NL2030606B1 - Monoclonal antibody of tenuazonic acid (tea) and enzyme-linked immunosorbent assay (elisa) thereof - Google Patents

Abstract

The present disclosure discloses a monoclonal antibody capable of specifically recognizing tenuazonic acid (TEA) and an enzyme— linked immunosorbent assay (ELISA) thereof. In the present disclosure, hapten (TEA—CMO) is prepared, and coupled with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) separately to obtain an immunogen (TEA—CMO—KLH) and a coating antigen (TEA— CMO—BSA); and a monoclonal antibody of the TEA is obtained through animal immunization and hybridoma technology, to establish an icELISA with the monoclonal antibody of the TEA. The method is a rapid detection method for TEA residues, with a detection limit (IC…) of 1.00 ng/mL, a half maximal inhibitory concentration (ICw) of 18.50 ng/mL and a linear detection range of 3.56—96.24 ng/mL. The method can directly detect the TEA with desirable specificity and, high sensitivity, and, has no cross—reactivity‘ with other 9 Alternaria toxin structural analogs. (+ Fig. 1)

Description

P1017 /NLpd MONOCLONAL ANTIBODY OF TENUAZONIC ACID (TEA) AND ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) THEREOF

TECHNICAL FIELD The present disclosure belongs to the technical field of tox- in detection. More specifically, the present disclosure relates to a monoclonal antibody of tenuazonic acid (TEA) and an enzyme- linked immunosorbent assay (ELISA) thereof.

BACKGROUND ART Tenuazonic Acid (TEA), as one of the main toxic metabolites of Alternaria, has a molecular structure of CsHi30:N (as shown in Table 2) with a molecular weight of 197. At present, more than 70 Alternaria toxins are known to have obvious toxicity, where the TEA, as an only nitrogen-containing metabolite, has the highest toxicity. Moreover, studies have shown that the TEA has various types of toxicities, which brings a huge potential threat to human health. Therefore, it is particularly important to establish a rapid, simple and sensitive detection method of the TEA. In addition, there are few reports on TEA rapid detection, and detection objects are mostly TEA derivatives that have a rela- tively cumbersome derivative process, thereby bringing a lot of inconvenience to the detection of actual samples.

SUMMARY To overcome the above shortcomings of the prior art, the pre- sent disclosure provides a monoclonal antibody of TEA and an ELISA thereof. The ELISA, due to accuracy, sensitivity, rapidity and simplicity, can efficiently detect the TEA. The present disclosure provides a certain theoretical guidance for developing rapid de- tection products for the TEA, and is of great significance to food safety monitoring. A purpose of the present disclosure is to provide a monoclo- nal antibody of TEA.

A second purpose of the present disclosure is to provide use of a monoclonal antibody of TEA in detecting the TEA.

A third purpose of the present disclosure is to provide an ELISA of the TEA.

The foregoing objective of the present disclosure is imple- mented by the following technical solution: A monoclonal antibody of TEA is provided, where a TEA mono- clonal antibody is obtained through animal immunization and hy- bridoma technology using a product TEA-CMO-KLH obtained by cou- pling hapten TEA-CMO to keyhole limpet hemocyanin (KLH) as an im- munogen; the TEA-CMO has a structure shown as follows: MeN \

SY Se y ss me

TEAM Compared with the prior art, the present disclosure achieves the following beneficial effects.

(1) In the present disclosure, the monoclonal antibody against the TEA has a desirable specificity, and has no cross- reactivity with other structural analogs; the established ELISA can directly detect the TEA with convenience and rapidness.

(2) In the present disclosure, a standard curve of the estab- lished indirect ELISA (iELISA) has a detection limit (ICs) of 1.00 ng/mL, a half maximal inhibitory concentration (IC) of 18.50 ng/mL and a linear detection range of 3.56-96.24 ng/mL. Moreover, the method has a recovery rate of addition in juice and beer sam- ples of 85.5-119.7%, and has a desirable correlation with detec- tion results of a high performance liquid chromatography (HPLC) method (R*=0.9132), indicating that the established icELISA can be applied to the detection of actual samples.

(3) In the present disclosure, the monoclonal antibody pre- pared by the immunogen TEA-CMO-KLH is used as a coating antigen to establish the icELISA for TEA detection, which provides a rapid and efficient detection method for a TEA content in foods. The method, due to stability, reliability and relatively low cost, is of great significance for developing TEA rapid detection kits and colloidal gold test strips.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a standard curve of the TEA.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present disclosure is further illustrated with reference to the specific examples, which are not intended to limit the pre- sent disclosure in any form. Unless otherwise specified, the rea- gents, methods and equipment used in the present disclosure are conventional in the art. Unless otherwise specified, the reagents and materials used in the present disclosure are commercially available. Example 1 Preparation and characterization of a TEA immunogen

1. Preparation of hapten for the TEA 50 mg of TEA (molecular weight (Mw)=197.1) and 50 mg of car- boxymethoxylamine hemihydrochloride (CMO) (Mw=109.3) were dis- solved separately in 4.0 mL and 2.0 mL of mixed solvents {metha- nol: pyridine: water = 1: 4: 1}; a CMO solution was slowly added to a TEA solution, mixed well and subjected to reflux heating in an oil bath at 70°C for 15 h. After the reaction was over (it was monitored whether the TEA was completely reacted, and the reaction was continued if not), a mixture was subjected to vacuum drying, and 3.0 mL of a 2 mol/L HCl solution was added to dissolve a dried substance. A composition in an aqueous phase was extracted with dichloromethane several times, organic phases were combined, and vacuum concentration was conducted to obtain a light brown oily substance. The oily substance was purified by dichloromethane/n- hexane reprecipitation to obtain a light brown powder, namely the hapten TEA-CMO. The TEA had a structure as follows: HO 0 “ed NPE. Sg ì N

LL A

2. Preparation of an immunogen and a coating antigen for the

TEA

0.1 mmoL of the hapten (TEA-CMO) was dissolved in 0.5 mL of a N,N-dimethyl formamide (DMF) solvent, 0.0309 g of 0.15 mmoL dicy- clohexylcarbodiimide (DCC) and 0.0173 g of 0.15 mmol N- hydroxysuccinimide (NHS) were added under stirring, and stirring was continued for reaction overnight at 4°C; centrifugation was conducted to obtain a supernatant, recording as a solution A. 0.02 g of a carrier protein (KLH/BSA/OVA) was weighed and dissolved in 2 mL of a 0.01 mol/L phosphate-buffered saline (PBS) at a pH value of 7.4 fully to obtain a solution B. Under magnetic stirring, the solution A was slowly added dropwise into the solution B, and re- action was conducted for 12 h at 4°C. After the reaction, centrif- ugation was conducted to obtain a supernatant, namely a complete antigen solution coupled with the carrier protein; and the antigen solution was dialyzed with a PBS solution for 3 days at 4°C (where a dialysate was replaced 3 times per day to remove small molecule impurities) to obtain the immunogen and the coating antigen. The immunogen and the coating antigen were divided into 1.5 mL centri- fuge tubes at a concentration of 1 mg/mL, and stored in a refrig- erator at -20°C for later use.

Example 2 Preparation of a TEA monoclonal antibody

1. Animal immunization When female Balb/C mice aged 6 months were immunized for the first time, 0.1 mL/mouse of an immunogen was injected, and 1 mg/mL of immunogen was added with an equal volume of a Freund's complete adjuvant; a fully emulsified solution was injected subcutaneously into the back and abdomen of the mice at multiple points, with each point injecting about 200 pl; for a second immunization 21 d later, 0.1 mL/mouse of the immunogen was injected, and the immuno- gen was emulsified with a Freund's incomplete adjuvant; from then on, the immunizations were boosted twice every 14 d, generally a total of 5 immunizations were conducted. In this experiment, after the 3th immunization and the 5th immunization, blood was collected 7 d later for antiserum titration. 3 days before cell fusion, the mice were subjected to booster immunization, and injected directly with 0.1 mL of the immunogen (without adjuvant). After the mice were immunized, numbering, management and recording for animals were conducted, and the health of the mice was monitored.

2. Analysis of antiserum effect (1) On the 7th day after the 3rd and 5th booster immuniza- tions, 200 uL of blood was collected from a tail of the mouse, in- 5 cubation was conducted at 37°C for 30 min, and centrifugation was conducted at 6,000 r/min for 10 min, and a supernatant was added with an equal volume of glycerol, and stored at -20°C for later use.

(2) The titer and specificity of the antiserum were deter- mined using an iELISA method, and the method included the follow- ing steps: 31, coating: 1 mg/mL of a coating antigen was diluted 1,000 times with a coating buffer, and a blank control group was set up, diluted coating antigen was added to a 96-well microtiter plate at a concentration of 100 uL/well, and incubation was conducted over- night in a 37°C water bath.

S2, washing: liquid in the wells was poured out, parameters of a plate washer were set and 300 pL of washing liquid was added to each well, the plate was washed twice, and the washing liquid was removed by spin drying.

S3, blocking: 120 pL of a blocking solution was added to each well, blocking was conducted at 37°C for 3 h, liquid in the well was removed by spin-drying, and the plate was dried in an oven at 37°C for 1 h.

S4, addition of antiserum and a standard product: the antise- rum was subjected to gradual dilution; a TEA standard product was dissolved in an appropriate amount of methanol to prepare a 1.0 mg/mL standard product solution, and stored at 4°C for later use. Titer column: 50 pL of a blank diluent and 50 pL of a gradiently diluted antiserum were added to each well, and a primary antibody diluent was added to the last two wells as a blank control; inhi- bition column: 50 pL of the standard solution and 50 pL of the gradiently diluted antiserum were added to each well, and a stand- ard diluent was added to the last two wells as a blank control; incubation was conducted at 37°C for 40 min, and the plate was washed 5 times.

35, addition of a secondary antibody: 100 uL of horseradish peroxidase (HRP)-goat anti-mouse immunoglobulin G (IgG) (after PBS dilution by 5,000 times) was added to each well, reaction was con- ducted in a constant-temperature water bath at 37°C for 40 min, and washed 5 times.

S6, color development: a solution A and a solution B in a color development kit were mixed in equal volumes, 100 pL of a mixed solution was added to each well, color development was con- ducted in a constant-temperature water bath at 37°C for 10 min, and 50 pL of a stop solution (10% H,S0,) was added to each well.

S7, reading measurement: an absorbance (optical density, OD) was read with a microplate reader at a wavelength of 450 nm.

(3) An antiserum dilution factor with an absorbance value of

1.0-1.5 was selected as an antiserum titer, and an antiserum ef- fect was derived from an inhibition rate. A calculation formula of the inhibition rate was as follows: ALA, ie Rh , AE en, a Inhibition rate ( 9%) rns x 100 ie Ie A * Ishida column The effect of antibody could be expressed by an inhibition rate on a certain concentration of a drug: under a same drug con- centration, a higher inhibition rate lead to a higher sensitivity of the antibody to the drug.

3. Cell fusion and screening of positive hybridomas (1) Resuscitation of myeloma cells: the myeloma cells were taken out from liquid nitrogen, quickly thawed in a 37°C water bath, and centrifugation was conducted at 1,000 r/min for 7 min after thawing; a supernatant was treated in an ultra-clean work- bench, about 1 mL of a complete culture medium was added to cell pellets, and the cells were blown off; the cells were pipetted us- ing a pipette and mixed well with a complete medium, put into a 9 cm petri dish, and expanded to 5-6 dishes, where the medium was changed 1 or 2 times; the cells in each petri dish were capable of cell fusion when filling a bottom.

(2) Feeder cell preparation: 1 d before the cell fusion, eye- balls were removed to take blood, and Kunming mice were sacri-

ficed; after soaking in 80% alcohol for 5 min, the mice were transferred to an ultra-clean workbench for dissection. The abdo- men was carefully cut (do not cut abdominal muscles), then the ab- dominal skin was peeled off to expose the abdominal muscles; the peritoneum was lifted with tweezers, a small opening was made in the peritoneum, and 3 mL of a hypoxanthine-aminopterin-thymidine (HAT) complete medium was injected; the cells were pipette back and forth repeatedly using a pipette to suck and rinse, and a me- dium containing feeder cells was taken out from the abdominal cav- ity using a pipette, and mixed with the medium in the petri dish; this step was repeated 2-3 times to ensure that enough feeder cells were obtained.

(3) Preparation of spleen cells: bleeding out was conducted on orbits of Balb/c mice that have been immunized several times and pass blood tests, the serum was collected, and scaked in 80% alcohol for 5 min, the body surface was disinfected, and the mice were transferred to an ultra-clean workbench for dissection. The spleen was taken out aseptically, white connective tissues around the spleen were removed with scissors, and the spleen was rinsed with a Roswell Park Memorial Institute (RPMI)-1640 basal medium, and placed in a petri dish for later use. The medium was aspirated by a disposable syringe, the removed spleen was held with tweezers by left hand, a syringe was inserted into the spleen by right hand to slowly inject the medium to wash out the cells in the spleen.

The step was repeated until the spleen changed from dark red to transparent, and the spleen was discarded. The mixed medium was collected into a 50 mL centrifuge tube; centrifugation was con- ducted at 1,000 r/min for 8 min after sealing. The supernatant was discarded after centrifugation for later use.

(4) Cell fusion: myeloma cells and immune spleen cells free of the supernatant by centrifugation were mixed in a centrifuge tube at a ratio of about 1:5, 25 mL of a basal medium was added, and centrifugation was conducted at 1,000 r/min for 8 min after sealing. The supernatant was discarded after centrifugation for later use. The supernatant was discarded from the myeloma cells and the immune spleen cells mixed by centrifugation, and excessive medium was removed by a pipette from the centrifuge tube. The ex-

cessive residual medium may affect the effect of polyethylene gly- col (PEG). The precipitated cells were flicked gently with fin- gers, and the centrifuge tube was placed in warm water at 37°C;

0.8 mL of PEG preheated to 37°C was pipetted by a pipette tip, the PEG was slowly added to the precipitated cells within 1 min, where the cells were gently, but not too vigorously stirred with the pi- pette tip to mix well when each drop of PEG was added; standing was allowed for 1 min, and 10 mL of a preheated complete medium was added within 2 min along the tube wall under gently stirring (do not blow away to separate the PEG). The centrifuge tube was sealed, centrifugation was conducted at 1,000 r/min for 10 min; after the centrifugation, a supernatant was discarded, a HAT medi- um was added by gently aspirating and dispensing with an elbow pi- pette, and stirred gently; the HAT medium in the centrifuge tube was transferred to mix with about 75 mL of a fresh HAT medium, and a mixed medium was evenly added tol10 96-well culture plates con- taining feeder cells prepared the day before. The volume of HAT medium with feeder cells was kept the same as that of the HAT me- dium containing fusion cells in each well. The HAT medium used in each plate was about 24 mL.

(5) Screening of positive hybridomas: HAT medium was used within 7-10 d after fusion, and an HT medium was used to replace the HAT medium for transition for one week, and the complete medi- um was used according to cell proliferation state after 14 d. When the cells grew adherently to 1/3 of the plate well (usually about 12 d after cell fusion), a supernatant was pipetted from the mul- ti-well culture plate, specific antibodies in the medium were de- tected by iELISA, and titer and inhibition rate were determined; the positive hybridoma cells with a high titer and strong drug in- hibition were selected, positive wells with the best fusion effect were screened out and marked. Under aseptic conditions, cells that grew in clusters in the positive wells were selected by a micro- scope, and transferred to a 96-well culture plate spread with feeder cells in advance, where each original well was cloned into 8 wells; after the cells adhered to the wall and grew up to 1/2 to 1/3 of the bottom of the well, a supernatant was taken for icE- LISA; titer and inhibition rate were also used as quantitative in-

dicators, and cells that were highly positive were sub-cloned by a limiting dilution method; the step was repeated for 3-4 rounds (the cells in the positive wells selected in each round needed to be expanded and frozen for later use), until each well of each plate was positive and the detected titer and inhibition rate were similar. Finally, a hybridoma cell line had been successfully es- tablished, which was capable of stably secreting uniform antibod- ies. Single-cell clones were selected, and all positive ones were transferred to a 24-well cell culture plate or a cell culture dish for expansion culture, and frozen in time.

Example 3 Mass production of TEA monoclonal antibody

1. Antibody secretion and expression After obtaining a clone of hybridoma cells that secrete a specific monoclonal antibody, the monoclonal antibody was usually produced in large quantities by in vitro culture and a method for inducing monoclonal antibodies in animals in vivo.

2. Antibody purification A purification method used in the present disclosure was a caprylic acid-ammonium sulfate method, and specific steps were as follows: (1) Ascites was centrifuged at 12,000 r/min at 4°C for 15 min to remove impurities.

(2) The ascites and an acetate buffer solution were mixed in a volume ratio of 1:2, and n-octanoic acid was added under stir- ring at room temperature. 33 pL of the n-octanoic acid was added to each milliliter of the ascites to adjust the pH to 4.8.

(3) The ascites was mixed by stirring at room temperature for min, and standing was allowed at 4°C for 2 h to fully precipi- tate.

30 (4) Centrifugation was conducted at 4°C, 12,000 r/min for 15 min, a precipitation was discarded.

(5) After a supernatant was filtered through a sand core fun- nel or a 125 um nylon mesh, 1/10 volume of a 0.1 mol/L PBS (pH

7.4) was added, and the pH value was adjusted to 7.4 with a 2 mol/L NaOH solution.

(6) 0.277 g/mL ammonium sulfate was added within 30 min under ice bath to make a saturation of 45%.

(7) Standing was allowed at 4°C for not less than 1 h, then centrifugation was conducted at 4°C, 10,000 r/min for 30 min, and a supernatant was discarded.

(8) The precipitate was dissolved with an appropriate amount of PBS with pH of 7.4, and dialyzed in 5 L of a PBS (0.01 mol/L, PH 7.4) at 4°C for 3 d, where a dialysate was replaced 3 times a day; after the dialysis was completed, a product was aliquoted and stored at -20°C for later use.

Example 4 Establishment of an icELISA for TEA

1. The icELISA specifically included the following reaction steps: Sl, Coating: a coating antigen was diluted to a suitable con- centration with a coating solution, and added to wells of a micro- titer plate at 100 uL/well, and reaction was conducted in a 37°C water bath overnight.

S2, washing: liquid in the wells was poured out, 300 pL of washing liquid was added to each well, the plate was washed twice, and the washing liquid was removed by spin drying.

53, blocking: 120 pL of a blocking solution was added to each well, blocking was conducted at 37°C for 3 h, liquid in the well was removed by spin-drying, and the plate was dried oven at 37°C for 1 h.

S4, sample loading and incubation: each well was added with 50 pL of antibodies diluted to a certain multiple and 50 pL of a drug diluent; a product was mixed well by shaking; reacting was conducted in a 37°C water bath for 40 min, 250 pL of washing lig- uid was added to each well, the plate was washed 5 times, and the washing liquid was removed by spin drying.

S5, addition of a secondary antibody: 100 uL of HRP-goat an- ti-mouse-goat anti-rabbit IgG (after dilution by 5,000 times) was added to each well, reaction was conducted in a water bath at 37°C for 40 min, the plate was washed 5 times and liquid was removed by spin-drying.

S6, color development: 100 pL of a color development solution was added to each well, color development was conducted in a water bath at 37°C for 10 min, and 50 pL of a stop solution (10% H:S0,) was added to each well.

57, determination: an absorbance of each well was measured at 2450 nm with an ELISA instrument. 38, calculation: the ICss of an inhibition curve was calculat- ed using a four-parameter fitting module of Origin8.6.

2. TEA standard solutions were prepared for 0.00098 ug/L,

0.00391 pg/L, 0.01563 ug/L, 0.0625 pg/L, 0.25 pg/L, 1 ug/L and 4 ug/L, and a standard curve of iELISA was established.

The method had a detection limit (IC) of 1.00 ng/mL, an ICs, of 18.50 ng/mL and a linear detection range of 3.56-96.24 ng/mL. Example 5 Determination of antibody cross-reactivity and de- termination of sample recovery rate

1. According to the optimal coating antigen concentration and the optimal antiserum dilution factor obtained in Example 4, icE- LISA experiment was conducted using structural analogues TEA, AME, AOH, ITeA, DON, AFBl, OA, OB, T-2 and ZEA as competing antigens; the specificity of a TEA monoclonal antibody was detected, and IC: and CR were listed in Table 1. Table 1 Antibody specificity of TEA icELISA (n=3) Compound Chemical structure ICs; (ng/mL) CR (%)

O

HN TEA 2 18.5 100 ©

OH

OH oo I AME 2) >1000 <0.1

O H i

OH wo I] ACH CO >1000 <0.1

H I 0

HN O ITeA 4 >1000 <0.1 CH, OH ’

oO DON Lik >1000 <0.1

H

H OH 0 == o 0 AFB1 / >1000 <0.1 0 HO Oo oO OA >1000 <0.1

N Oo H 1

H oO HO oO Oo OB >1000 <0.1

N 0 H

H 0 To T-2 Tr Y >1000 <0.1 0 H

OH O ZEA OL >1000 <0.1 HO NF 0 The experimental results show that the anti-TEA antibody has no cross-reactivity with the other 9 Alternaria toxin structural analogs (ICsy is not less than 1,000 ng/mL, and CR is not more than

0.1%). It shows that the monoclonal antibody has a relatively de- sirable specificity for TEA in practical use, and the icELISA de- tection method has a desirable specificity.

2. Determination of the sample recovery rate The spiked apple juice, grape juice and beer samples were re- covered for icELISA determination. The results are shown in Table

2. Table 2 Sample addition and recovery experiment of TEA icE-

LISA (n=3) Sam TBA-icCLEIA ple Addition value Measured value Recovery rate Variable fug/L (kg)] [ng/L (kg)] ( X+8D) (%) coefficient

(€)

Apple 20 OO I7.0982.14 BSD LZS juice 50 56.21+4.52 112.4 8.0

80 72.361t6.25 90.5 8.6 Grape 20 23.93+1.95 118.7 8.2 juice 50 56.40+7.95 100.7 14.1

80 8.97x1.02 89.7 11.4 Beer 20 18.0612.42 20.3 13.4

50 59.0018.53 ii8 14.5

80 86.91+10.66 108.6 12.3

The experimental results show that the three samples have a recovery rate of 85.5-119.7%, and CV<15%. The method has a desira- ble accuracy, indicating that the monoclonal antibody and icELISA detection method can detect the TEA in actual samples.

Claims (5)

CONCLUSIONS A monoclonal antibody capable of specifically recognizing tenuazonic acid (TEA), wherein a monoclonal TEA antibody is obtained by animal immunization and hybridoma technology using a product TEA-CMO-KLH, obtained by hapten coupling TEA-CMO to keyhole limpet hemocyanin (KLH) as an immunogen. The monoclonal antibody according to claim 1, wherein a preparation method of the hapten TEA-CMO comprises the following steps: separately dissolving TEA and carboxymethoxylamine hemihydrochloride (CMO) of the same mass in a mixed solvent A in a mass-volume ratio from (23-27) mg : (1-2) ml, adding a CMO solution to a TEA solution and mixing well; heating a mixture under reflux at 60-80°C for 10-20 hours, drying under vacuum, adding 2-4 ml of 1-3 mol/L HCl to dissolve the mixture, and performing of extraction with dichloromethane and vacuum concentration to obtain a light brown oily substance; and performing reprecipitation by dichloromethane/n-hexane to purify the oily substance to obtain a TEA-CMO powder; wherein the mixed solvent A is prepared by mixing methanol, pyridine and water in a volume ratio of 1 : (3-5) : 1. The monoclonal antibody according to claim 1, wherein a method of preparation of the immunogen comprises the following steps: S11: dissolving the hapten in N,N-dimethylformamide (DMF), adding dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide ( NHS) with stirring, and stirring overnight at 4°C; performing centrifugation to obtain a supernatant and taking it up as a solution A; wherein the hapten, DCCI and NHS have a molar ratio of 1 : (1.2-1.5) : (1.2-1.5); S12: completely dissolving a carrier protein in a 0.01 mol/L phosphate buffered saline (PBS) to obtain a solution B; wherein the carrier protein and the PBS have a mass to volume ratio of 1 mg : (0.1-0.2) ml; and S13: adding the solution A to the solution B dropwise with stirring, and conducting the reaction at 4°C for 10-15 hours; performing centrifugation to obtain a supernatant, and dialyzing the supernatant with a PBS solution at 4°C for 2 - 4 d (replacing a dialysate 3 times a day) to remove an immunogen from the TEA obtain. Use of the monoclonal antibody according to any one of claims 1 to 3 in directly detecting the TEA or in preparing a kit for directly detecting the TEA. An enzyme-linked immunosorbent assay (ELISA) of TEA, wherein the ELISA is an indirect competitive ELISA (icELISA) mediated by the monoclonal antibody of the TEA according to any one of claims 1-3.