Monoclonal antibody against the surface protein 1 of the merozoite of the malaria parasite plasmodium and its use

Abstract

The application belongs to the field of biological products, and relates to a kind of anti-theileria equiperda schizont surface protein 1 monoclonal antibody and its application.The application specifically discloses a monoclonal antibody with light chain variable region and heavy chain variable region amino acid sequences shown in SEQ ID NO.8 and SEQ ID NO.6 respectively.The monoclonal antibody can specifically bind to the extracellular domain of theileria equiperda schizont surface protein 1.The monoclonal antibody can be used to detect theileria equiperda positive serum by competitive ELISA method, and has good sensitivity and specificity.The monoclonal antibody has clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of veterinary bioproducts and relates to a monoclonal antibody against merozoon surface protein 1 of Tyleil equine and its application. Background Technology

[0002] Giridiasis is caused by Theileria equi and Babesia caballi, transmitted by ticks, and has a global distribution. In my country, it is classified as a Class III animal disease.

[0003] There is an urgent need to establish sensitive and specific diagnostic methods. EMA1 (merozoite surface protein 1) is an important surface antigen of *Thief's horse*, exhibiting high immunogenicity and conservation. The preparation of its monoclonal antibody could provide key reagents for the development of competitive ELISA (cELISA), enabling efficient and low-cost serological detection and aiding in epidemiological investigations and control. However, there are currently no reports on highly efficient cELISA detection of *Thief's horse*. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8 and P9;

[0005] P1: Monoclonal antibody

[0006] The monoclonal antibody maintains specific binding activity to the extracellular domain of merozoon surface protein 1 of *Ethylene equina* or to *Ethylene equina* merozoon surface protein 1.

[0007] The monoclonal antibody includes a monoclonal antibody heavy chain and a monoclonal antibody light chain;

[0008] The monoclonal antibody heavy chain includes heavy chain FR1, heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3 and heavy chain FR4;

[0009] The monoclonal antibody light chain includes light chain FR1, light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3 and light chain FR4;

[0010] The amino acid sequence of the heavy chain CDR1 is shown in positions 26-33 of SEQ ID NO. 6;

[0011] The amino acid sequence of the heavy chain CDR2 is shown in positions 51-58 of SEQ ID NO. 6;

[0012] The amino acid sequence of the heavy chain CDR3 is shown in positions 97-107 of SEQ ID NO. 6;

[0013] The amino acid sequence of the light chain CDR1 is shown in positions 27-32 of SEQ ID NO. 8;

[0014] The amino acid sequence of the light chain CDR2 is shown in positions 50-52 of SEQ ID NO. 8;

[0015] The amino acid sequence of the light chain CDR3 is shown in positions 89-97 of SEQ ID NO. 8;

[0016] P2: Genetically engineered monoclonal antibody

[0017] The genetically engineered monoclonal antibody maintains specific binding activity to the extracellular domain of merozoite surface protein 1 or merozoite surface protein 1 of *Ethylene equina*.

[0018] The genetically engineered monoclonal antibody includes a genetically engineered monoclonal antibody heavy chain and a genetically engineered monoclonal antibody light chain.

[0019] The genetically engineered monoclonal antibody heavy chain includes the monoclonal antibody heavy chain described in P1 and a tag peptide and / or signal peptide used for protein isolation and purification.

[0020] The genetically engineered monoclonal antibody light chain includes the monoclonal antibody light chain described in P1 and a tag peptide and / or signal peptide for protein isolation and purification.

[0021] P3: Antibody derivatives

[0022] The antibody derivative maintains specific binding activity to the extracellular domain of merozoite surface protein 1 or to merozoite surface protein 1 of *E. equisetifolia*.

[0023] The antibody derivative is selected from: the monoclonal antibody described in fluorescently labeled P1;

[0024] The monoclonal antibody described in P1 labeled with a chemiluminescent enzyme;

[0025] The genetically engineered monoclonal antibody described in fluorescently labeled P2;

[0026] The genetically engineered monoclonal antibody described in P2, labeled with a chemiluminescent enzyme;

[0027] P4: RNA assembly

[0028] The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA;

[0029] The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2.

[0030] The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2.

[0031] P5: Gene Combination

[0032] The gene combination includes monoclonal antibody heavy chain genes and monoclonal antibody light chain genes;

[0033] The coding sequence of the monoclonal antibody heavy chain gene can encode the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2.

[0034] The coding sequence of the monoclonal antibody light chain gene can encode the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2.

[0035] P6: Gene Engineering Vector

[0036] The genetic engineering vector is a combination of a monoclonal antibody heavy chain genetic engineering vector and a monoclonal antibody light chain genetic engineering vector, or a monoclonal antibody genetic engineering vector.

[0037] The monoclonal antibody heavy chain gene engineering vector encodes the expressible monoclonal antibody heavy chain RNA described in P4;

[0038] The monoclonal antibody light chain gene engineering vector encodes expressible P4 monoclonal antibody light chain RNA;

[0039] The monoclonal antibody gene engineering vector encodes expressible monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA as described in P4.

[0040] P7: Cells

[0041] The cells contain the monoclonal antibody heavy chain gene engineering vector and the monoclonal antibody light chain gene engineering vector described in P6; or

[0042] The cells contain the monoclonal antibody gene engineering vector described in P6;

[0043] P8: Reagent Kit

[0044] The kit contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the genetically engineered vector described in P6, or the cells described in P7;

[0045] P9: Composition

[0046] The composition contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the genetically engineered vector described in P6, or the cell described in P7.

[0047] In some embodiments, the amino acid sequence of the extracellular domain is shown in SEQ ID NO.2;

[0048] The surface protein 1 of the merozoite of *Ethylophora equina* contains a peptide with the amino acid sequence shown in SEQ ID NO. 2.

[0049] The amino acid sequence of the variable region of the monoclonal antibody heavy chain is shown in SEQ ID NO. 6;

[0050] The amino acid sequence of the variable region of the monoclonal antibody light chain is shown in SEQ ID NO. 8; or

[0051] In the monoclonal antibody or the genetically engineered monoclonal antibody, the heavy chain subtype is IgG1 and the light chain subtype is Kappa.

[0052] The second aspect of the present invention provides the use of the monoclonal antibody, genetically engineered monoclonal antibody or antibody derivative described in the first aspect of the present invention in the preparation of a formulation for recognizing a first biological material;

[0053] The first biological material is selected from:

[0054] The extracellular domain of equinetella merozoon surface protein 1 expressed in prokaryotes or eukaryotes;

[0055] Fusion proteins expressed in prokaryotes or eukaryotes that contain a protein tag for protein isolation and purification and the extracellular domain of Matheylorhizium merozoon surface protein 1;

[0056] Prokaryotic, eukaryotic, or extracted from *Ethylethia equina* cells, *Ethylethia equina* merozoon surface protein 1;

[0057] A prokaryotic or eukaryotic fusion protein containing a protein tag for protein isolation and purification and *Ethylethia mustiole* merozoon surface protein 1; and

[0058] Cells of *Ethylethia equinoides* containing merozoite surface protein 1, their lysates, or their inactivated products.

[0059] In some embodiments, the amino acid sequence of the extracellular domain is as shown in SEQ ID NO.2; or

[0060] The merozoite surface protein 1 of *Ethylethia esculenta* contains a peptide with the amino acid sequence shown in SEQ ID NO.2.

[0061] A third aspect of the present invention provides a kit containing the antibody derivative and enzyme-labeled plate described in the first aspect of the present invention;

[0062] The reaction wells of the ELISA plate are coated with the first protein;

[0063] The first protein is selected from:

[0064] The extracellular domain of equinetella merozoon surface protein 1 expressed in prokaryotes or eukaryotes;

[0065] Fusion proteins expressed in prokaryotes or eukaryotes that contain a protein tag for protein isolation and purification and the extracellular domain of Matheylorhizium merozoon surface protein 1;

[0066] Prokaryotically expressed, eukaryotically expressed, or extracted from *Ethylethia equina* cells, *Ethylethia equina* merozoon surface protein 1; and

[0067] A fusion protein expressed in prokaryotes or eukaryotes containing a protein tag for protein isolation and purification and a merozoan surface protein 1 from *Tetraylus equina*.

[0068] In some embodiments, the amino acid sequence of the extracellular domain is shown in SEQ ID NO.2;

[0069] The *Ethylophora equina* merozoite surface protein 1 contains a peptide with the amino acid sequence shown in SEQ ID NO. 2; or

[0070] The antibody derivative is either a horseradish peroxidase-labeled monoclonal antibody or a horseradish peroxidase-labeled genetically engineered monoclonal antibody.

[0071] A fourth aspect of the present invention provides a method for detecting antibodies against the surface protein 1 of *Thealeria equina* merozoites for non-diagnostic purposes, the method comprising the following steps:

[0072] S1: Coating the solid surface with a second biomaterial to obtain a coated solid surface;

[0073] The second biological material is selected from:

[0074] The extracellular domain of equinetella merozoon surface protein 1 expressed in prokaryotes or eukaryotes;

[0075] Fusion proteins expressed in prokaryotes or eukaryotes that contain a protein tag for protein isolation and purification and the extracellular domain of Matheylorhizium merozoon surface protein 1;

[0076] Prokaryotic, eukaryotic, or extracted from *Ethylethia equina* cells, *Ethylethia equina* merozoon surface protein 1;

[0077] A fusion protein expressed in prokaryotes or eukaryotes containing a protein tag for protein isolation and purification and a merozoan surface protein 1 from *Ethylene equina*.

[0078] S2: The sample to be tested and the monoclonal antibody linked with the characterization label are added simultaneously or sequentially to the coated solid surface and incubated to obtain the incubated solid surface.

[0079] The monoclonal antibody is the monoclonal antibody described in the first aspect of the present invention or a genetically engineered monoclonal antibody.

[0080] The characterization marker is a fluorescent group or a chemiluminescent enzyme;

[0081] S3: Characterize the characterization marker that is indirectly bound to the incubated solid surface, and quantitatively or semi-quantitatively determine the content of the antibody against the surface protein 1 of *Ethylene equina merozoon* in the sample to be tested based on the fluorescence intensity or enzyme-catalyzed luminescence intensity of the characterization marker.

[0082] In some embodiments, the amino acid sequence of the extracellular domain is shown in SEQ ID NO.2;

[0083] The surface protein 1 of the merozoite of *Ethylophora equina* contains a peptide with the amino acid sequence shown in SEQ ID NO. 2.

[0084] The solid surface is the inner surface of the pores of the microporous plate;

[0085] The sample to be tested was horse serum.

[0086] In S2, equine Theileria-negative horse serum is added in parallel with the sample to be tested as a negative control;

[0087] In S2, equine theileria-positive horse serum is added in parallel with the sample to be tested as a positive control; or

[0088] The monoclonal antibody linked with the characterization label is either a horseradish peroxidase-labeled monoclonal antibody or a horseradish peroxidase-labeled genetically engineered monoclonal antibody. In step S3, the TMB substrate chromogenic solution is added to the surface of the incubating solid phase, and the OD of the incubating solid phase surface is measured. 450nm The content of the antibody against the surface protein 1 of *Tetraylus equina* merozoon in the sample to be tested is determined by quantitative or semi-quantitative calculation.

[0089] In some embodiments, the antibody against the surface protein 1 of the merozoite is a monoclonal antibody, polyclonal antibody, or antiserum stimulated by a third biological material;

[0090] The third biomaterial is selected from:

[0091] The extracellular domain of equinetella merozoon surface protein 1 expressed in prokaryotes or eukaryotes;

[0092] Fusion proteins expressed in prokaryotes or eukaryotes that contain a protein tag for protein isolation and purification and the extracellular domain of Matheylorhizium merozoon surface protein 1;

[0093] Prokaryotic, eukaryotic, or extracted from *Ethylethia equina* cells, *Ethylethia equina* merozoon surface protein 1;

[0094] A prokaryotic or eukaryotic fusion protein containing a protein tag for protein isolation and purification and *Ethylethia mustiole* merozoon surface protein 1; and

[0095] Cells of *Ethylethia equinoides* containing merozoite surface protein 1, their lysates, or their inactivated products.

[0096] In some embodiments, the amino acid sequence of the extracellular domain is as shown in SEQ ID NO.2; or

[0097] The merozoite surface protein 1 of *Ethylethia esculenta* contains a peptide with the amino acid sequence shown in SEQ ID NO.2. Attached Figure Description

[0098] Figure 1 To compete for ELISA clinical sample testing results.

[0099] Figure 2 To compete for specific detection results from ELISA.

[0100] Figure 3 To compete for ELISA sensitivity test results. Detailed Implementation

[0101] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0102] Materials and instruments not described in this invention are conventional materials and instruments in the art. Operational details not described in this invention are conventional operations in the art. The software used in this invention is operated by conventional methods in accordance with the software provider's instructions. The reagent kits used in this invention are operated by conventional methods in accordance with the reagent kit's instruction manual.

[0103] The nucleic acid sequences shown in this invention are all written from left to right in the direction from 5' to 3', and the protein sequences are written from left to right in the direction from N-terminus to C-terminus.

[0104] Applications of detecting antibodies against *Ethylethia equina* merozoite surface protein 1 for non-diagnostic purposes include, but are not limited to: studying the epidemiological patterns of *Ethylethia equina* and studying the immune patterns of *Ethylethia equina*.

[0105] Example 1: Expression and purification of recombinant EMA1 protein

[0106] I. Plasmid Preparation

[0107] Positive whole blood of *Ethylethia equina*: Whole blood of horses infected with the Xinjiang strain of *Ethylethia equina*.

[0108] The coding sequence of the extracellular region of merozoon surface protein 1 (EMA1) of this *Ethylethia equina* from position 31 to 786 is shown in SEQ ID NO. 1.

[0109] The amino acid sequence of the extracellular region of merozoite surface protein 1 (EMA1) of this *Ethylethia equina* is shown in SEQ ID NO. 2.

[0110] Using whole blood DNA from the aforementioned positive *Thealeria equina* strain as a template, the EMA1 gene in whole blood was amplified using primers EMA1-F (SEQ ID NO. 3) and EMA1-R (SEQ ID NO. 4). BamHI restriction enzyme sites were added to primer EMA1-F, and NotI restriction enzyme sites were added to primer EMA1-R. The amplified product and the pET-30a vector (purchased from Novagen) were double-digested with restriction enzymes BamHI and NotI, respectively. The digested products were then ligated using DNA ligase to construct the recombinant prokaryotic expression plasmid pET-30a-EMA1, which was transformed into Rosetta (DE3) competent cells. The plasmid in the transformed cells was amplified using the inherent primers on the pET-30a vector; the length was correct, verifying successful transformation.

[0111] II. Protein Expression

[0112] The highest soluble expression level was achieved at an induction temperature of 25℃, an IPTG concentration of 0.5 mM, and an induction time of 8 h. The protein was purified using a Ni-NTA agarose gel pre-packed column to obtain purified recombinant EMA1 protein. The purified protein can be used for animal immunization to prepare monoclonal antibodies.

[0113] Example 2: Preparation and Characterization of Monoclonal Antibodies

[0114] I. Inoculation of mice

[0115] The purified recombinant EMA1 protein solution (1 mg / ml, solvent: sterile PBS (0.01 mol / L, pH 7.4)) prepared in Example 1 was mixed with an equal volume of Freund's complete adjuvant and emulsified to obtain composition 1.

[0116] The purified recombinant EMA1 protein solution (1 mg / ml, solvent: sterile PBS (0.01 mol / L, pH 7.4)) prepared in Example 1 was mixed with an equal volume of Freund's incomplete adjuvant and emulsified to obtain composition 2.

[0117] Four 6-week-old female BALB / c mice were subcutaneously immunized with Composition 1 at a dose of 0.1 ml per mouse.

[0118] Two weeks later, each mouse underwent two booster immunizations subcutaneously with Composition 2 at a dose of 0.1 ml of Composition 2 per mouse, with the same dose administered two weeks apart. One week after the second booster immunization, blood was collected from each mouse by tail amputation, serum was separated, and serum antibody titers were detected using an indirect ELISA method (microplate-coated EMA1 protein). The mouse with the highest titer was selected for hybridoma preparation.

[0119] The specific steps of the indirect ELISA method are as follows:

[0120] The EMA1 protein used in the indirect ELISA was prepared in Example 1.

[0121] The EMA1 antibody production effect was determined using an indirect ELISA antibody detection method to assess the reactivity of the antibody with the EMA1 protein. The detection procedure is as follows:

[0122] 1. Coating: The EMA1 protein antigen prepared in Example 1 of this invention was diluted to 1 μg / ml with antigen coating buffer (carbonate buffer, pH 8.0), added to a 96-well plate, 100 µl / well, and coated overnight at 4°C.

[0123] 2. Washing: Wash 5 times with 300µl / well of PBST (phosphate-buffered saline containing 0.05v / v% Tween 20, pH 7.4, the same below), 1 minute each time.

[0124] 3. Blocking: Block with 5 w / v% (5 g / 100 ml) skim milk (solvent is PBS), 200 µl / well, incubate at 37°C for 2 hours.

[0125] 4. Washing: Same as 2.

[0126] 5. Add antibody: Dilute each sample of mouse serum to be tested 1:100 with PBS, add 100 µl / well to a 96-well plate, and incubate at 37°C for 2 hours.

[0127] 6. Washing: Same as 2.

[0128] 7. Enzyme-labeled antibody: Dilute the enzyme-labeled antibody (horseradish peroxidase-labeled goat anti-mouse IgG antibody, concentration 0.5 mg / ml, purchased from KPL Company, catalog number 5450-0011) with PBS at a volume ratio of 1:10000, 100 µl / well, and incubate at 37°C for 45 minutes.

[0129] 8. Washing: Same as 2.

[0130] 9. Color development: Add 50µl of TMB substrate color development solution (purchased from Ingenic) to each well and react at 37°C in the dark for 10 minutes.

[0131] 10. Termination: Add 50µl of stop solution (2M H2SO4) to each well.

[0132] 11. Reading: OD measurement should be completed within 5 minutes of termination. 450nm value.

[0133] Calculate the S / N value using the following formula: S / N value = sample OD 450nm / Negative control mean OD 450nm .

[0134] The criteria for judgment are: an S / N value higher than 2.1 is considered positive, and a value lower than 2.1 is considered negative.

[0135] II. Hybridoma Preparation and Antibody Screening

[0136] Mice with the highest serum titers after immunization were used to fuse hybridoma cells. Seven days after fusion, monoclonal cell lines were screened using an indirect ELISA method (the difference being that in step 5, the hybridoma supernatant was diluted 1:1; other procedures were the same as in step 1). OD200 cells were selected. 450nm A well containing a single cell cluster with a high concentration of HT culture medium was added to the well. After cell counting, the cells were aspirated and seeded into a 96-well plate. After three subclonings, the cells were expanded and cultured. The hybridoma cells that secreted EMA1 monoclonal antibodies were named 1A18, and the monoclonal antibody secreted by the hybridoma was named 1A18.

[0137] Five mice were taken and injected intraperitoneally with 500 μL of Freund's incomplete adjuvant per mouse; 3 days later, they were injected with 1×10 6 One A18 monoclonal cell line was collected; after 7-10 days, ascites fluid was collected, centrifuged (10000 rpm, 15 min), and filtered. The ascites fluid was purified according to the Protein L HP instructions: 5-fold dilution with PBS, equilibrated with binding buffer, 5 mL of the sample was loaded, eluted, and stored in 20% ethanol.

[0138] III. Monoclonal Antibody Typing

[0139] The monoclonal antibody 1A18 was identified by typing using a mouse immunoglobulin typing kit (supplier: Southern Biotech, catalog number 5300-05). The results showed that the heavy chain type of the monoclonal antibody was IgG1 and the light chain type was Kappa chain.

[0140] IV. Monoclonal Antibody Gene Sequencing

[0141] Total RNA was extracted from hybridoma cell line 1A18, and cDNA was obtained by reverse transcription. The variable regions (VH) and (VL) of the heavy and light chains of monoclonal antibody 1A18 were amplified using RACE technology, cloned into sequencing vectors, and sequenced. Full-length nucleic acid and amino acid sequences of the variable heavy and light chains, including CDR and framework regions, were obtained. The FR and CDR domains were analyzed using the IMGT method.

[0142] The nucleotide coding sequence of the variable region of the heavy chain of monoclonal antibody 1A18 is shown in SEQ ID NO.5.

[0143] Among them, FR1 is located at positions 1-75; CDR1 is located at positions 76-99; FR2 is located at positions 100-150; CDR2 is located at positions 151-174; FR3 is located at positions 175-288; CDR3 is located at positions 289-321; and FR4 is located at positions 322-354.

[0144] The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 1A18 is shown in SEQ ID NO.6.

[0145] Among them, FR1 is located at positions 1-25; CDR1 is located at positions 26-33; FR2 is located at positions 34-50; CDR2 is located at positions 51-58; FR3 is located at positions 59-96; CDR3 is located at positions 97-107; and FR4 is located at positions 108-118.

[0146] The nucleotide coding sequence of the light chain variable region of monoclonal antibody 1A18 is shown in SEQ ID NO.7.

[0147] Among them, FR1 is located at positions 1-78; CDR1 is located at positions 79-96; FR2 is located at positions 97-147; CDR2 is located at positions 148-156; FR3 is located at positions 157-264; CDR3 is located at positions 265-291; and FR4 is located at positions 292-321.

[0148] The amino acid sequence of the variable region of the light chain of monoclonal antibody 1A18 is shown in SEQ ID NO.8.

[0149] Among them, FR1 is located at positions 1-26; CDR1 is located at positions 27-32; FR2 is located at positions 33-49; CDR2 is located at positions 50-52; FR3 is located at positions 53-88; CDR3 is located at positions 89-97; and FR4 is located at positions 98-107.

[0150] Example 3: Establishment of a Competitive ELISA Model

[0151] To evaluate the application prospects of monoclonal antibody 1A18 as an EMA1 antibody detection reagent, a cELISA method based on 1A18 was established. Three samples of horse serum positive for *Theileria equine* and five samples of horse serum negative for *Theileria equine* antibody were selected to determine the performance of EMA1 detection and establish the cELISA method.

[0152] The EMA1 antibody blocking rate was determined using a cELISA antibody detection method for EMA1 protein. The detection procedure is as follows:

[0153] 1. Coating: Dilute the EMA1 protein antigen prepared in Example 1 to 1 μg / ml with antigen coating buffer (carbonate buffer, pH 8.0), add 100 µl / well to a 96-well plate, and coat overnight at 4°C.

[0154] 2. Washing: Wash 5 times with 300µl / well of PBST (phosphate-buffered saline containing 0.05v / v% Tween-20, pH 7.4, the same below), 1 minute each time.

[0155] 3. Blocking: Block with 5 w / v% (5 g / 100 ml) BSA (solvent is PBS), 200 µl / well, incubate at 37 °C for 2 hours.

[0156] 4. Washing: Same as 2.

[0157] 5. Competitive incubation: Dilute the HRP-labeled 1A18 monoclonal antibody (1 mg / ml) at a volume ratio of 1:1000 with PBS, and then use this as a diluent to dilute each sample of serum to be tested at a volume ratio of 1:1. After thorough mixing, add 100 μl / well to a 96-well plate and incubate at 37°C for 45 minutes.

[0158] 6. Washing: Same as 2.

[0159] 7. Color development: Add 100 μl of TMB substrate color development solution (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., catalog number TMB-S-004) to each well for color development, and react at 37℃ in the dark for 10 minutes.

[0160] 8. Termination: Add 50µl of stop solution (2M H2SO4) to each well.

[0161] 9. Reading: OD measurement should be completed within 5 minutes of termination. 450nm value.

[0162] The inhibition rate % is calculated using the following formula: [(mean OD of negative control)] 450nm Value - Sample OD 450nm Value) × 100% / Negative control mean OD 450nm value].

[0163] The experimental results show that 1A18 can be competitively bound to horse serum positive for Theileria equine, and exhibits a high inhibition rate.

[0164] Using the established detection method, 200 clinical horse serum samples, confirmed by the commercially available VMRD Theileria equine cELISA antibody detection kit, were tested. The final cut-off value for this method was determined to be 40%. See attached results. Figure 1 .

[0165] Example 4: Competitive ELISA Specific Detection

[0166] The specificity of the cELISA constructed in Example 3 was evaluated using positive sera against several other major equine diseases. Using the conditions described in Example 3, equine serum positive for Theileria equine, Equine Infectious Anemia Virus (EIAV), Equine Arteritis Virus (EAV), Equine Herpesvirus (EHV-4), Equine Influenza Virus (EIV), Streptococcus equi (S. equi), and Salmonella abortus (S. abortus equi) were used as primary antibodies for detection, with parallel detection conditions. The detection results are shown below. Figure 2 Therefore, it can be seen that all serum test results other than those of horse serum that tested positive for Tylenolium equine were negative.

[0167] The results showed that the 1A18-based cELISA had good specificity for the diagnosis of *Ethylethia equina* and no cross-reactivity with positive sera from other pathogens.

[0168] Example 5: Competitive ELISA Sensitivity Detection

[0169] To evaluate the detection sensitivity of the competitive ELISA (cELISA) method based on the EMA1 antibody of *Ethylethia equina* constructed using the 1A18 monoclonal antibody, serial dilution was used to detect *Ethylethia equina*-positive clinical serum samples confirmed by the gold standard.

[0170] Following the optimal cELISA reaction conditions established in Example 3, *Ethylethia equina* positive serum was serially diluted 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, with three replicates for each dilution. Negative serum controls and blank controls were also included, and all detection conditions were kept consistent. The detection results are as follows: Figure 3 As shown, the inhibition rate gradually decreased with increasing serum dilution. When the serum was diluted to 1:32, the inhibition rate was still higher than the critical value (40%); when diluted to 1:64 or higher, the inhibition rate was lower than the critical value, and the result was considered negative.

[0171] The results showed that the cELISA method based on 1A18 established in this study had a limit of detection of 1:32 dilution for positive clinical serum of *Ethylethia equina*, and had good detection sensitivity, which could meet the detection needs of low-level antibodies in clinical samples.

[0172] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A biomaterial, said biomaterial being any one of P1, P3, P4, P5, P6, P7, P8 and P9; P1: Monoclonal antibody The monoclonal antibody maintains specific binding activity to the extracellular domain of merozoon surface protein 1 of *Ethylene equina* or to *Ethylene equina* merozoon surface protein 1. The monoclonal antibody includes a monoclonal antibody heavy chain and a monoclonal antibody light chain; The monoclonal antibody heavy chain includes heavy chain FR1, heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3 and heavy chain FR4; The monoclonal antibody light chain includes light chain FR1, light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3 and light chain FR4; The amino acid sequence of the heavy chain CDR1 is shown in positions 26-33 of SEQ ID NO. 6; The amino acid sequence of the heavy chain CDR2 is shown in positions 51-58 of SEQ ID NO. 6; The amino acid sequence of the heavy chain CDR3 is shown in positions 97-107 of SEQ ID NO. 6; The amino acid sequence of the light chain CDR1 is shown in positions 27-32 of SEQ ID NO. 8; The amino acid sequence of the light chain CDR2 is shown in positions 50-52 of SEQ ID NO. 8; The amino acid sequence of the light chain CDR3 is shown in positions 89-97 of SEQ ID NO. 8; P3: Antibody derivatives The antibody derivative maintains specific binding activity to the extracellular domain of merozoite surface protein 1 or to merozoite surface protein 1 of *E. equisetifolia*. The antibody derivative is selected from: the monoclonal antibody described in fluorescently labeled P1; The monoclonal antibody described in P1 labeled with a chemiluminescent enzyme; P4: RNA assembly The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA; The monoclonal antibody heavy chain RNA can be translated to the monoclonal antibody heavy chain described in P1; The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1; P5: Gene Combination The gene combination includes monoclonal antibody heavy chain genes and monoclonal antibody light chain genes; The coding sequence of the monoclonal antibody heavy chain gene can encode the monoclonal antibody heavy chain described in P1; The coding sequence of the monoclonal antibody light chain gene can encode the monoclonal antibody light chain described in P1; P6: Gene Engineering Vector The genetic engineering vector is a combination of a monoclonal antibody heavy chain genetic engineering vector and a monoclonal antibody light chain genetic engineering vector, or a monoclonal antibody genetic engineering vector. The monoclonal antibody heavy chain gene engineering vector encodes the monoclonal antibody heavy chain RNA that can be expressed in P4; The monoclonal antibody light chain gene engineering vector encodes the monoclonal antibody light chain RNA that can be expressed in P4; The monoclonal antibody gene engineering vector encodes the monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA that can be expressed in P4; P7: Cells The cells contain the monoclonal antibody heavy chain gene engineering vector and the monoclonal antibody light chain gene engineering vector described in P6; or The cells contain the monoclonal antibody gene engineering vector described in P6; P8: Reagent Kit The kit contains the monoclonal antibody described in P1, the antibody derivative described in P3, the gene engineering vector described in P6, or the cells described in P7; P9: Composition The composition contains the monoclonal antibody described in P1, the antibody derivative described in P3, the genetic engineering vector described in P6, or the cell described in P7.

2. The biomaterial as described in claim 1, characterized in that, The monoclonal antibody heavy chain includes the monoclonal antibody heavy chain described in P1 and a tag peptide and / or signal peptide for separating and purifying the protein. The monoclonal antibody light chain comprises the monoclonal antibody light chain described in P1 and a tag peptide and / or signal peptide for separating and purifying the protein.

3. The biomaterial as described in claim 1, characterized in that, The amino acid sequence of the extracellular domain is shown in SEQ ID NO. 2; The surface protein 1 of the merozoite of *Ethylophora equina* contains a peptide with the amino acid sequence shown in SEQ ID NO.

2. The amino acid sequence of the variable region of the monoclonal antibody heavy chain is shown in SEQ ID NO. 6; The amino acid sequence of the variable region of the monoclonal antibody light chain is shown in SEQ ID NO. 8; or In the monoclonal antibody, the heavy chain subtype is IgG1, and the light chain subtype is Kappa.

4. The use of the monoclonal antibody or antibody derivative according to any one of claims 1-3 in the preparation of a formulation for recognizing a first biological material; The first biological material is selected from: The extracellular domain of equinetella merozoon surface protein 1 expressed in prokaryotes or eukaryotes; Fusion proteins expressed in prokaryotes or eukaryotes that contain a protein tag for protein isolation and purification and the extracellular domain of Matheylorhizium merozoon surface protein 1; Prokaryotic, eukaryotic, or extracted from *Ethylethia equina* cells, *Ethylethia equina* merozoon surface protein 1; A prokaryotic or eukaryotic fusion protein containing a protein tag for protein isolation and purification and *Ethylethia mustiole* merozoon surface protein 1; and Cells of *Ethylethia equinoides* containing merozoite surface protein 1, their lysates, or their inactivated products.

5. The application as described in claim 4, characterized in that, The amino acid sequence of the extracellular domain is shown in SEQ ID NO.2; or The merozoite surface protein 1 of *Ethylethia esculenta* contains a peptide with the amino acid sequence shown in SEQ ID NO.

2.

6. A kit comprising the antibody derivative and ELISA plate according to any one of claims 1-3; The reaction wells of the ELISA plate are coated with the first protein; The first protein is selected from: The extracellular domain of equinetella merozoon surface protein 1 expressed in prokaryotes or eukaryotes; Fusion proteins expressed in prokaryotes or eukaryotes that contain a protein tag for protein isolation and purification and the extracellular domain of Matheylorhizium merozoon surface protein 1; Prokaryotically expressed, eukaryotically expressed, or extracted from *Ethylethia equina* cells, *Ethylethia equina* merozoon surface protein 1; and A fusion protein expressed in prokaryotes or eukaryotes containing a protein tag for protein isolation and purification and a merozoan surface protein 1 from *Tetraylus equina*.

7. The kit according to claim 6, characterized in that, The amino acid sequence of the extracellular domain is shown in SEQ ID NO.2; The *Ethylophora equina* merozoite surface protein 1 contains a peptide with the amino acid sequence shown in SEQ ID NO. 2; or The antibody derivative is a horseradish peroxidase-labeled monoclonal antibody.

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