CN115521920B - Testosterone propionate monoclonal antibody hybridoma cell strain and application thereof - Google Patents
Testosterone propionate monoclonal antibody hybridoma cell strain and application thereof Download PDFInfo
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Abstract
The invention relates to a testosterone propionate (Tp) monoclonal antibody hybridoma cell strain and application thereof. The preservation number of the hybridoma cell strain is CGMCC No.45120. The invention mixes and emulsifies the complete antigen of testosterone propionate and equivalent Freund's adjuvant, and immunizes BALB/c mice through subcutaneous multipoint injection at the neck and back. The first immunization was performed with complete Freund's adjuvant, multiple booster immunization was performed with incomplete Freund's adjuvant, and the last immunization was performed with testosterone propionate complete antigen. Will have high potency and low IC 50 The spleen cells of the mice are fused with myeloma cells of the mice by a PEG method, and a selection medium is adopted to screen out hybrid cells after the two cells are fused; and screening cells by an indirect competitive ELISA method and subcloning for three times to finally obtain a testosterone propionate monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity to testosterone propionate.
Description
Technical Field
The invention relates to the field of food safety immunodetection, in particular to a testosterone propionate monoclonal antibody hybridoma cell strain and application thereof.
Background
Testosterone propionate (Testosterone propionate, tp) is a steroid assimilating hormone (Anabolic steroids, acs) and has the main effects of promoting the formation of male sexual organs and the development and maintenance of secondary sexual characteristics, and in addition, has the effects of protein assimilation, promoting epiphyseal fusion, stimulating erythrocyte proliferation and the like. Can promote protein anabolism, excite bone marrow hematopoietic function, and stimulate hematopoiesis. Testosterone, methyltestosterone and testosterone phenylpropionate all belong to steroid assimilating hormone, and have effects of promoting protein synthesis, promoting bone growth and erythropoiesis, etc.
In recent years, steroid anabolic hormones have been frequently used in animal husbandry. Anabolic hormone is a semisynthetic hormone drug obtained by structural modification of natural androgens. It can promote cell growth and differentiation, expand muscle and raise feed conversion rate, so that it is added to the raising process of food animal by illegal molecule. Abuse of anabolic hormones can pose direct and potential hazards to human and animal health, social and ecological environments. Therefore, many countries or organizations have regulated or banned the use of assimilating hormones in edible animal farming by legislation since the 80 s of the 20 th century. However, due to the driving of interest, assimilating hormones are still abused in large quantities worldwide. The foreign documents on detection of anabolic hormone residues in urine, blood and tissues are many, and the foreign documents are focused on analyzing the urine of living animals or high-concentration target tissues collected at slaughter sites. The use of steroid assimilating hormones for growth promotion purposes was prohibited from the beginning of the 20 th century in the 90 th year in China, and related monitoring work was started. However, no mature method for confirming the detection of steroid hormone residues in animal muscles is currently available in China.
Therefore, the establishment of a rapid and effective method for detecting the testosterone propionate content has important significance and market value. At present, the content of testosterone propionate is detected by a high performance liquid chromatography method, but the detection method is complicated and complicated, and the detection limit is too high. In order to maintain the interests of vast consumers, it is necessary to establish a high-efficiency and rapid detection method for Tp, and the enzyme-linked immunosorbent assay (ELISA) pretreatment is simple, the cost is low, the rapid detection of a large number of samples can be realized, and the purity requirement on the samples is not high during the detection. Therefore, it is necessary to establish an efficient immunological detection method, however, an important premise for establishing this method is to screen out a highly specific monoclonal monomer against testosterone propionate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a testosterone propionate monoclonal antibody hybridoma cell strain, and an antibody prepared by the hybridoma cell strain has better specificity and detection sensitivity on testosterone propionate, can be used for simultaneously detecting testosterone propionate, testosterone, methyltestosterone and testosterone phenylpropionate, and can be used for establishing an immunological detection method for detecting four assimilation hormones.
The technical scheme of the invention is as follows:
the first object of the present invention is to provide a hybridoma cell line secreting testosterone propionate monoclonal antibody, wherein the hybridoma cell line is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45120 and the preservation address of Beicheng Kogyo area Beicheng xi Lu No. 1 and No. 3 in 2022, 03 month.
The second object of the present invention is to provide a method for producing the hybridoma cell line, comprising the steps of:
(1) Preparing a testosterone propionate hapten and a testosterone propionate complete antigen, emulsifying the obtained testosterone propionate complete antigen with Freund's adjuvant to obtain an immunogen 1, and emulsifying the testosterone propionate complete antigen with incomplete Freund's adjuvant to obtain an immunogen 2;
(2) Performing first subcutaneous immunization on the animal by using the immunogen 1 obtained in the step (1), and performing booster immunization by using the immunogen 2;
(3) Collecting blood from the tail 1mm-2mm of the animal subjected to the immunization process in the step (2), and screening out the mice with highest titer in serum of the collected mice and the best testosterone propionate inhibition by enzyme-linked immunosorbent assay (ic-ELISA);
(4) Performing sprinting immunization on the animal screened in the step (3), wherein the sprinting immunization adopts a complete antigen of testosterone propionate without Freund's adjuvant;
(5) And (3) fusing spleen cells and myeloma cells of the animals subjected to the sprint immunization in the step (4) to obtain the hybridoma cell strain secreting the testosterone propionate monoclonal antibody.
In one embodiment of the present invention, in step (1), the testosterone propionate hapten has a formula of:
in one embodiment of the invention, the testosterone propionate complete antigen is prepared by the following method: dissolving testosterone propionate hapten, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in an organic solvent, stirring for reaction to obtain an activation solution, and adding the activation solution into a protein solution for coupling for reaction to obtain the testosterone propionate complete antigen.
In one embodiment of the invention, the molar ratio of testosterone propionate hapten, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1:2:2.
In one embodiment of the invention, the coupling protein is selected from bovine serum albumin, chicken serum albumin, human serum albumin, keyhole limpet hemocyanin or synthetic polylysine.
In one embodiment of the present invention, in the steps (2) and (4), the first subcutaneous immunization is separated from the boost by one month, the boost is separated from the boost by 21 days, and the boost is separated from the sprint by 18-21 days.
In one embodiment of the present invention, in the steps (2) and (4), the dose of the first subcutaneous immunization is 100 μg/dose, the dose of the booster immunization is 50 μg/dose, and the dose of the sprint immunization is 25 μg/dose.
In one embodiment of the invention, in step (3), the immunization procedure comprises 1 first subcutaneous immunization, at least 3 booster immunizations, and 1 sprint immunization.
In one embodiment of the present invention, in the step (5), spleen cells and myeloma cells of the BALB/c mice after sprint immunization are fused, the fused cells are screened and cultured by HAT medium, positive cell pores are detected by indirect ELISA, the inhibition effect of the positive cell pores is further determined by indirect competition ELISA, the positive cell pores with the best inhibition are subcloned by limiting dilution, and finally hybridoma cell strains capable of secreting testosterone propionate monoclonal antibodies with high sensitivity are screened out.
In one embodiment of the invention, in step (5), the cell fusion is performed 3 days after the termination of the sprint immunization.
In one embodiment of the present invention, in step (5), the cell fusion is performed by a polyethylene glycol (PEG 4000) method.
In one embodiment of the present invention, in step (5), the medium is RPMI-1640 medium.
In one embodiment of the invention, in step (5), the number of subcloning is at least 3.
The third object of the invention is to provide the application of the hybridoma cell strain in preparing testosterone propionate monoclonal antibody.
In one embodiment of the invention, the preparation method of the testosterone propionate monoclonal antibody comprises the steps of taking animals, injecting paraffin oil into the abdominal cavity, injecting hybridoma cell strain with the preservation number of CGMCC No.45120 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and preserving the obtained monoclonal antibody at low temperature.
At the bookIn one embodiment of the invention, the method is to take 8-10 week old BALB/c mice, each mouse is intraperitoneally injected with 1mL of paraffin oil, and each mouse is intraperitoneally injected with 1X 10 after 7 days 6 Collecting ascites from 7 th day of hybridoma cell strain with preservation number of CGMCC No.45120, purifying the ascites by octanoic acid-ammonium sulfate method, and preserving the obtained monoclonal antibody at-20deg.C.
A fourth object of the present invention is to provide a testosterone propionate monoclonal antibody secreted by the hybridoma cell line of claim 1.
The fifth object of the invention is to provide the hybridoma cell strain and the application of the testosterone propionate monoclonal antibody in detecting one or more of testosterone propionate, testosterone, methyltestosterone and testosterone phenylpropionate.
It is a sixth object of the present invention to provide a composition comprising said hybridoma cell line and/or said testosterone propionate monoclonal antibody.
A seventh object of the present invention is to provide a kit comprising one or more of the hybridoma cell line, the testosterone propionate monoclonal antibody, and the composition.
An eighth object of the present invention is to provide the use of the composition and the kit for detecting one or more of testosterone propionate, testosterone, methyltestosterone and testosterone phenylpropionate.
The hybridoma cell strains are classified and named as monoclonal cell strains.
The technical scheme of the invention has the following advantages:
the monoclonal antibody secreted by the hybridoma cell strain provided by the invention has better specificity and detection sensitivity (IC) to testosterone propionate 50 A value of 0.72 ng/mL) and testosterone (IC) can be detected simultaneously 50 With a value of 5.33 ng/mL), methyltestosterone (IC) 50 A value of 12.71 ng/mL) and testosterone phenylpropionate (IC) 50 The value was 7.80 ng/mL). Can realize the effects of testosterone propionate, testosterone, methyltestosterone and the like in chicken and porkThe simultaneous detection of testosterone phenylpropionate provides a raw material for the immunodetection of four homohormone residues of testosterone propionate, testosterone, methyltestosterone and testosterone phenylpropionate in food, and has practical application value.
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In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIG. 1 is a standard curve of testosterone propionate inhibition by testosterone propionate monoclonal antibodies according to example 3 of the present invention;
FIG. 2 is a standard curve of testosterone inhibition by testosterone propionate monoclonal antibody according to example 3 of the present invention;
FIG. 3 is a standard curve of testosterone propionate monoclonal antibody versus methyltestosterone inhibition in example 3 of the present invention;
FIG. 4 is a standard curve of testosterone propionate monoclonal antibody versus testosterone phenylpropionate inhibition in example 3 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The following examples relate to the following media:
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 B120.005, sodium bicarbonate 2000.
The solution was prepared 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 double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use.
Phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12 H 2 O, dissolving in 800mL of pure water, adjusting pH to 7.2-7.4 with NaOH or HCl, and fixing volume to 1000mL.
PBST: PBS containing 0.05% Tween 20.
Antibody dilution: PBS containing 0.1% gelatin.
TMB color development liquid: and (3) solution A: na (Na) 2 HPO 4 ·12H 2 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The solution B is prepared from the following components in percentage by weight: 1 to obtain TMB color development liquid, and mixing immediately.
The detection method involved in the following examples is as follows:
the testosterone propionate inhibition rate detection method comprises the following steps: the most appropriate antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.01. Mu.g/mL, 0.03. Mu.g/mL, 0.1. Mu.g/mL and 0.3. Mu.g/mL with Carbonate Buffer (CBS), and the antibody was diluted to 0.01. Mu.g/mL, 0.03. Mu.g/mL, 0.1. Mu.g/mL and 0.3. Mu.g/mL with antibody dilution. After selecting the optimal working point, testosterone propionate standard was diluted to 8 concentrations (0 ng/mL,0.034ng/mL,0.103ng/mL,0.309ng/mL,0.926ng/mL,2.778ng/mL,8.333ng/mL and 25 ng/mL), and according to the IC-ELISA procedure, the testosterone propionate standard inhibition curve was obtained by plotting OrigingPro 8.5 (the results are shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4), and IC was calculated 50 。
According to the invention, a mouse is immunized by using a testosterone propionate complete antigen, a HAT selective medium is used for culturing by cell fusion, and cell supernatant is screened by using an ic-ELISA, so that a hybridoma cell strain with high secretion specificity antibody against testosterone propionate is finally obtained.
EXAMPLE 1 Synthesis of hapten and complete antigen
Because the testosterone propionate small molecule has no immunogenicity, can not stimulate mice to generate immune response, and then generate antibodies, testosterone propionate is coupled to protein through protein connection technology, so that the testosterone propionate is immunogenic; the commonly used active groups in the protein coupling technology are amino, carboxyl, hydroxyl, sulfhydryl and the like, and the testosterone propionate is derived in view of the fact that the molecular structural formula of the testosterone propionate does not contain the active groups.
Derivatization of hapten: the product with carboxyl is obtained by the oximation reaction of testosterone propionate, namely hapten Tp, and the hapten Tp is coupled with carrier protein by a carbodiimide method to obtain testosterone propionate artificial antigen; the method comprises the following steps:
synthesis of testosterone hapten (hapten Tp):
the synthetic route is as follows:
(1) The compound testosterone propionate (Tp) 0.5g was added to a 25mL round bottom flask with 5mL methanol, 5mL pyridine, 1g O- (carboxymethyl) hydroxylamine hemi-hydrochloride in the round bottom flask;
(2) Magnetically stirring in a water bath at 70deg.C, and refluxing with a condensing device overnight;
(3) The reaction solution was cooled to room temperature, the pH was adjusted to acidity with 1M hydrochloric acid, extraction was performed by adding ethyl acetate, and the upper layer was dried with nitrogen to obtain testosterone propionate Hapten (Hapten Tp).
Synthesis of testosterone propionate complete antigen:
1.8mg of testosterone propionate hapten is weighed and dissolved in 400 mu LN, N-Dimethylformamide (DMF), stirred and reacted for 10min, and after complete dissolution, 1.3mg of N-hydroxysuccinimide (NHS) and 2.1mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) are added successively, and the mixture is activated at room temperature for 4h to 6h to obtain an activation solution. 6mg BSA was taken, dissolved (called protein solution) with 2mL of 0.01M carbonate buffer (CB, pH=9.0), and the activated solution was slowly added dropwise to the protein solution, followed by coupling reaction overnight at room temperature; then, the unreacted small molecule Hapten is removed by dialysis with 0.01M PBS solution to obtain the complete antigen Hapten Tp-BSA of testosterone propionate, and the complete antigen is identified by an ultraviolet absorption scanning method.
EXAMPLE 2 preparation of a hybridoma cell line secreting testosterone propionate monoclonal antibody
1. Immunization of animals
After mixing and emulsifying the testosterone propionate complete antigen and the equivalent Freund's adjuvant, BALB/c mice were subjected to subcutaneous multipoint injection immunization (except sprint immunization) at the back of the neck. The first immunization is carried out by using complete Freund's adjuvant, and the dosage is 100 mug/dose; multiple boosting with incomplete Freund's adjuvant and halving the dose to 50 μg/dose; the sprint immunity is directly diluted by normal saline without adjuvant, and then the dosage is halved to 25 mug/patient. One month is separated from the first immunization and the second immunization, 21 days is separated from the multiple boosting, and 18-21 days is separated from the final boosting. The immune effect of the mice is observed by an indirect competition enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the serum of the mice are detected.
2. Cell fusion
Three days after sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, as follows:
a. taking out eyeballs of a mouse to obtain blood, killing the mouse by a cervical dislocation method, immediately putting the mouse into 75% alcohol for disinfection, soaking for about 5 minutes, taking out the spleen of the mouse by aseptic operation, moderately grinding the spleen by a syringe rubber head, obtaining spleen cell suspension by a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 minutes), washing the spleen cells for three times by using an RPMI-1640 culture medium, and diluting the spleen cells to a certain volume and counting for later use after the last centrifugation;
b. collecting SP 2/0 And (3) cells: 7-10 days before fusion, SP was isolated 2/0 Tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium in 5% CO 2 Culturing in an incubator. Pre-fusion requirement SP 2/0 The number of the tumor cells reaches1-4×10 7 Guarantee of pre-fusion SP 2/0 Tumor cells are in logarithmic growth phase. During fusion, collecting tumor cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
c. fusion process 7min: 1min, 1mL of PEG4000 was added dropwise to the cells from slow to fast; and (2) standing for 2 min. Dripping 1mL of RPMI-1640 culture medium in the period of 1min for 3min and 4 min; dripping 2mL of RPMI-1640 culture medium in the period of 1min at the 5 th and 6 th min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture was incubated at 37℃for 5min. Centrifuging (800 rpm,10 min), removing supernatant, gently tapping cells, and adding RPMI-1640 selective medium (HAT medium) containing 20% fetal bovine serum and 2%50 XHAT to 96-well cell plate at 200 μl/well, placing at 37deg.C, 5% CO 2 Culturing in an incubator.
3. Cell screening and cell strain establishment
Half-changing the fused cells by HAT medium on the 3 rd day after cell fusion; full exchange with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum, 1% 100×ht on day 5; cell supernatants were taken on day 7 for screening. Screening is carried out in two steps: the first step is to screen out positive cell holes by using an ic-ELISA method, and the second step is to select testosterone propionate as a standard substance, and to measure the inhibition effect of positive cells by using the ic-ELISA method. Cell holes with better inhibition on testosterone propionate standard are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method after seven days. Three subcloning were performed as described above, and testosterone propionate monoclonal antibody cell line 4E1 was finally obtained.
EXAMPLE 3 preparation and identification of testosterone propionate monoclonal antibodies
Taking 8-10-week-old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; intraperitoneal injection of 1X 10 per mouse after 7 days 6 And collecting ascites from the seventh day by using testosterone propionate hybridoma cells, and purifying the ascites by an octanoic acid-saturated ammonium sulfate method. Under the condition of meta-acid, the n-octanoic acid can precipitate other hetero proteins except IgG immunoglobulin in ascites, and then the mixture is centrifuged and the precipitate is discarded; precipitating Ig with an equal amount of saturated ammonium sulfate solutionThe monoclonal antibody of the G type is centrifuged, the supernatant is discarded, dissolved in 0.01M PBS (pH 7.4), and then dialyzed and desalted, and finally the purified monoclonal antibody is stored at-20 ℃.
(1) Coating: the coating antigen Hapten Tp-BSA was diluted 3-fold from 1. Mu.g/mL with 0.05M carbonate buffer, pH9.6, 100. Mu.L/well, and reacted at 37℃for 2 hours.
(2) Washing: the plate solution was poured off and washed 3 times with wash solution for 3min each.
(3) Closing: after drying, 200. Mu.L/well of blocking solution was added thereto and reacted at 37℃for 2 hours. And (5) drying for standby after washing.
(4) Sample adding: the antisera was diluted from 1:1000 in a double ratio and added to the coated wells at each dilution, 100. Mu.L/well, and reacted at 37℃for 30min; after extensive washing, add 1:3000 dilution of HRP-goat anti-mouse IgG, 100. Mu.L/well, reaction at 37℃for 30min.
(5) Color development: and taking out the ELISA plate, fully washing, adding 100 mu L of TMB color developing solution into each hole, and carrying out light-shielding reaction for 15min at 37 ℃.
(6) Termination and measurement: 50. Mu.L of stop solution was added to each well to terminate the reaction, and the OD of each well was measured with a microplate reader 450 Values, standard curves were plotted as shown in fig. 1, 2, 3 and 4.
IC for determining monoclonal antibody testosterone propionate by IC-ELISA 50 The method comprises the following steps: 0.72ng/mL; testosterone IC 50 The method comprises the following steps: 5.33ng/mL; IC of methyltestosterone 50 The method comprises the following steps: 12.71ng/mL; IC of phenylpropionic acid testosterone 50 The method comprises the following steps: 7.80ng/mL, indicating testosterone propionate; testosterone, methyltestosterone and testosterone phenylpropionate have good sensitivity, and can be used for immunoassay detection of four assimilation hormones of testosterone propionate, testosterone, methyltestosterone and testosterone phenylpropionate.
EXAMPLE 4 determination of Cross-reactivity of testosterone propionate monoclonal antibody
IC of monoclonal antibodies to analogs (testosterone propionate, testosterone, methyltestosterone, and testosterone phenylpropionate) using IC-ELISA 50 Value (specific IC 50 The values are shown in example 3), and the Cross-reactivity (CR) is calculated according to the following formula.
The calculation formula is as follows: CR (%) =ic 50 (object)/IC 50 (analog) ×100%
The cross-reactivity of the monoclonal antibodies was determined by measuring structural analogs of the target, and the cross-reactivity measurements are shown in Table 1. As can be seen from Table 1, monoclonal antibodies to testosterone propionate crossed testosterone propionate, testosterone, methyltestosterone, and testosterone phenylpropionate at 100%,13.51%,5.66%, and 9.23%. According to the cross reaction rate, the testosterone propionate monoclonal antibody is a group-selective monoclonal antibody, and can detect various assimilation hormones of testosterone propionate, testosterone, methyltestosterone and testosterone phenylpropionate at the same time.
TABLE 1 determination of monoclonal antibody Cross-reactivity
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (7)
1. A hybridoma cell strain secreting testosterone propionate monoclonal antibody is characterized in that the hybridoma cell strain is preserved in China general microbiological culture Collection center (CGMCC) at the year 2022 and the year 03, and has a preservation number of CGMCC No.45120 and a preservation address of North Chenxi Lu No. 1 and No. 3 in the Korean region of Beijing city.
2. Use of the hybridoma cell line of claim 1 for the preparation of testosterone propionate monoclonal antibodies.
3. A testosterone propionate monoclonal antibody, characterized in that it is secreted by the hybridoma cell line of claim 1.
4. Use of the hybridoma cell line of claim 1 or the testosterone propionate monoclonal antibody of claim 3 for the preparation of a formulation for detecting one or more of testosterone propionate, testosterone, methyltestosterone and testosterone benzoate.
5. A composition comprising the hybridoma cell line of claim 1 and/or the testosterone propionate monoclonal antibody of claim 3.
6. A kit comprising one or more of the hybridoma cell line of claim 1, the testosterone propionate monoclonal antibody of claim 3, and the composition of claim 5.
7. Use of a composition according to claim 5 or a kit according to claim 6 for the preparation of a formulation for the detection of one or more of testosterone propionate, testosterone, methyltestosterone and testosterone phenylpropionate.
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