CN113151188B - Hybridoma cell strain capable of secreting diphenhydramine monoclonal antibody and application thereof - Google Patents

Hybridoma cell strain capable of secreting diphenhydramine monoclonal antibody and application thereof Download PDF

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CN113151188B
CN113151188B CN202110459860.1A CN202110459860A CN113151188B CN 113151188 B CN113151188 B CN 113151188B CN 202110459860 A CN202110459860 A CN 202110459860A CN 113151188 B CN113151188 B CN 113151188B
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diphenhydramine
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胥传来
刘杰
匡华
刘丽强
宋珊珊
胡拥明
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Abstract

A hybridoma cell strain secreting diphenhydramine monoclonal antibody and application thereof belong to the field of cosmetic safety immunoassay. The hybridoma cell strain SHS5B11 secreting diphenhydramine monoclonal antibody is classified and named as monoclonal cell strain, the preservation date is 2020, 9 and 27 days, and the preservation number is CGMCC No.20787. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity to diphenhydramine. The invention synthesizes diphenhydramine complete antigen, mixes and emulsifies the diphenhydramine complete antigen and equivalent Freund's adjuvant, immunizes BALB/c mice through back subcutaneous injection, and obtains hybridoma cell strain SHS5B11 through screening and three times of subcloning of indirect competitive enzyme-linked immunosorbent assay. The monoclonal antibody secreted by the cell strain has better detection sensitivity (IC) on diphenhydramine 50 The value is 2.93 ng/mL), can be used for detecting the residue of diphenhydramine in cosmetics.

Description

Hybridoma cell strain capable of secreting diphenhydramine monoclonal antibody and application thereof
Technical Field
The invention relates to a hybridoma cell strain secreting diphenhydramine monoclonal antibody and application thereof, belonging to the field of cosmetic safety immunoassay.
Background
Diphenhydramine is an antihistamine, is widely applied to the field of medicines, and has good effects on allergic reactions such as skin itch, allergic rhinitis, urticaria, contact dermatitis and the like. Diphenhydramine has the effects of sterilizing and removing acnes and has certain antiallergic effect, so that diphenhydramine can be added into acne-removing cosmetics to achieve the effect of removing acnes. Because the raw materials used in the cosmetics or the substances in the process flow such as propylene glycol can promote the absorption of diphenhydramine by human body, when the diphenhydramine content in the human body is too high, the central nerve can be strongly inhibited, and the human body can generate symptoms such as lethargy, dizziness, headache, nausea, vomiting, epigastric discomfort and the like. The 'cosmetic hygiene standard' in China and the European Union cosmetic laws clearly stipulate diphenhydramine as a forbidden substance in cosmetics. At present, the diphenhydramine detection method is mainly instrument detection, and gas chromatography, liquid chromatography and gas chromatography-mass spectrometry are commonly used. Despite the high sensitivity and specificity of these chromatography-based methods, there are some disadvantages such as the need for thorough sample cleanup, high solvent consumption, expensive equipment and skilled personnel. Therefore, a fast and simple method for analyzing diphenhydramine residues is needed.
The enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, has the advantages of simple pretreatment of samples during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for the field rapid detection of a large number of samples, thereby being widely applied to the analysis of forbidden substances of cosmetics. The precondition for detecting diphenhydramine by using an enzyme-linked immunosorbent assay is that a monoclonal antibody with high specificity and high sensitivity to diphenhydramine is obtained, so that the key is to find a method for preparing the monoclonal antibody with high specificity and high sensitivity to diphenhydramine. The inventor tries to prepare the diphenhydramine monoclonal antibody through the hybridoma cell, but in the process of preparing the hybridoma cell strain capable of secreting the diphenhydramine monoclonal antibody, how to prepare diphenhydramine hapten and diphenhydramine complete antigen and how to make a mouse generate strong immunity need further research; further research is needed on how to successfully secrete diphenhydramine monoclonal antibody from the prepared hybridoma cell strain; how to make the secreted diphenhydramine monoclonal antibody have strong specificity and high sensitivity also needs further research.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain secreting diphenhydramine monoclonal antibody and application thereof. The diphenhydramine monoclonal antibody secreted by the hybridoma cell strain has better detection sensitivity (IC) on diphenhydramine 50 The value is 2.93 ng/mL), can be used for establishing an immunological detection method of diphenhydramine and detecting whether cosmetics contain diphenhydramine.
The technical scheme of the invention is as follows: a hybridoma cell strain SHS5B11 secreting diphenhydramine monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), china academy of sciences, china institute of microbiology, no. 3, of Xilu No. 1, beijing, chaoyang, and the date of deposition is 2020, 9 months and 27 days, and the number of deposition is CGMCC No.20787.
The invention provides a preparation method of a hybridoma cell strain secreting a diphenhydramine monoclonal antibody, which comprises the following steps:
(1) Preparing diphenhydramine hapten and diphenhydramine complete antigen, and preparing the obtained diphenhydramine complete antigen into Freund adjuvant and incomplete Freund adjuvant;
(2) Injecting the Freund's adjuvant into BALB/c mouse via back subcutaneous injection for multiple times of immunization, wherein complete Freund's adjuvant is adopted for the first immunization, and incomplete Freund's adjuvant is adopted for boosting immunization;
(3) Collecting blood from the mice subjected to the immunization process, detecting the serum immune titer and the immune suppression capacity of the mice through indirect ELISA, and screening the mice with high diphenhydramine antibody content in the serum to obtain immunized mice;
(4) Performing last boosting immunization on the screened mice by using incomplete Freund adjuvant, and performing sprint immunization by intraperitoneal injection, wherein the sprint immunization is performed by using diphenhydramine complete antigen without Freund adjuvant;
(5) Fusing splenocytes of BALB/c mice subjected to sprint immunization with myeloma cells, culturing the fused cells through a culture medium, detecting positive cell holes by using indirect ELISA, further determining the inhibition effect of the positive cell holes by using an indirect competitive ELISA method, subcloning the positive cell holes with the best inhibition by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain SHS5B11 capable of secreting diphenhydramine monoclonal antibodies.
In one embodiment of the present invention, the first immunization and the booster immunization in the steps 2 and 4 are separated by one month, the booster immunization is separated by 21 days, and the booster immunization and the sprint immunization are separated by 18 to 21 days.
In one embodiment of the present invention, the primary immune dose in the steps 2 and 4 is 100 μ g/mouse, the booster immune dose is 50 μ g/mouse, and the sprint immune dose is 25 μ g/mouse.
In one embodiment of the present invention, the immunization process in steps 2 and 4 comprises 1 first immunization, 4 booster immunizations and 1 sprint immunization.
In one embodiment of the present invention, the blood collection in step 3 is performed on day 7 after the 3 rd boosting procedure.
In one embodiment of the present invention, the cell fusion in step 5 is performed 3 days after the completion of the sprint immunization.
In one embodiment of the present invention, the cell fusion in step 5 is performed by a polyethylene glycol (PEG 4000) method.
In one embodiment of the present invention, the medium in the step 5 is RPMI-1640 medium.
In one embodiment of the present invention, the number of subclonings in step 5 is 3.
The molecular formula of the diphenhydramine hapten in the step (1) is as follows:
Figure BDA0003041874950000031
the preparation method of the diphenhydramine hapten comprises the following steps: weighing 2- (benzhydryloxy) -N-methylethylamine, dissolving in pyridine, adding succinic anhydride into the reaction solution, magnetically stirring for reaction, and adding ultrapure water to terminate the reaction to obtain a derivative product; extracting with chloroform for three times, mixing chloroform phases, and blowing nitrogen to obtain diphenhydramine hapten.
Further, the steps are as follows: weighing 50mg of 2- (benzhydryloxy) -N-methylethylamine, dissolving in 5mL of pyridine, adding 25mg of succinic anhydride into the reaction solution, magnetically stirring at 50 ℃ for reaction for 4 hours, and adding 10mL of ultrapure water to terminate the reaction to obtain a derivative product; and extracting for three times by using trichloromethane, combining the trichloromethane, and blowing nitrogen to obtain the diphenhydramine hapten.
The preparation method of diphenhydramine complete antigen comprises diphenhydramine immunogen and diphenhydramine coating antigen;
diphenhydramine immunogen was prepared as follows: dissolving diphenhydramine hapten and N-hydroxysuccinimide NHS in N, N-dimethylformamide DMF, and stirring at room temperature to react to obtain diphenhydramine hapten solution; weighing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, fully dissolving with DMF, adding into diphenhydramine hapten solution, and stirring at room temperature to react to obtain solution A1; taking BSA, and diluting with carbonate buffer CBS to obtain solution B1; slowly adding the solution A1 into the solution B1 drop by drop, and reacting overnight at room temperature; dialyzing with PBS solution, removing unreacted small molecule hapten to obtain diphenhydramine immunogen, and identifying by ultraviolet absorption scanning method;
the diphenhydramine coating antigen is prepared as follows: dissolving diphenhydramine hapten and N-hydroxysuccinimide NHS in anhydrous N, N-dimethylformamide DMF, and stirring at room temperature for reaction to obtain diphenhydramine hapten solution; dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in anhydrous DMF, adding into diphenhydramine hapten solution, and stirring at room temperature to react to obtain solution A2; diluting chicken egg albumin OVA with carbonate buffer CBS to obtain B2 solution; slowly adding the solution A2 into the solution B2 dropwise for reaction to obtain a reaction solution; and dialyzing the reaction solution by using a PBS solution, and removing unreacted small molecule hapten to obtain the diphenhydramine coating antigen.
The immunogen is prepared by the following specific steps: weighing 5.5mg diphenhydramine hapten and 4.2mg N-hydroxysuccinimide NHS, dissolving in 300 mu L N and N-dimethylformamide DMF, and stirring at room temperature for 10min to obtain diphenhydramine hapten solution; weighing 6.9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, fully dissolving the EDC in 100 mu L of DMF, adding the solution into diphenhydramine hapten solution, and stirring at room temperature for 6-8h to obtain solution A1; taking 8mg BSA, diluting to 4mg/mL with 0.01M carbonate buffer CBS to obtain solution B1, dropwise adding the solution A1 into the solution B1 slowly, and reacting at room temperature overnight; then dialyzing with 0.01M PBS solution to remove unreacted biomolecule hapten to obtain diphenhydramine immunogen. And (5) obtaining diphenhydramine immunogen by using small molecule hapten.
The preparation method of the coating antigen comprises the following specific steps: dissolving diphenhydramine hapten of 3.0mg and N-hydroxysuccinimide NHS of 2.3mg in anhydrous N, N-dimethylformamide DMF of 300 mu L, stirring and reacting for 10min at room temperature to obtain diphenhydramine hapten solution; dissolving 3.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in 100 mu L of anhydrous DMF, adding the solution into diphenhydramine hapten solution, and stirring at room temperature to react for 6-8h to obtain A2 liquid; diluting 10mg of chicken ovalbumin OVA with 1mL of carbonate buffer CBS with the concentration of 0.01mmol/L to obtain solution B2; slowly adding the A2 solution dropwise into the B2 solution for reaction to obtain a reaction solution; and dialyzing the reaction solution by using a PBS solution, and removing unreacted small molecule hapten to obtain the diphenhydramine coating antigen.
The invention provides a hybridoma cell strain SHS5B11 secreting diphenhydramine monoclonal antibody, a preparation method of the hybridoma cell strain SHS5B11 secreting diphenhydramine monoclonal antibody, and application of the hybridoma cell strain SHS5B11 secreting diphenhydramine monoclonal antibody.
The invention provides a diphenhydramine monoclonal antibody which is obtained by secreting a hybridoma cell strain with the preservation number of CGMCC No.20787.
The invention provides a preparation method of the diphenhydramine monoclonal antibody, which comprises the steps of taking a BALB/c mouse, injecting paraffin oil into the abdominal cavity, injecting a hybridoma cell strain SHS5B11 with the preservation number of CGMCC No.20787 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and preserving the obtained monoclonal antibody at low temperature.
In one embodiment of the invention, the method comprises taking BALB/c mice 8-10 weeks old, injecting 1mL paraffin oil into the abdominal cavity of each mouse, injecting 1X 10 paraffin oil into the abdominal cavity of each mouse after 7 days 6 Collecting ascites from 7 days, purifying the ascites by octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 deg.C.
The invention provides an application of the diphenhydramine monoclonal antibody or the preparation method of the diphenhydramine monoclonal antibody in the aspect of identifying diphenhydramine; in particular to the detection of diphenhydramine residue in cosmetics.
The invention has the beneficial effects that: the diphenhydramine monoclonal antibody obtained by the invention has better detection sensitivity (IC) on diphenhydramine 50 A value of 2.93 ng/mL); the diphenhydramine monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection.
Biological material sample preservation: a hybridoma cell strain SHS5B11 secreting diphenhydramine monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), china academy of sciences, china institute of microbiology, no. 3, of Xilu No. 1, beijing, chaoyang, and the date of deposition is 2020, 9 months and 27 days, and the number of deposition is CGMCC No.20787.
Drawings
FIG. 1 shows the inhibition standard curve of diphenhydramine monoclonal antibody of the present invention.
Detailed Description
The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.
The media involved in the following examples are as follows: RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B hydrochloride 12.005, and sodium bicarbonate 2000.
The reagents involved in the following examples are as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to fix the volume to 1000mL, and storing at 4 ℃ for later use;
phosphate Buffered Saline (PBS): 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12H 2 Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and metering the volume to 1000mL;
PBST: PBS containing 0.05% tween 20;
antibody dilution: PBS containing 0.1% gelatin;
TMB color development liquid: solution A: na (Na) 2 HPO 4 ·12H 2 18.43g of O, 9.33g of citric acid and pure water with constant volume of 1000mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. And the volume ratio of the solution B to the solution B is 5:1 to obtain the TMB color developing solution which is mixed at present.
The detection methods referred to in the following examples are as follows:
the diphenhydramine inhibition rate detection method comprises the following steps: the most suitable antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.01, 0.03, 0.1 and 0.3. Mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03, 0.1, 0.3 and 1. Mu.g/mL with antibody diluent. Best choiceAfter the working point is optimized, the diphenhydramine standard is diluted to 8 concentrations (0.05,0.15,0.45,1.35,4, 12, 36, 110 ng/mL), the IC-ELISA procedure is followed, and finally originPro 8.5 is used for drawing (the result is shown in figure 1), so as to obtain the diphenhydramine standard inhibition curve, and IC is calculated 50 。
Example 1: synthesis of diphenhydramine hapten
The diphenhydramine micromolecules have no immunogenicity, and can not stimulate mice to generate immune response so as to generate antibodies, so the diphenhydramine is coupled to protein by a protein connection technology to obtain the immunogenicity; the active groups commonly used in the protein coupling technology comprise amino, carboxyl, hydroxyl, sulfydryl and the like, and the structural formula of diphenhydramine does not contain the active groups, so that derivatization is needed, and the specific steps are as follows:
50mg of 2- (benzhydryloxy) -N-methylethylamine was weighed and dissolved in 5mL of pyridine, and then 25mg of succinic anhydride was added to the reaction solution, and the reaction was magnetically stirred at 50 ℃ for 4 hours, followed by addition of 10mL of ultrapure water to terminate the reaction, to obtain a derivative product. And extracting for three times by using trichloromethane, combining the trichloromethane, and blowing nitrogen to obtain the diphenhydramine hapten.
Example 2: synthesis of diphenhydramine immunogen
Weighing 5.5mg diphenhydramine hapten and 4.2mg N-hydroxysuccinimide (NHS), dissolving in 300 mu L N and N-Dimethylformamide (DMF), and stirring at room temperature for 10min for reaction to obtain diphenhydramine hapten solution; then 6.9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is weighed, fully dissolved by 100 microliter of DMF, added into diphenhydramine hapten solution, and stirred and reacted for 6-8h at room temperature (called solution A). Diluting 8mg BSA with 0.01M Carbonate Buffer (CBS) to 4mg/mL (called solution B), slowly adding solution A into solution B dropwise, and reacting at room temperature overnight; and dialyzing with 0.01M PBS solution to remove unreacted small molecule hapten to obtain diphenhydramine immunogen, and identifying by ultraviolet absorption scanning method.
Example 3: synthesis of diphenhydramine coatingen
Dissolving diphenhydramine hapten of 3.0mg and N-hydroxysuccinimide (NHS) of 2.3mg in anhydrous N, N-Dimethylformamide (DMF) of 300 mu L, and stirring and reacting for 10min at room temperature to obtain diphenhydramine hapten solution; dissolving 3.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 100 mu L of anhydrous DMF, adding the solution into diphenhydramine hapten solution, and stirring at room temperature to react for 6-8h to obtain solution A; diluting 10mg of chicken Ovalbumin (OVA) with 1mL of Carbonate Buffer Solution (CBS) with the concentration of 0.01mmol/L to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction to obtain a reaction solution; and dialyzing the reaction solution by using a PBS solution to remove unreacted small molecule hapten to obtain the coating antigen.
Example 4: preparation of hybridoma cell strain secreting diphenhydramine monoclonal antibody
(1) Obtaining of mouse immunity: mixing and emulsifying a diphenhydramine complete antigen and an equivalent amount of Freund's adjuvant, and performing neck-back subcutaneous multipoint injection immunization (except for sprint immunization) on a BALB/c mouse; complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, the thorny immunity is directly diluted by normal saline and then injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse; one month is separated between the first immunization and the second boosting immunization, 21 days are separated between the multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization; the titer and the inhibition of the mouse serum are detected by observing the immune effect of the mouse by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).
(2) Cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:
a. cutting tail, taking blood, killing the mouse by cervical dislocation, immediately placing the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out the spleen of the mouse by aseptic operation, properly grinding by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain spleen cell suspension, collecting, centrifuging (1200rpm, 8min), washing the spleen cells for three times by using RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;
b. collecting SP2/0 cells: 7-10 days before the fusion, the SP2/0 tumor cells were subjected to 5% CO using a medium containing 10% FBS (fetal bovine serum) RPMI-1640 2 Culturing in an incubator, and before fusion, requiring SP2/0 tumor cell number to reach (1-4) x 10 7 Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion, collecting the tumor cells during fusion, suspending the tumor cells in RPMI-1640 basic culture solution, and counting the cells;
c. the fusion process is 7min: 1min, 1mL of PEG 4000 is added to the cells dropwise from slow to fast; standing for 2 min; at 3min and 4min, 1mL of RPMI-1640 culture medium is added dropwise within 1 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then carrying out warm bath at 37 ℃ for 5min; centrifuging (800rpm, 8min), discarding the supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding to a 96-well cell plate at 200. Mu.L/well, incubating at 37 ℃ and 5% CO 2 Culturing in an incubator.
3. Cell screening and cell strain establishment: on day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected and screened.
The screening is divided into two steps: the first step is to screen out positive cell holes by an ic-ELISA method, and the second step is to select diphenhydramine as a standard substance and to test the inhibition effect of the positive cells by the ic-ELISA method.
Selecting cell holes with good inhibition to diphenhydramine standard substance, subcloning by limiting dilution method, and detecting by the same method after seven days.
And subcloning for three times according to the method to finally obtain the diphenhydramine monoclonal antibody cell strain SHS5B11.
Example 5: preparation and identification of diphenhydramine monoclonal antibody
Taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10 6 Diphenhydramine hybridoma SHS5B11, collecting ascites from the seventh day, and removing ascitesAntibody purification was performed by the octanoic acid-saturated ammonium sulfate method.
Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
IC determination of diphenhydramine monoclonal antibodies using indirect competitive ELISA 50 The value is 2.93ng/mL, which shows that the reagent has good sensitivity to diphenhydramine and can be used for diphenhydramine immunoassay detection.
Example 6: application of diphenhydramine monoclonal antibody
The monoclonal antibody prepared from hybridoma cell strain SHS5B11 through in vivo ascites is applied to ELISA addition recovery test of diphenhydramine, and the specific steps are as follows:
(1) Coating a 96-well enzyme label plate with coating antigen with the concentration of 0.3 mu g/mL diluted by Carbonate Buffer Solution (CBS), coating 100 mu L of the enzyme label plate in each well at 37 ℃ for 2h, washing the plate with PBST washing liquor three times, wherein 200 mu L of the washing liquor is used in each well, and the washing liquor is patted dry after 3min each time;
(2) Sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3min, and drying;
(3) Respectively preparing 0.05,0.15,0.45,1.35,4, 12, 36 and 110ng/mL diphenhydramine standard solutions by using Phosphate Buffer Solution (PBS), respectively adding the standard solutions and the extract of a sample to be detected into the closed enzyme label plate, wherein each hole is 50 mu L, repeating 3 holes for each sample, adding 50 mu L of anti-diphenhydramine monoclonal antibody diluted to 0.3 mu g/mL into each hole, reacting at 37 ℃ for 0.5h, and washing and drying the plate;
(4) Mu.l of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at 1;
(5) Adding 100 μ L of TMB developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 2M H into each well 2 SO 4 Stopping solution, 450And measuring the light absorption value at nm.
The inhibition standard curve of diphenhydramine monoclonal antibody to diphenhydramine is shown in figure 1. The diphenhydramine monoclonal antibody secreted by the hybridoma cell strain SHS5B11 has better detection sensitivity (IC) on diphenhydramine 50 The value is 2.93 ng/mL), can be used for establishing an immunological detection method of diphenhydramine and detecting whether the cosmetics contain diphenhydramine or not.

Claims (4)

1. A hybridoma cell strain SHS5B11 secreting diphenhydramine monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), china academy of sciences, china institute of microbiology, no. 3, of Xilu No. 1, beijing, chaoyang, and the date of deposition is 2020, 9 months and 27 days, and the number of deposition is CGMCC No.20787.
2. A diphenhydramine monoclonal antibody is characterized in that: the hybridoma cell strain SHS5B11 with the preservation number of CGMCC No.20787 as claimed in claim 1.
3. The use of the diphenhydramine monoclonal antibody of claim 2, characterized in that: the method is applied to the detection of diphenhydramine residues.
4. The use of the diphenhydramine monoclonal antibody of claim 3, characterized in that: the detection field is the detection of diphenhydramine residues in cosmetics.
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US5155212A (en) * 1986-05-21 1992-10-13 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens, antibodies and reagent kit
CN102659939A (en) * 2012-04-26 2012-09-12 嘉兴九七九生物技术有限公司 Diphenylhydantoin immunogen, diphenylhydantoin specificity-resistant antibody and diphenylhydantoin detection reagent

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Publication number Priority date Publication date Assignee Title
US5155212A (en) * 1986-05-21 1992-10-13 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens, antibodies and reagent kit
CN102659939A (en) * 2012-04-26 2012-09-12 嘉兴九七九生物技术有限公司 Diphenylhydantoin immunogen, diphenylhydantoin specificity-resistant antibody and diphenylhydantoin detection reagent

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