addition recovery values can indicate that the detection results measured by this method have high accuracy, and the coefficient of variation of the detection results is low, indicating that the detection results are stable. This shows that the prepared anti-heavy metal copper ion monoclonal antibody can be used for rapid and accurate detection of heavy metal copper ions.

In the above Examples 10-12, the artificial antigen Cu-L ₂ -BSA was replaced with Cu-L ₁ -BSA, the original Cu-L ₂ -OVA coated with Cu-L ₁ -OVA was replaced, or the artificial antigen Cu- Replace L _{2 -BSA with Cu-L 3 -BSA, replace the original Cu-L 2 -OVA with Cu-L 3 -OVA ,} or replace the artificial antigen Cu-L ₂ -BSA with Cu-L ₄ -BSA, Coating the original Cu-L ₂ -OVA was replaced by Cu-L ₄ -OVA, using the same preparation method above, different hybridoma cell lines, anti-heavy metal copper ion monoclonal antibodies, and indirect competition ELISA method can be used to prepare copper ions. test.

In the above-mentioned embodiment 1-12, the bovine serum albumin BSA is replaced with keyhole limpet hemocyanin KLH or other carrier proteins, and the same preparation method as above can also be used to prepare copper ion artificial antigens, hybridoma cell lines, single Antibodies were cloned and copper ions were detected using an indirect ELISA.

Claims (5)

1. A hybridoma cell line that secretes anti-heavy metal copper ion monoclonal antibodies, which is preserved in the General Microbiology Center of the China Microorganism Culture Collection and Administration Commission, and the preservation number is CGMCC No.16388. 2. An anti-heavy metal copper ion monoclonal antibody, characterized in that it is secreted and produced by the hybridoma cell line whose deposit number is CGMCC No. 16388. 3. The application of hybridoma cell line CGMCC No. 16388 or the anti-heavy metal copper ion monoclonal antibody secreted by it in copper ion immunoassay. 4. a preparation method of a hybridoma cell line secreting anti-heavy metal copper ion monoclonal antibody, is characterized in that, this method is used for preparing the described hybridoma cell line of claim 1, comprises the following steps: (1) Synthesis of copper ion artificial antigen: Weigh 5-8mg of 2-S-(3-aminobenzene)-1,4,7 triazacyclononane-1,4,7-triacetic acid, dissolve in 2mL0.01mol·L ^-1 , pH 7.4 4 -Hydroxyethylpiperazine ethanesulfonic acid HEPES solution, obtained chelating agent solution, this solution is A solution; Weigh 70.33 mg of copper nitrate and dissolve it in 5 mL of ultrapure water to prepare a copper nitrate solution with a concentration of 7.5×10 ^-2 mol·L ^-1 , which is solution B; Add 150-200 μL of solution B to solution A, and react in the dark for 3-5 hours at room temperature, this reaction solution is solution C; Add 500-800 μL, 20 mmol·L ^-1 glutaraldehyde solution to solution C, react overnight at room temperature in the dark, and this reaction solution is solution D; Weigh 20-30 mg of bovine serum albumin BSA or chicken ovalbumin OVA, dissolve it in 3 mL of HEPES, and mix with magnetic stirring at room temperature. This reaction solution is E solution; Add solution D to solution E, react in the dark for 24 hours at room temperature, then add 150-200 μL sodium borohydride solution, and react in the dark for 1 hour at room temperature; The reaction solution was first dialyzed with an 8KD dialysis bag for 3 to 5 times, then centrifuged with a 30KD ultrafiltration centrifuge tube at 7000 to 9000 r·min ^-1 for 3 to 5 times, and washed with 5 to 10 mL of 0.01 mol·L ^-1 , pH 7.4 The HEPES solution was reconstituted in 2000 ℃, and then it was repackaged and stored at -20°C to obtain the heavy metal copper artificial antigen Cu-L ₂ -BSA or Cu-L ₂ -OVA, where L ₂ represents 2-S-(3-aminobenzene)- 1,4,7 triazacyclononane-1,4,7-triacetic acid; The synthetic route is: (2) Immunization of mice: BALB/C mice were selected for immunization, the prepared heavy metal copper artificial antigen Cu-L ₂ -BSA was mixed and emulsified with an equal volume of Freund's complete adjuvant, and then injected into the abdomen and axilla at multiple points, and then boosted at regular intervals. For immunization, blood was collected to measure the titer after 3 booster immunizations. When the titer was no longer significantly increased, the final immunization was performed, and cell fusion was performed 3 days after the final immunization; (3) Cell fusion and cell line screening: The mouse spleen cells were fused with mouse myeloma cells SP2/0 in a ratio of 5 to 10:1 by the polyethylene glycol method, added to the culture medium, and the antibody-positive holes were screened by indirect ELISA. The cells were cloned 3-4 times by limiting dilution method, and the hybridoma cell line 6F9 was obtained after screening. 5. the preparation method of anti-heavy metal copper ion monoclonal antibody, is characterized in that, this method is used to prepare the described monoclonal antibody of claim 2, comprises the following steps: BALB/C mice were injected intraperitoneally with 0.3ml/mice of pristane, and 7-10 days later, the screened 6F9 monoclonal cells were injected in the same way. After the abdominal cavity of the mice was obviously distended, the ascites was extracted aseptically, and the oil was removed by centrifugation. After precipitation, mouse ascites McAb was obtained; After purification of the ascites fluid, purified monoclonal antibodies were obtained.