CN115960840A - Hybridoma cell strain BBBE1H1, anti-benzo [ a ] pyrene monoclonal antibody produced by same and application - Google Patents

Abstract

The invention relates to a hybridoma cell strain BBBE1H1 and anti-benzo [ a ] produced by the same]A pyrene monoclonal antibody and application. The hybridoma cell strain BBBE1H1 provided by the invention can be used for preparing high-titer anti-benzo [ a]The titer of the pyrene monoclonal antibody measured by enzyme-linked immunosorbent assay (ELISA) can reach 1.2 multiplied by 10 ⁵ . The invention provides anti-benzo [ a]Pyrene monoThe cloned antibody has high sensitivity and good specificity, and is p-benzo [ a]50% inhibitory concentration IC of pyrene ₅₀ It was 0.013ng/mL.

Description

Hybridoma cell strain BBBE1H1, anti-benzo [ a ] pyrene monoclonal antibody produced by same and application

Technical Field

The invention relates to a hybridoma cell strain BBBE1H1 and an anti-benzo [ a ] pyrene monoclonal antibody generated by the same.

Background

Benzo [ a ] pyrene (BaP), also known as 3, 4-benzopyrene, is a fused aromatic hydrocarbon containing a benzene ring. It is yellow powder with stable property at room temperature, has strong carcinogenic effect, and can induce lung cancer, respiratory tract cancer, gastric cancer, digestive tract cancer, bladder cancer and skin cancer. Benzo [ a ] pyrene also has teratogenicity and mutagenicity, and it can affect offspring through maternal passage through placenta, thereby causing embryo malformation or death and reduced immune function of young animals. The source of benzo [ a ] pyrene in food mainly has two aspects, namely, the waste gas generated by incomplete combustion enters the food such as vegetables, fruits, grains, aquatic products and the like through water sources, atmosphere and soil; secondly, when the scorching phenomenon occurs in the smoking, baking and frying processes, the content of benzopyrene is increased by 10 to 20 times compared with that of common food. Therefore, benzo [ a ] pyrene pollution is a major risk hazard threatening the quality safety of agricultural products and the development of agricultural industry, and China has made a limited amount of regulation on benzo [ a ] pyrene in food.

The existing detection method of benzo [ a ] pyrene is mainly based on laboratory confirmatory detection technology of large-scale instruments. The immunological detection method is based on antigen-antibody specific recognition, and converts an immune signal into a detectable signal through labeling technologies such as enzyme, fluorescence and the like, so that the content of a target object is qualitatively or quantitatively displayed. The method has the advantages of simple sample treatment, high sensitivity, strong specificity, short experimental period and the like, wherein the sensitivity and the specificity of the antibody directly determine the key of the accuracy of the immunoassay.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a hybridoma cell strain BBBE1H1, an anti-benzo [ a ] pyrene (BaP) monoclonal antibody produced by the same and application thereof.

The invention provides a variable region sequence of a monoclonal antibody specifically binding to benzo [ a ] pyrene, wherein a heavy chain variable region of the monoclonal antibody specifically binding to benzo [ a ] pyrene is an amino acid sequence shown by SEQ ID NO. 3 in a sequence table or a conservative mutant obtained by adding, deleting, replacing or modifying conservative mutation by one or more amino acids; the light chain variable region is an amino acid sequence shown as SEQ ID NO. 4 in the sequence table or a conservative mutant obtained by conservative mutation through one or more amino acid additions, deletions, substitutions or modifications. The variable region sequence of the monoclonal antibody provided by the invention can be specifically combined with benzo [ a ] pyrene.

The invention also provides a coding gene sequence of the variable region sequence of the monoclonal antibody specifically binding to benzo [ a ] pyrene.

Preferably, as an embodiment of the invention, the antibody of the monoclonal antibody specifically binding to benzo [ a ] pyrene is an anti-benzo [ a ] pyrene monoclonal antibody secreted by hybridoma cell strain BBBE1H1.

Provides a hybridoma cell strain, wherein the hybridoma cell strain BBBE1H1 is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 4 months and 3 days, the preservation address is China, wuhan university, and the preservation number is CCTCC NO: C201882. The gene sequence of the heavy chain variable region coding gene sequence of the anti-benzo [ a ] pyrene monoclonal antibody shown in SEQ ID NO. 1 in the sequence table and the gene sequence of the light chain variable region coding gene sequence of the anti-benzo [ a ] pyrene monoclonal antibody shown in SEQ ID NO. 2 in the sequence table.

The anti-benzo [ a ] pyrene monoclonal antibody is secreted and produced by hybridoma cell strain BBBE1H1 with the preservation number of CCTCC NO: C201882. The heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 3 in the sequence table; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 4 of the sequence table.

The invention provides anti-benzo [ a]The pyrene monoclonal antibody has high sensitivity and high specificity. Which is p-benzo [ a]50% inhibitory concentration, IC, of pyrene (BaP) ₅₀ 0.013ng/mL; and benzo [ a ]]Anthracene, benzo [ b ]]Fluoranthene, benzo [ e ]]Pyrene, benzo [ ghi]Perylene, benzo [ j ]]Fluoranthene, benzo [ k ]]The cross reaction of fluoranthene, chrysene, fluoranthene and pyrene is less than 15%.

Application of the anti-benzo [ a ] pyrene monoclonal antibody in preparing an immunodetection benzo [ a ] pyrene product or benzo [ a ] pyrene affinity column.

According to the scheme, the immune detection benzo [ a ] pyrene product is benzo [ a ] pyrene immune detection test paper or a benzo [ a ] pyrene immune detection ELISA kit.

Provides benzo [ a ] pyrene immunomagnetic beads, which comprise magnetic beads and anti-benzo [ a ] pyrene monoclonal antibodies coupled with the anti-benzo [ a ] pyrene monoclonal antibodies based on COOH groups on the magnetic beads.

According to the scheme, the mass ratio of the anti-benzo [ a ] pyrene monoclonal antibody to the magnetic beads in the immunomagnetic beads is 2:1 to 1:5.

providing a preparation method of benzo [ a ] pyrene immunomagnetic beads, and cleaning the magnetic beads to remove impurities; adding an anti-benzo [ a ] pyrene monoclonal antibody for coupling, and performing magnetic separation; sealing the coupled magnetic beads by adding a sealing liquid; and after the sealing reaction is finished, carrying out magnetic separation, discarding the supernatant, and storing for later use.

According to the scheme, the method comprises the following steps of,

provides a benzo [ a ] pyrene immunoadsorbent, which comprises a solid phase carrier and an anti-benzo [ a ] pyrene monoclonal antibody coupled with the solid phase carrier.

An immunoaffinity column loaded with the above-described [ a ] pyrene immunoadsorbent is provided.

Provides a preparation method of benzo [ a ] pyrene immunoadsorbent, which comprises the following steps:

a) Substrate treatment

Redissolving and activating the CNBr activated agarose gel matrix powder;

b) Ligand coupling

Dissolving an anti-benzo [ a ] pyrene monoclonal antibody to be coupled by using a coupling buffer solution to obtain an antibody solution, and quickly transferring the activated sepharose gel matrix obtained in the step a) into the antibody solution for coupling;

c) Ligand blocking

Blocking all residual reactive groups;

d) Removing excess ligand that has not been coupled after coupling;

and further a method for obtaining the immunoaffinity column:

and (3) loading the benzo [ a ] pyrene immunoadsorbent into a column to obtain the benzo [ a ] pyrene immunoaffinity column loaded with the benzo [ a ] pyrene immunoadsorbent.

The hybridoma cell strain BBBE1H1 provided by the invention is obtained by a two-step screening method, and the method comprises the following specific steps: a BALB/c mouse is immunized by benzo [ a ] pyrene complete antigen BaP-BSA for 4-6 times, 1/2 times of complete antigen BaP-BSA of the previous immunization dose is used for boosting immunization, cell fusion is carried out after 3 days, after HAT semisolid culture medium is adopted for culture for 1-2 weeks, a single cell colony is transferred to a 96-well cell culture plate for culture, when the cell grows to 1/2 of a well, an indirect competition ELISA method is adopted for screening, a benzo [ a ] pyrene standard product is used as a competition source, a well with higher absorbance and inhibition rate is selected, a limiting dilution method is adopted for 4-5 times of subcloning, and finally, a hybridoma cell strain BBBE1H1 is obtained through screening.

The invention further provides a preparation method of the anti-benzo [ a ] pyrene monoclonal antibody, which comprises the following steps: and (3) injecting the hybridoma cell strain BBBE1H1 obtained in the above step into the abdomen of a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting the mouse in a centrifuge tube after ascites grows out, and purifying the mouse by adopting an octanoic acid-ammonium sulfate method to obtain the anti-benzo [ a ] pyrene monoclonal antibody.

According to the scheme, the method for purifying the antibody by adopting the octanoic acid-ammonium sulfate method comprises the following specific operations: filtering ascites of mice with double-layer filter paper, centrifuging at 4 deg.C and 12000r/min for more than 15min, sucking supernatant, mixing the obtained ascites supernatant with 4 times volume of acetate buffer solution, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid required by each milliliter of ascites is 30-35 μ L, mixing at room temperature for 30-60min, and standing at 4 deg.C for more than 2h. 12000r/min, centrifuging at 4 ℃ for more than 30min, discarding the precipitate, filtering the obtained supernatant by double-layer filter paper, adding phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4 according to the volume of 1/10 filtrate, adjusting the pH of the mixed solution to 7.4 by using 2mol/L sodium hydroxide solution, slowly adding ammonium sulfate in an ice bath until the final concentration of the ammonium sulfate is 0.277g/mL, standing at 4 ℃ for more than 2h, then centrifuging at 12000r/min and the temperature of 4 ℃ for more than 30min, discarding the supernatant, resuspending the obtained precipitate by using phosphate buffer solution with the molar concentration of 0.01mol/L and the pH of 7.4 according to the volume of 1/10 of the original ascites volume, filling the precipitate into a dialysis bag, dialyzing for two days by using 0.01mol/LPBS, dialyzing for two days by using a PB, taking out the protein solution in the dialysis bag, centrifuging, collecting the supernatant, discarding the precipitate, pre-freezing at-70 ℃, and freeze-drying in a freeze dryer. Collecting freeze-dried powder, namely the purified anti-benzo [ a ] pyrene monoclonal antibody;

the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water to 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate to reach a constant volume of 100 mL.

The invention has the beneficial effects that:

(1) The hybridoma cell strain BBBE1H1 provided by the invention can be used for preparing high-valence benzo [ a]The pyrene monoclonal antibody has the titer of 1.2 multiplied by 10 measured by an enzyme-linked immunosorbent assay (ELISA) ⁵ 。

(2) The invention provides anti-benzo [ a ]]The pyrene monoclonal antibody has high sensitivity and good specificity. Which is to benzo [ a ]]50% inhibitory concentration, IC, of pyrene (BaP) ₅₀ The concentration was 0.013ng/mL. Para-benzo [ a]Anthracene, benzo [ b ]]Fluoranthene, benzo [ e ]]Pyrene, benzo [ ghi ] benzene]Perylene, benzo [ j ]]Fluoranthene, benzo [ k ]]The cross reaction of fluoranthene, chrysene, fluoranthene and pyrene is less than 15%.

(3) The monoclonal antibody for resisting benzo [ a ] pyrene provided by the invention can be applied to determination of benzo [ a ] pyrene content.

Detailed Description

Example 1: screening of hybridoma cell line BBBE1H1

1. Immunization of animals

Female BALB/c mice 6 weeks old were immunized with the laboratory prepared benzo [ a ] pyrene complete antigen, baP-BSA. For the first immunization, benzo [ a ] pyrene complete antigen is emulsified with equal volume of Freund complete adjuvant and injected subcutaneously in five points of the back of the mouse neck. The second immunization was carried out 21 days later, emulsified with equal volume of benzo [ a ] pyrene complete antigen using Freund's incomplete adjuvant, and injected intraperitoneally into mice. The third immunization was performed 2 weeks apart from the second immunization in the same manner as the first immunization, and the fourth immunization was performed 3 weeks after the third immunization in the same manner as the second immunization, which was also intraperitoneal injection. The 4 immunization doses were identical and were 100. Mu.g per mouse. 8-10 days after each immunization of the first 3 times, tail cutting and blood sampling are carried out, serum is separated, and the serum titer of the mice is detected by indirect ELISA. 8 days after 3 rd immunization, cutting off the tail, collecting blood, and selecting mice corresponding to serum with relatively high titer and sensitivity for the last boosting immunization, wherein the immunization dose is 1/2 times of the previous immunization dose.

2. Cell fusion

After 3 days of boosting immunity, adopting 50% polyethylene glycol PEG (molecular weight 1450) as a fusion agent, and carrying out cell fusion according to a conventional method, wherein the method comprises the following specific steps: the mice were sacrificed by decapitation under aseptic conditions, the spleen was removed, crushed with a homogenizer, and the spleen cells were separated with a filter screen, mixed with murine myeloma cells SP2/0 at a ratio of 5:1-10:1, centrifuging at 1000rpm for 5min, resuspending the mixed cells in RPMI-1640 basic culture medium, centrifuging at 1000rpm for 5min, and discarding the supernatant. Adding 50% PEG 1mL, 1 minute for each time, adding 20mL of resuspended cells in RPMI-1640 basic culture medium adherent thereto, centrifuging, discarding the supernatant, resuspending the fused cells at the bottom of the tube in 20mL of 1% HAT-containing cell complete medium, adding the suspended cells to 80mL of semisolid medium, mixing well, adding to 6-well cell culture plates, 1-2 mL/well, and standing in a 37 ℃ carbon dioxide incubator. The complete cell culture medium containing 1% HAT contains 20% (volume percent) fetal bovine serum, 75% (volume percent) RPMI-1640 basic medium, 1% (weight percent) L-glutamine, 1% (volume percent) HEPES,1% (volume percent) diabody (10000 units per ml penicillin and 10000 micrograms per ml streptomycin), 1% (volume percent) growth factor (clone easy) and 1% (weight percent) hypoxanthine-aminopterine-thymidine, HAT and methylcellulose, which are purchased from sigma-Aldrich.

Screening and cloning of cell lines

And (3) picking out the clone from the culture medium by using a micropipette when the cell colony grows to be visible by naked eyes 1-2 weeks after the cell fusion, transferring the clone to a 96-hole cell culture plate, culturing by using HAT liquid, and sucking culture supernatant for detection when the cell grows to 2/3 of the bottom of the hole. Adopting a two-step screening method, adopting an indirect ELISA method in the first step to screen out the anti-benzo [ a]Positive wells for pyrene but not anti-carrier protein BSA; in the second step, the positive holes screened in the first step are detected by adopting an indirect competitive ELISA method, and benzo [ a ] is used]Pyrene as a competitor, selectedSelecting the wells with higher absorbance and sensitivity (higher absorbance means higher final measurement value of the positive control well which is the well with 0 competitive antigen, and higher sensitivity means IC of the concentration of competitive antigen when the inhibition rate is 50% ₅₀ Smaller value), performing subcloning by using a limiting dilution method, detecting by using the same two-step method after subcloning, and repeating the subcloning for 4-5 times to obtain a hybridoma cell strain BBBE1H1. The hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 4 and 3 months, wherein the preservation address is China, wuhan and Wuhan university, and the preservation number is CCTCC NO: C201882.

Example 2: and (3) determining the sequence of the variable region of the anti-benzo [ a ] pyrene monoclonal antibody hybridoma cell strain BBBE1H1 antibody.

(1) Extracting total RNA: extracting total RNA capable of generating hybridoma cell strain BBBE1H1 by adopting a total RNA extraction kit of Tiangen company according to an instruction;

(2) Synthesizing cDNA: taking the total RNA obtained in the step 1 as a template, taking oligo (dT) 15 as a primer and adopting SuperScript as an auxiliary primer ^TM -2II reverse transcriptase instructions for reverse transcription to synthesize first strand cDNA; primer oligo (dT) 15 was purchased from Invitrogen;

(3) Cloning of variable region genes by PCR: designing primers according to conserved sites of mouse antibody gene sequences in GENBANK, and amplifying antibody heavy chain and light chain variable region genes by using CDNA as a template. The PCR procedure was: amplification is carried out for 30 cycles at 94 ℃ for 30s, at 58 ℃ for 45s, at 72 ℃ for 1min, and finally extension is carried out for 10min at 72 ℃. After the PCR product is separated by agarose gel electrophoresis of 1 percent (weight percentage), a kit is used for purifying and recovering DNA fragments, the DNA fragments are connected in a vector pMD18-T, escherichia coli DH5 alpha competent cells are transformed, positive clones are picked up and sent to Suzhou hong fast biotechnology limited company for sequencing. Wherein the sequences of the primers are respectively as follows: the heavy chain variable region primers are 5' -CAG GTS MAR CTG MAG GAG TCW G-3' (22 mer) and 5-sand CAG GGG CCA GTG GAT AGA CAG ATG GGG-sand 3' (28 mer), wherein S, M, R and W are merged bases, M = A/C, R = A/G, S = G/C, W = A/T, and the light chain variable region primer is 5-sand GAC ATC AAG ATG ACC CAG TCT CCA-sand 3' (24 mer) and 5-sand CCG TTT TAT TTC CAG CTT GGT CCC-sand 3' (24 mer).

Results of the gene sequences obtained: the heavy chain variable region coding gene sequence has the length of 360bp and is shown as SEQ ID NO. 1, the heavy chain variable region coded by the gene sequence is deduced to consist of 120 amino acids according to the obtained gene sequence, and the sequence is shown as SEQ ID NO. 3. The light chain variable region coding gene sequence has the length of 321bp and is shown as SEQ ID NO. 2, the light chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 107 amino acids, and the sequence is shown as SEQ ID NO. 4.

Example 3: preparation, purification, subtype and characteristic identification of anti-benzo [ a ] pyrene monoclonal antibody

Injecting the anti-benzo [ a ] pyrene monoclonal antibody hybridoma cell strain BBBE1H1 obtained in the example 1 into a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the concrete operation is as follows: filtering mouse ascites with double-layer filter paper, centrifuging at 4 deg.C at 12000r/min for more than 15min, sucking supernatant, mixing the obtained ascites supernatant with 4 times volume of acetate buffer solution, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid required by each ml of ascites is 30-35 μ L, mixing at room temperature for 30-60min, and standing at 4 deg.C for more than 2h. 12000r/min, centrifuging at 4 ℃ for more than 30min, discarding the precipitate, filtering the obtained supernatant by double-layer filter paper, adding phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4 according to the volume of 1/10 filtrate, adjusting the pH of the mixed solution to 7.4 by using 2mol/L sodium hydroxide solution, slowly adding ammonium sulfate in an ice bath until the final concentration of the ammonium sulfate is 0.277g/mL, standing at 4 ℃ for more than 2h, then centrifuging at 12000r/min and the temperature of 4 ℃ for more than 30min, discarding the supernatant, resuspending the obtained precipitate by using phosphate buffer solution with the molar concentration of 0.01mol/L and the pH of 7.4 according to the volume of 1/10 of the original ascites volume, filling the precipitate into a dialysis bag, dialyzing for two days by using 0.01mol/LPBS, dialyzing for two days by using a PB, taking out the protein solution in the dialysis bag, centrifuging, collecting the supernatant, discarding the precipitate, pre-freezing at-70 ℃, and freeze-drying in a freeze dryer. Collecting freeze-dried powder, namely the purified anti-benzo [ a ] pyrene monoclonal antibody;

the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water to 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate to reach a constant volume of 100 mL.

The subtype of the anti-benzo [ a ] pyrene monoclonal antibody secreted by the hybridoma cell strain BBBE1H1 is identified to be IgG1 by using a commercial subtype identification kit.

The titer of the antibody obtained by ascites purification of the mice can reach 1.2 multiplied by 10 by the conventional non-competitive enzyme-linked immunosorbent assay (ELISA) ⁵ I.e. antibody dilution 1.2X 10 ⁵ The solution test result is positive. Determination of para-benzo [ a ] by conventional indirect competitive ELISA]The pyrene sensitivity IC50 was 0.013ng/mL. The specificity of the antibody can be evaluated by the cross-reactivity. Measuring BBBE1H1 monoclonal antibody by indirect competition ELISA method, and detecting BaP and benzo [ a ]]Anthracene, benzo [ b ]]Fluoranthene, benzo [ e ]]Pyrene, benzo [ ghi]Perylene, benzo [ j ]]Fluoranthene, benzo [ k ]]Preparing standard solutions with a series of concentrations from fluoranthene, chrysene, fluoranthene and pyrene, respectively adding the standard solutions and an antibody with the same volume into an ELISA plate, incubating for 1h at 37 ℃, and performing other steps with an indirect competitive ELISA method. OD value B/B measured by enzyme-labeling instrument at 450nm with the above standard substance concentration as abscissa ₀ As an ordinate, a competitive inhibition curve was plotted, and the IC of BaP and other analogues was calculated ₅₀ The value ratio is used to determine the cross reaction rate. The calculation formula is as follows:

CR％＝(IC ₅₀ BaP/IC ₅₀ other analogues) × 100.

The cross reaction of the BBBE1H1 monoclonal antibody provided by the invention with other structural analogues benzo [ a ] anthracene, benzo [ b ] fluoranthene, benzo [ e ] pyrene, benzo [ ghi ] perylene, benzo [ j ] fluoranthene, benzo [ k ] fluoranthene, chrysene, fluoranthene and pyrene is less than 15%, and the part of the cross reaction is as low as less than 1%.

The detailed results are shown in table 1:

TABLE 1 Cross-reactivity of BBBE1H1 with other structural analogs

The affinity of BBBE1H1 was determined using an indirect non-competitive ELISA:

coating an enzyme label plate with BaP-OVA at the concentration of 2.0, 1.0, 0.5 and 0.25 mu g/mL for 2 hours at the temperature of 37 ℃ at the concentration of 100 mu L/hole; after blocking with blocking solution for 1h, the antibody diluted with PBS (dilution factor 1. To determine OD ₄₅₀ The values are plotted on the ordinate and the logarithm of the antibody concentration (mol/L) is plotted on the abscissa, and 4S-shaped curves of 4 concentrations are plotted. The maximum OD value at the top of each S-curve, i.e., ODmax, was found, and the antibody concentration corresponding to 50% ODmax of each curve was found. Any two of the 4 concentrations are combined in a group according to the formula Ka = (n-1)/2 (n [ Ab']t-[Ab]t) calculating the affinity constant of the antibody, wherein [ Ab']t、[Ab]t is the antibody concentration corresponding to the two 50% maximum OD values in each group, n is the fold of the envelope antigen concentration in each group (including three ratios of 1. The six Ka values are averaged to obtain the benzo [ a ]]The affinity of pyrene mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) method can reach 1.6 multiplied by 10 ⁹ L/moL。

Example 4: preparing benzo [ a ] pyrene immunomagnetic beads:

a, cleaning: weighing 2.5mg of magnetic bead powder modified with carboxyl into a 15mL centrifuge tube, adding 2mL of cleaning solution to clean twice, placing the centrifuge tube in a magnetic frame to carry out magnetic separation, discarding the supernatant, then cleaning the centrifuge tube for 2 times by using coupling buffer solution, carrying out magnetic separation, and discarding the supernatant.

b, coupling: adding 4mL of coupling buffer solution into the washed magnetic beads, adding 2.5mg of anti-benzo [ a ] pyrene monoclonal antibody, placing the mixture in a shaking table at 8 ℃, carrying out overnight coupling at 200r/min, and placing the mixture in a magnetic rack for magnetic separation after the reaction is finished.

c, sealing: and adding Tris-HCl buffer solution into the coupled magnetic beads, washing twice, then adding 4mL of Tris-HCl confining solution, and reacting for 2 hours at the temperature of 200r/min in a shaking table at 8 ℃.

d, preservation: after the completion of the blocking reactionMagnetic separation, discarding supernatant, and 0.01% of NaN ₃ The volume of the PBS solution is 5mL, and the PBS solution is stored in a refrigerator at 4 ℃ for later use. The method is used for the subsequent sample pretreatment.

Example 5: the establishment of the benzo [ a ] pyrene ic-ELISA detection method comprises the following steps:

1. sample pretreatment:

(1) Taking a 10mL plastic centrifuge tube as a reaction container, accurately weighing 1g vegetable oil sample, adding 5mL n-hexane solution, performing ultrasonic treatment for 10min, adding 5mL n-hexane solution, and performing ultrasonic treatment for 10min. Taking 1mL of the extract, using 9mL of ddH ₂ O is added to the volume of 10mL, vortexed for 2min, and the benzo [ a ] coupled with the benzopyrene monoclonal antibody is added]Pyrene immunomagnetic beads for specifically binding to benzo [ a ] in sample]Pyrene, vortex for 14 min and discard supernatant.

(2) 1mL of methanol solution was added, vortex-mixed for 2 minutes, and the benzo [ a ] pyrene bound to the magnetic beads was eluted, and diluted 5-fold with a detection solution (0.5% Tween 20/PBS, pH 7.4) as a test solution for loading detection by ELISA method.

2. The specific operation steps are as follows:

the benzo [ a ] pyrene complete antigen was coated on a 96-well microplate and allowed to stand overnight at 4 ℃. PBST plates were washed three times, 3.5% -5% skimmed milk powder was added and blocked for 1h at 37 ℃. Adding 50 mu l of benzo [ a ] pyrene monoclonal antibody with proper working concentration and gradient diluted benzo [ a ] pyrene standard solution respectively, and incubating for 1h at 37 ℃. And (4) washing the plate for three times by using the PBST, adding a secondary antibody marked by HRP, washing the plate for three times by using the PBST, stopping color development, and reading an OD value by using an enzyme-labeling instrument.

The concentration of the benzo [ a ] pyrene complete antigen is 1 mu g/mL; the concentration of the benzo [ a ] pyrene monoclonal antibody is 0.1 mu g/mL.

The antibody diluent had a salt ion concentration of 10mM, pH of 7.4, and a Tween 20 concentration of 0.05%. ic-ELISA detection of anti-benzo [ a ] s of the invention]Sensitivity of pyrene monoclonal antibody (to benzo [ a ]]50% inhibitory concentration, IC, of pyrene (BaP) ₅₀ ) The concentration was 0.013ng/mL.

Obtaining the content of benzo [ a ] pyrene in the sample liquid to be detected based on the obtained relation curve between the OD value and the concentration of benzo [ a ] pyrene:

benzo [ a ] pyrene (BaP) standards were added to the blank edible vegetable oil matrix samples in a range of concentrations to give final concentrations of 200, 66.6, 22.2, 7.4, 2.4, 0.8, 0.2ng/mL, pre-treated as per step 1, and after testing in step 2, a calibration curve of Bx/B0 as a function of BaP (log 10) concentration was plotted by four parameter logistic regression using Origin 8.6 (Origin Lab Corporation, north ampton, MA, USA), with each data point being the average of three independent measurements.

3. Test for recovery with addition of standard

In order to evaluate the accuracy of the established benzo [ a ] pyrene ic-ELISA (indirect competitive ELISA) method, a matrix standard curve is established, and a standard recovery experiment is carried out on a blank soybean oil sample. The method comprises the following specific steps: the sample is added with benzo [ a ] pyrene standard substance with three concentrations of 5ng/mL, 50ng/mL and 100ng/mL respectively, extraction is carried out according to the sample pretreatment method, the ic-ELISA method established in the step 2 is adopted to draw a standard curve, the content value of the added standard sample is measured, and the result shows that the added standard recovery rates of the samples with three concentrations are 104.1%,89.6% and 96.8% respectively.

4. Actual sample detection

6 collected soybean oil samples are detected by adopting an established ic-ELISA method and are compared with a liquid chromatography for verification. The results of the tests showed that (Table 2), ic-ELISA has a high correlation with HPLC, the correlation curve is y =1.0481x-0.2124 ² Is 0.9974.

TABLE 2 actual sample testing

Example 6: preparing the benzo [ a ] pyrene immunoaffinity column:

preparation of benzo [ a ] pyrene immunoaffinity column

Preparation of the substrate

The desired 10g of CNBr-activated Sepharose lyophilized matrix powder (4-5 mL of swollen matrix can be formed per gram of lyophilized matrix powder) was weighed out and dissolved in 20% methanol-water. The matrix will swell immediately and then be placed in a sand core funnel and washed 3 times with 20% methanol-water.

Antibody conjugation

a coupling buffer (0.2 mol/L NaHCO) was used ₃ pH8.3) were washed 2-3 times with the agarose gel. Dissolving the above-mentioned anti-benzo [ a ] to be coupled with a coupling buffer]The pyrene monoclonal antibody has an antibody concentration of 20mg/mL. The agarose gel and the antibody solution were added to a triangular flask, placed on a temperature-controlled shaker, and reacted at 10 ℃ and 250rpm with shaking for 20 hours.

b calculation of coupling ratio: 2,000rpm, the agarose gel was centrifuged to the bottom of the tube, the supernatant was transferred to a new centrifuge tube, and the protein content value of the supernatant was determined. The calculated coupling ratio was 98.5%.

And (3) sealing: transfer the substrate to 0.1mol/L Tris-HCl buffer. The reaction mixture was placed on a temperature-controlled shaker and shaken at 10 ℃ and 250rpm for 4 hours to block any residual reactive groups.

And (3) storage: the gel was washed 3 times with 0.01mol/L PBS to remove excess ligand that was not coupled after coupling, using 5-10 gel volumes of PBS buffer each time. Then using 0.01% of 3-5 times of the gel volume to make NaN ₃ -PBS wash 1 time and use 0.01% NaN ₃ PBS preservation.

Column assembling: after the column tube is cleaned by ultrapure water, the column tube is arranged in a lower sieve plate for cleaning again. Each column was loaded with 0.5mL of the immunoaffinity adsorbent prepared in the previous step. And after the column is filled, installing a plug, filling the column tube with 0.01mol/L PBS, then installing an upper sieve plate, slightly pushing the upper sieve plate downwards, and slightly pressing the immunoaffinity adsorbent between the upper sieve plate and the lower sieve plate without compacting. 0.01% of NaN using sterile filtration ₃ Column chromatography with PBS and use of 0.01% NaN ₃ PBS storage, until the benzopyrene affinity column is filled and equilibrated, ready for use.

Claims (10)

1. The hybridoma cell strain BBBE1H1 is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 4 and 3 months, and the preservation address is China, wuhan and Wuhan university with the preservation number of CCTCC NO: C201882.

2. An anti-benzo [ a ] pyrene monoclonal antibody, which is characterized in that: it is secreted and produced by hybridoma cell strain BBBE1H1 with the preservation number of CCTCC NO: C201882.

3. The use of the anti-benzo [ a ] pyrene monoclonal antibody of claim 2 in the preparation of an immunoassay for benzo [ a ] pyrene products or benzo [ a ] pyrene affinity columns.

4. Use according to claim 3, characterized in that: the immunoassay benzo [ a ] pyrene product is benzo [ a ] pyrene immunoassay test paper or a benzo [ a ] pyrene immunoassay ELISA kit.

5. Benzo [ a ] pyrene immunomagnetic beads, characterized in that: comprising magnetic beads and the anti-benzo [ a ] pyrene monoclonal antibody of claim 2 based on coupling of COOH groups on the magnetic beads to amino groups of the anti-benzo [ a ] pyrene monoclonal antibody.

6. The method for preparing benzo [ a ] pyrene immunomagnetic beads according to claim 5, wherein: cleaning the magnetic beads to remove impurities; adding an anti-benzo [ a ] pyrene monoclonal antibody for coupling, and performing magnetic separation; sealing the coupled magnetic beads by adding a sealing liquid; and after the sealing reaction is finished, carrying out magnetic separation, discarding the supernatant, and storing for later use.

7. Benzo [ a ] pyrene immunoadsorbent characterized by: the immunoadsorbent comprises a solid support and the anti-benzo [ a ] pyrene monoclonal antibody of claim 2 coupled to the solid support.

8. An immunoaffinity column loaded with the benzo [ a ] pyrene immunoadsorbent of claim 7.

9. The method for preparing benzo [ a ] pyrene immunoadsorbent of claim 7, wherein: the preparation steps are as follows:

a) Substrate treatment

Redissolving and activating the CNBr activated agarose gel matrix powder;

b) Ligand coupling

Dissolving an anti-benzo [ a ] pyrene monoclonal antibody to be coupled by using a coupling buffer solution to obtain an antibody solution, and quickly transferring the activated sepharose gel matrix obtained in the step a) into the antibody solution for coupling;

c) Ligand blocking

Blocking all residual reactive groups;

d) Excess ligand not coupled after coupling is removed.

10. A variable region sequence of a monoclonal antibody specifically binding benzo [ a ] pyrene, wherein the heavy chain variable region of the monoclonal antibody specifically binding benzo [ a ] pyrene is an amino acid sequence shown in SEQ ID NO. 3 in a sequence table or a conservative mutant obtained by conservative mutation through addition, deletion, substitution or modification of one or more amino acids; the light chain variable region is an amino acid sequence shown as SEQ ID NO. 4 in the sequence table or a conservative mutant obtained by conservative mutation through one or more amino acid additions, deletions, substitutions or modifications.