Claims:1. An isolated monoclonal antibody that specifically binds TACP protein or an antigenic fragment thereof , comprising :
a. A variable heavy chain (VH) complementarity determining region 1 (CDR1) region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 1 or 4 and a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 2 or 5, and a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 3 or 6, and a variable light chain (VL) CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 7; a VL CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 8, a VL CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 9; or
b. A VH CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 13 or 16; a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 14 or 17, a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 15 or 18, and a VL CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 19; a light chain CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 20, a light chain CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 21.

2. The monoclonal antibody as claimed in claim 1a, wherein the heavy chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 10 or 11.

3. The monoclonal antibody as claimed in claim 1a, wherein the light chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 12.

4. The monoclonal antibody as claimed in claim 1b, wherein the heavy chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 22 or 23.

5. The monoclonal antibody as claimed in claim 1b, wherein the light chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 24.

6. The monoclonal antibody as claimed in claim1, wherein it is a recombinant monoclonal antibody.

7. A method of detecting TACP protein or an antigenic fragment thereof, in a sample , the method comprising the steps of contacting the sample with the monoclonal antibody as claimed in claim 1.

8. A kit for detecting the TACP protein or an antigenic fragment thereof, in a sample , the kit comprising at least one monoclonal antibody or antigen - binding fragment of the monoclonal antibody as claimed in claim 1.

9. The kit according to claim 8, wherein the kit is a sandwich assay kit.

10. The kit according to claim 8, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

11. A method of detecting the TACP protein in a sample , the method comprising the step of contacting the monoclonal antibody as claimed in claim 1 with the sample, followed by detection of the antibody bound to the TACP protein or fragment thereof.
, Description:FIELD OF INVENTION
The current invention relates to monoclonal antibodies directed to Theileria annulata cysteine proteinase (TACP), production thereof, and immunoassay for detecting TACP. The present invention relates to methods and compositions for recombinant expression of Theileria annulata cysteine proteinase, monoclonal antibodies directed to it, and a kit for detecting Theileria annulata using these monoclonal antibodies.

BACKGROUND
Tropical theileriosis (Mediterranean coast fever) is an endemic disease of cattle in Mediterranean basin and parts of Asia. Piroplasmid parasite Theileria annulata is a Theileriosis disease causative agent in cattle and buffaloes in India and other tropical countries. Theileria annulata infects monocytes/macrophages and B lymphocytes, and causes severe lymphoproliferative disease in ruminants. Moreover, infection by T. annulata leads to the permanent proliferation of cell population through deregulating signalling pathways of host cells. It manifests severely in cross bred cattle and pure breed Bos taurus while the desi indigenous cattle and buffaloes do not exhibit clinical symptoms though infected. Cross breeding programsare being undertaken in India to increase the milk production by crossing the indigenous cattle with the Bos taurus Jersey &Holstein-Friesian. Theileriosis is an intracellular protozoan parasite transmitted by ticks of genus Hyalomma. The disease has severe adverse effects on the infected cattle, as compared to other diseases caused by tick born parasites, such as Babesiosis, Anaplasmosis and it causes huge economic losses per annum to the rural farmers due to reduced milk yield and high cost of treatment of animals infected with the disease. The clinical symptoms of the disease include high temperature, anaemia, depression, lacrimation, salivation, nasal discharge, swelling of superficial lymphnodes and sometimes diarrhoea. Buparvaquone is the only anti-theilerial drug used for treatment of the Theileriosis, but it is quite expensive, and can be a drain on earnings of low to middle income farmers. Theileriosis is fatal to young calves and the adult cattle also succumb to infection if the disease is not diagnosed and treated in a timely manner.
The symptomatic clinical diagnosis, smear based microscopic test and nucleotide specific PCR based protocol that are regularly used for Theileria detection are reliable. Diagnosis of Theileriosis by field veterinarians is usually done by observing the clinical symptoms which are mostly misleading. In the laboratory confirmative diagnosis is carried out by observing piroplasms in RBCs and Koch’sBlue Bodies in the lymphocytes. Most of the times developmental stages are not found in the blood smear of the animals suffering from chronic disease. Moreover, it requires skilled laboratory personnel to detect these stages in the blood smear.
Theileria annulata cysteine proteinase (TACP) is the most common pathogenic protease of C1A family involved in apicomplexan life cycle processes such as host cell invasion, parasite nutrition, intracellular hemoglobin degradation and cytoskeletal rupture. The antigen-antibody reaction process is of interest for the detection and diagnosis of Theileriosis in cattle and buffaloes. Recombinant expression and purification of TACP in E.coli was done to achieve generation of highly specific and sensitive antibodies towards T.annulata. Furthermore, the antibody production was done against this antigen for detection and diagnosis of Theileriosis. TACP protein of Theileria annulata is not a very well studied protein, and hence its characteristics are not well understood. We are the first to purify and isolate this recombinant enzyme from Theileria annulata. The protein expression level was too low as compare to other papain-like cysteine protease family. Non specific degradation was seen while expression and purification. The protease stability was the other issue during raising of antibody. The aim of the current invention is to generate highly specific antibodies towards T.annulata TACP to enable detection of this parasite in any sample.
The current invention is directed to monoclonal antibodies that bind to Theileria annulata cysteine proteinase with high affinity, and diagnostic kits for detecting infection in cattle using these antibodies/ kits. These antibodiesare generated using mouse immunization experiments conducted with the recombinant expression of Theileria annulata cysteine proteinase.

SUMMARY

The invention is to be understood as not being limited to the particular embodiments described herein.
The current invention encompasses a highly specific and sensitive monoclonal antibody specific to TACP which contains heavy chain (VH) and light chain (VL) variable domain and heavy chain (CH) and light chain (CL) constant domain.
In one embodiment, the current invention encompasses at least one monoclonal antibody directed to Theileria annulata cysteine proteinase (TACP).
In one embodiment, the present invention encompasses at least one monoclonal antibody which binds Theileria annulata cysteine proteinase. In one embodiment, the at least one monoclonal antibody binds TACP with nanomolar affinity. In one embodiment, the titre of the antibodies disclosed herein is 3.2ng/ml.
An isolated monoclonal antibody that specifically binds TACP protein or an antigenic fragment thereof , comprising :
A variable heavy chain (VH) complementarity determining region 1 (CDR1) region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 1 or 4 and a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 2 or 5, and a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 3 or 6, and a variable light chain (VL) CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 7; a VL CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 8, a VL CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 9; In one embodiment, the heavy chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 10 or 11, when the variable heavy chain (VH) complementarity determining region 1 (CDR1) region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 1 or 4 and a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 2 or 5, and a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 3 or 6, and a variable light chain (VL) CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 7; a VL CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 8, a VL CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 9;
In one embodiment, the light chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 12, when the variable heavy chain (VH) complementarity determining region 1 (CDR1) region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 1 or 4 and a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 2 or 5, and a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 3 or 6, and a variable light chain (VL) CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 7; a VL CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 8, a VL CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 9;.
or the monoclonal antibody disclosed herein comprises:
A VH CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 13 or 16; a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 14 or 17, a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 15 or 18, and a VL CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 19; a light chain CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 20, a light chain CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 21.
In one embodiment, the heavy chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 22 or 23 when the VH CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 13 or 16; a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 14 or 17, a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 15 or 18, and a VL CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 19; a light chain CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 20, a light chain CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 21.

In one embodiment, the light chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 24 when the VH CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 13 or 16; a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 14 or 17, a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 15 or 18, and a VL CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 19; a light chain CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 20, a light chain CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 21.

In one embodiment, the monoclonal antibody as disclosed herein is a recombinant monoclonal antibody.
In one embodiment, the current invention encompasses a method of detecting TACP protein or an antigenic fragment thereof in a sample , the method comprising the steps of contacting the sample with the monoclonal antibody as disclosed herein.
In one embodiment, the current invention encompasses kit for detecting the TACP protein or an antigenic fragment thereof in a sample , the kit comprising at least one monoclonal antibody or antigen - binding fragment of the monoclonal antibody as claimed in claim 1.
In one embodiment, the kit is a sandwich assay kit.
In one embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.
In one embodiment, the current invention encompasses a method of detecting the TACP protein in a sample, the method comprising the step of contacting the monoclonal antibody disclosed herein with the sample, followed by detection of the antibody bound to the TACP protein or fragment thereof.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCES:
Fig. 1: shows the titre of the sera used for fusion. X-axis denotes optical density at 450 nm of the sera against the antigen, Y-axis denotes the dilution of sera.
Fig. 2: Fig. 2A shows sandwich assay done for detection of TACP where X-axis denotes the concentration of antigen in ng/ml quantity while the Y-axis is the signal produced in the ELISA.
Fig. 2B shows Sandwich ELISA for detection of TACP in cattle serum samples. Total 12 positive and 12 negative serum sample from cow assayed on Sandwich ELISA developed using C2 as capture Antibody and Rabbit polyclonal as detection antibody. The cut off value for positive sample is >0.4. X-axis is the sample while Y-axis is the signal of the sample at 450nm in spectrophotometer.
Fig. 3 shows western blot of animal (Theileria annulate +/-ve ) sera samples.
Fig. 4: Monoclonal antibody GNG-TACP-C2 and GNG- TACP-G6 cross-reactivity analysis with different proteins.
Fig. 5 shows TACP protein, 23kDa band.
Fig. 5B shows SDS-PAGE gel showing a single TACP 8kDa Band

DETAILED DESCRIPTION
The current invention encompasses monoclonal antibodies directed to Theileria annulata cysteine proteinase (TACP), production thereof and immunoassays for detecting TACP. The present invention relates to methods and compositions for recombinant expression of Theileria annulata cysteine proteinase, monoclonal antibodies directed to it and diagnosis of Theileriosis disease in a rapid and sensitive manner.
Piroplasmids are tick-transmitted obligatory hemoprotozoan parasites including species of the genus Babesia, Theileria, and Cytauxzoon. Many species are of veterinary significance, either due to their negative economic impact on livestock industries or due to the diseases they cause in pets and occasionally in wild life . Examples of the former group include Babesia bovis and/or B. bigemina causing bovine babesiosis of cattle; Theileria annulata, Theileria parva, and Theileria orientalis that cause tropical theileriosis, East Coast Fever, and oriental theileriosis of cattle, respectively; and Babesia ovis causing ovine babesiosis of sheep.

DEFINITIONS
Theileria annulata cysteine proteinase (TACP): C1A cysteine-proteinases (C1A-Cp) are papain-like enzymes implicated in pathogenic and vital steps of the parasite life cycle such as nutrition and host cell egress. The sequence of the 23kDa portion of the TACP protein is given in SEQ ID NO:33.
TACP herein includes the entire protein (SEQ ID NO:31) as well as fragments or peptides derived from the protein, including the antigenic domain (23 kDa) of TACP enzyme(Protein accession no. P25781.2).
As used herein, “antibody” includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., dispecific antibodies). The term “antibody” also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')2, Fab, Fv and rIgG). The term also refers to recombinant single chain Fv fragments (scFv).
Also, the term “monoclonal antibody” as used herein, refers to an antibody molecule that has been obtained from a substantially identical antibody clone, which shows single-binding specificity and affinity for a specific antigen.

Typically, an immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region (the regions are also known as “domains”). Light chain and heavy chain variable regions contain three hypervariable regions called “complementarity-determining regions” or “CDRs” and four “framework” regions. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.

The term “sample” as used herein, may be a tissue, a cell, whole blood, serum, plasma, autoptical sample of tissue (brain, skin, lymph node, spinal cord), supernatant of cell culture, eukaryotic cell and bacterial expression system, but may not be limited to these examples. The presence of TACP or its reactive 23kDa antigen (SEQ ID NO: 33) can be detected by reacting the manipulated or non-manipulated sample with the monoclonal antibody of the present invention.

The “antigen-antibody complex” as used herein, refers to a complexes formed by binding of the TACP protein or its antigenic fragment to the monoclonal antibody described in the present invention. Formation of such antigen-antibody complex may be detected by a method selected from a group consisting of colorimetric method, electrochemical method, fluorometric method, luminometry, particle counting method, visual assessment and scintillation counting method. However, the method is not limited to the above examples and has a variety of applications.
Specific binding of a monoclonal antibody to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art.
Any technique known in literature to detect the amount of bound monoclonal antibody or fragment can be used in the current invention. For instance, the detection can be direct or indirect, and may generate a fluorescent or chromogenic signal. Direct detection typically involves the use of a monoclonal antibody or fragment that is conjugated to a label. Indirect detection utilizes a labelled secondary antibody raised against the host species of the monoclonal antibody or fragment.
Commonly used labels for visualization of binding of antibody to epitope includes fluorophores and enzymes that convert soluble substrates into chromogenic end products.

As used herein, the term “contacting” refers to the introduction of a sample putatively containing TACP protein to a monoclonal antibody described herein, for example, by combining or mixing the sample with the TACP protein-binding compound. When the TACP protein is present in the sample, a TACP-antibody complex is then formed; such complex formation refers to the ability of an anti-TACP antibody to selectively bind to the TACP protein in order to form a stable complex that can be detected. Detection can be qualitative, quantitative, or semi-quantitative. Binding TACP protein in the sample to the anti-TACP antibody is accomplished under conditions suitable to form a complex. Such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art. Binding can be measured using a variety of methods standard in the art including, but not limited to, enzyme immunoassays (e.g., ELISA), immunoprecipitations, immunoblot assays and other immunoassays.

Embodiments:
The invention is to be understood as not being limited to particular embodiments described.
The current invention encompasses a highly specific and sensitive monoclonal antibody specific to TACP which contains heavy chain (VH) and light chain (VL) variable domain and heavy chain (CH) and light chain (CL) constant domain.
In one embodiment, the current invention encompasses at least one monoclonal antibody directed to Theileria annulata cysteine proteinase (TACP).
In one embodiment, the present invention encompasses at least one monoclonal antibody which binds Theileria annulata cysteine proteinase. In one embodiment, the at least one monoclonal antibody binds TACP with nanomolar affinity.
An isolated monoclonal antibody that specifically binds TACP protein or an antigenic fragment thereof, comprising:
A variable heavy chain (VH) complementarity determining region 1 (CDR1) region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 1 or 4 and a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 2 or 5, and a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 3 or 6, and a variable light chain (VL) CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 7; a VL CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 8, a VL CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 9; In one embodiment, the heavy chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 10 or 11, when the variable heavy chain (VH) complementarity determining region 1 (CDR1) region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 1 or 4 and a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 2 or 5, and a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 3 or 6, and a variable light chain (VL) CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 7; a VL CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 8, a VL CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 9;
In one embodiment, the light chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 12, when the variable heavy chain (VH) complementarity determining region 1 (CDR1) region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 1 or 4 and a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 2 or 5, and a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 3 or 6, and a variable light chain (VL) CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 7; a VL CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 8, a VL CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 9;.
or the monoclonal antibody disclosed herein comprises:
A VH CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 13 or 16; a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 14 or 17, a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 15 or 18, and a VL CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 19; a light chain CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 20, a light chain CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 21.
In one embodiment, the heavy chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 22 or 23 when the VH CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 13 or 16; a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 14 or 17, a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 15 or 18, and a VL CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 19; a light chain CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 20, a light chain CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 21.
.
In one embodiment, the light chain variable domain comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 24 when the VH CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 13 or 16; a VH CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 14 or 17, a VH CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 15 or 18, and a VL CDR1 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 19; a light chain CDR2 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 20, a light chain CDR3 region comprising an amino acid sequence with at least 98% identity to SEQ ID NO: 21.
.
In one embodiment, the monoclonal antibody as disclosed herein is a recombinant monoclonal antibody.
In one embodiment, the current invention encompasses a method of detecting TACP protein or an antigenic fragment thereof in a sample , the method comprising the steps of contacting the sample with the monoclonal antibody as disclosed herein.
In one embodiment, the current invention encompasses kit for detecting the TACP protein or an antigenic fragment thereof in a sample , the kit comprising at least one monoclonal antibody or antigen - binding fragment of the monoclonal antibody as claimed in claim 1.
In one embodiment, the kit is a sandwich assay kit.
In one embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.
In one embodiment, the current invention encompasses a method of detecting the TACP protein in a sample, the method comprising the step of contacting the monoclonal antibody disclosed herein with the sample, followed by detection of the antibody bound to the TACP protein or fragment thereof.
In one embodiment , the affinity of the antibodies disclosed herein was calculated based on titre of antibody which was about 3.2 ng/ml.
In one embodiment, the antibodies disclosed herein can detect as less as 3.2ng/ml of protein in a sample.
In one embodiment, the monoclonal antibodies disclosed herein are showing no reactivity to other protein. Hence, they are very high specificity.
A kit for detecting the TACP protein or an antigenic fragment thereof, the kit comprising at least one monoclonal antibody or antigen - binding fragment of claim 1.
In one embodiment, the current invention encompasses a method of diagnosing Theileria annulata in a rapid, specific and sensitive way. In one embodiment, the method of diagnosis encompasses the use of at least one highly specific monoclonal antibody that binds to TACP.
In one embodiment, the antibodyis bound to a label.
In one embodiment, the label is a radioisotope, bead, a ligand, a chemiluminescent molecule, a fluorescent molecule, or an enzyme.
In one embodiment, the antibody is bound to a therapeutic compound.

In one embodiment, the current invention encompasses a diagnostic kit for diagnosing infection by Theileriaannulata, wherein the kit comprises at least one TACP specific monoclonal antibody, and at least one reagent for detecting the monoclonal antibody bound to TACP. The reagent that detects the monoclonal antibody bound to TACP may be detectable by further binding to another reagent, or may be detected by any detectable reaction itself. The detectable reaction may be any reaction, examples of which include, but are not limited to, colorimetric, luminescent, photometric, andradioactive reaction.
In one embodiment, the diagnostic method disclosed herein provides a protein based method, which has high accuracy, high Sensitivity and Specificity. In one embodiment, the method of diagnosis disclosed herein is rapid, and decreases frequent veterinarian visits. It is cost effective, and decreases the mortality rate of animals because of early and accurate detection.
In one embodiment, the current invention encompasses a method of identifying a Theileria annulata infection by detecting any developmental stage of Theileria annulata, or a Theileria annulata antigen in a sample, the method comprising the steps of: (a) contacting the sample with at least one monoclonal antibody that binds immunologically to Theileria annulata antigen, and (b) determining binding of the monoclonal antibody to a Theileria annulata antigen in the sample, whereby binding of the monoclonal antibody indicates the presence of Theileria annulata antigen. The sample may be obtained from any animal. The sample may more specifically be obtained from a ruminant animal. The sample may be a tissue sample, a fluid sample or a fecal sample. The tissue sample may be from brain, spinal cord, placenta, lung, liver, muscle, connective tissue, vascular endothelium or gastrointestinal tract. The fluid sample may be from blood, serum, plasma, urine, milk, ascites, cerebrospinal fluid or foetal fluid.
The assay format may be a Western blot, a radioimmunoprecipitation, RIA, or an ELISA, including a sandwich ELISA. The method may employ a solid support such as a column, a dipstick, a filter or a microtiter dish. The ELISA may comprise detection of bound monoclonal antibody using a labelled anti-Ig antibody. The ELISA also may be a competitive assay. The assay also may involve quantification. The assay may further comprise determining antigenic profile of the Theileria annulata.
In one embodiment, the at least one anti- TACP antibody disclosed herein is raised by using the recombinant TACP antigen. In one embodiment, the monoclonal antibody is detectable by ELISA.
The monoclonal antibody of the present invention includes the antibody being produced by hybridoma and also any cell comprising the encoding gene of the antibody. In one embodiment, the current invention also encompasses the nucleotide sequence encoding the monoclonal antibody disclosed herein.
In one embodiment, antibodies of the present invention may be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.
Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as “antibody-directed imaging.” Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.
In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
One embodiment of the invention is a method of producing monoclonal antibodies against TACP. In one embodiment of the invention, TACP was recombinantly expressed in E.coli. In one embodiment of the invention, a high antigenic domain of TACP was recombinantly expressed. In one embodiment of the invention, a high antigenic domain of TACP (23kDa) with Protein accession no. P25781.2(SEQ ID NO: 33) was recombinantly expressed. In one embodiment of the invention, recombinant TACP produced was used for immunization to raise antibodies against TACP. In one embodiment of the invention, recombinant TACP produced was used for immunization of mice.
In one embodiment, A method of the present invention can be generally accomplished by:
(a) obtaining a sample from an animal; and
(b) detecting presence or absence of TACP protein in the sample
In one embodiment, the kit disclosed herein detects TACP protein in a sample using an assay selected from the group consisting of an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral-flow assay, a dipstick assay, an agglutination assay, a particulate-based assay, an immunoprecipitation assay, an immunodot assay, an immunoblot assay, an immunodiffusion assay, a phosphorescence assay, a flow-through assay, a chromatography assay, a PAGe-based assay, an electronic-sensory assay, a surface plasmon resonance assay and a fluorescence correlation spectroscopy assay.
Examples:
Example 1:
The customized truncated protein clones were developed by cloning in pET28 vectorand expressed in E.colion the basis of high to low antigenicity of the Theileriaannulata cysteine proteinase protein (TACP). Polyclonal sera were raised in mice against purified cysteine proteinases macromolecules. Standardized hybridoma technology platform of GNG (Genext Genomics, SOP: GNG_HYBRIDOMA_003_V5) was used for monoclonal antibody production and purification. The hybridoma platform is commonly used though for immunization we used phenylalanine and leucine also for obtaining functional Antibody.

Highly antigenic domain (23 kDa) of TACP enzyme was recombinantly expressed in E. coli using synthetic protein sequence given in SEQ ID NO: 33 (Fig. 5A-); Protein accession no. P25781.2). Briefly, C-terminal of protein sequence was embedded with six histidine tag. Expressed protein was purified by Ni-NTA chromatography. Phenylalanine and leucine at micromolar quantity mixed with purified protein and was used for generating antibodies.
Immunization:
One or more immunization schedules was used to generate the anti-TACP hybridomas. The first several fusions can be performed after the following exemplary immunization protocol, but other similar known protocols can be used. Several 14-20 week old female or male mice are immunized Intraperiotoneal (IP) or Intradermal (ID) and/or with 10 mg of recombinant TACP (23kDa, SEQ ID NO: 33) with 250µg pf phenylalanine and 200µg of leucine and antigen was aliquoted according to the immunization protocol and emulsified with anequal volume of complete Freund's adjuvant in a final volume of 100-400µl.

Each mouse can also optionally receive 25µg antigen. The mice in our experiment received 2µg of protein/ antigen. The mice were then be immunized 1-7, 5-12,10-18, 17-25 and/or 21-34 days later IP and finally 10µg of IV. Mice can be bled 12-25 and 25-40 days later by retro-orbital puncture without anti-coagulant. The blood was then allowed to clot at RT (room temperature) for one hour and the serum was collected and tittered using an TACP ELISA assay according to known methods. Fusions are performed when repeated injections do not cause titers to increase. At that time, the mice were given a final IV booster injection of 10µg TACP diluted in 50µl physiological saline. Three days later, the mice were euthanized by cervical dislocation and the spleens removed aseptically and immersed in 10 ml of Media (DMEM). The splenocytes were harvested by sterilely perfusing the spleen using homogenizer in a sterilized environment. The cells were washed thrice and counted using Trypan blue dye exclusion and resuspended in DMEM media containing 25 mM HEPES.

Cell Fusion:
Fusion was carried out at a 1:1to 1:10 ratio of murine myeloma cells to viable spleen cells according to known methods, e.g., as known in the art. As a non-limiting example, spleen cells and myeloma cells were collected washed and pelleted together. The pellet was then be slowly resuspended, over 60 seconds, in 1ml of 50% (w/v) PEG solution (Sigma) at 37ºC. The fusion was then be stopped by slowly adding 10.5 ml of DMEM Glutamax media (GIBCO) containing 25 mM HEPES (37ºC) over1 minute. The fused cells were centrifuged for 5 minutes at 500-1500 rpm. The cells were then resuspended in HAT medium, 1mM sodium pyruvate, 4 mM Glutamine, and then plated at 100 µl/well in fifteen 96-well flatbottom tissue culture plates. The plates were then placed in a humidified 37ºC incubator containing 5% CO2 for 7-10 days.

The fused cells (hybridomas) or recombinant cells were isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods.
Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

Example 2:
Detection of Mouse IgG Anti-TACP Antibodies in Mouse Serum
ELISA was used to screen mouse sera for mouse IgG antibodies specific for human TACP. Briefly, plates can be coated with TACP at 2 µg/ml in PBS overnight. After washing in PBST, the wells were blocked with 2% (w/v) BSA in PBS, 200 µl/well for 1 hour at RT. Plates wereused immediately or kept at 4ºC for future use. Mouse serum dilutions were incubated on the TACP coated plates at 100µl/well at RT for 1 hour. The plates were washed and then probed with 100µl/well HRP-labeled goat anti-Mouse IgG, Fc specific diluted 1:10,000 in 1X PBST for 1 hour at RT. The plates were again washed and 50 µl/well of TMB (3,3',5,5'-Tetramethylbenzidine) was added for 15 minutes at RT. Stop solution (4N sulfuric acid) was then added at 50µL/well and the OD's were read at 450 nm via an automated plate spectrophotometer.

Fig. 1 shows the titre of the sera used for fusion. X-axis denotes optical density at 450nm of the sera against the antigen, Y-axis denotes the dilution of sera.

Example 3: Determination of Anti-TACP antibody Reactivity

Hybridomas, generated as described above, were simultaneously assayed for reactivity to TACP, 23kDa (SEQ ID NO: 26) using ELISA. 50µl of Hybridoma supernatant was used to screen the positive antibodies clones. The resulting clonal populations can be expanded and cryopreserved in freezing medium (95% FBS, 5% DMSO) and stored in liquidnitrogen. Briefly, 96 well Nunc maxisorp plate were coated with 1µg/ml of recombinant TACP Protein in PBS buffer overnight at 4ºC and used for indirect ELISA. Next day, the plate was washed and blocked with 2% BSA prepared in 1X PBST and incubated for 1hour at 37ºC. 50µl supernatant from hybridoma were added to each well and incubated for 1hour at 37ºC. The plates were washed and anti-mouse HRPO (Horse Raddish peroxidase) conjugate was added at the 1: 10000 dilution and incubated for 1hour as the previous condition. The plate was finally washed and the substrate TMB (3,3',5,5'-Tetramethylbenzidine) was added to each well for 15min at room temperature. Stop Solution (1N HCl) was added at 50µl and OD was read at 450nm.Mouse anti-TACP antibody secreting hybridomas were expanded in cell culture and serially subcloned by limiting dilution.
Example 4: Selection of Clones and Subcloning:
Fusion of Anti-TACP splenocyte with myeloma cell line with current protocol produced total seven growth positive hybridomas with a cut off selection of OD >1.0. One of the clones was selected for further subcloning twice with limiting dilution (0.5cells/Well) to obtain stable cell line. Cell line name was given as 10G12 which was a positional name as per the plate map.10G12 clone was single cell diluted by limiting dilution methodin 96 well plate and the plate was screened after a week when50% confluency of the cells was reached. After subcloning of 10G12, the following clones were identified and found positive for TACP.We generated seven monoclonal antibodies of widerange of specificities against TACP protein after two round of sub cloning of 10G12 clone as mentioned in Table 1.
Table 1 depicts the selected clones from the subcloning of Hybridoma. Supernatant were added to recombinant TACP (antigen) coated at 1ug/mL followed by Goat Anti-Mouse Antibody.
Table 1
S.No Name of the Clone O.D (optical density)
1 GNG-TaCP-D12 1.6427
2 GNG-TaCP-A-F 1.623
3* GNG-TaCP-C2* 1.8813
4 GNG-TaCP-B12 1.496
5* GNG-TaCP-G6* 1.634
6 GNG-TaCP-A8 1.7622
7 GNG-TaCP-B10 1.5571
8. Positive Control
(Fusion Sera 1:1000) 3.5755

DNA SEQUENCE ANALYSIS
As a first step, C2 and G6 mAbs (monoclonal antibodies) that showed reactivity to TACP were scaled up and total RNA was isolated from the two hybridomas cell line that produce these mAbs. Each RNA sample was then used to prepare mouse Antibody heavy or light chain cDNA that included complete signal sequence, complete variable region sequence and part of the constant region sequence for each mAb. These DNA products were then amplified in PCR reaction and PCR amplified fragment was directly sequenced without first cloning the fragment. The heavy chain sequences were >90% identical to one of the five mouse germline gene . Similarly, the light chain cDNA sequences were either 100% or 98% identical to one of the mouse germline gene. These sequence results confirmed that the RNA molecules were transcribed into cDNA and sequenced encoded mouse antibody heavy and light chains. It should be noted that, because the variable regions were PCR-amplified using oligonucleotides that map to the 5'end of the signal sequence coding sequence, the first few amino acids of the signal sequence may not be the actual sequence of the original translation products but they do represent the actual sequences of the recombinant C2 and G6 mAbs.

Two antibody clones were scaled up (GNG-TaCP-C2, GNG-TaCP-G6, marked by asterisk in above Table 1 ) for production and purification of antibodies. Sequence of two selected antibodies was retrieved using sequencing with VH and VL primers. (Mouse IgG Library Primer Set, Progen cat: F2010).

Table 2: Total Primer tested from Degenerate primer list

Process step GNG-TACP-10G12.C2 GNG-TACP-10G12.G6
Light
Chain Heavy
Chain Light
chain Heavy
Chain
RNA extraction from hybridoma pellet v v
Reverse transcription v v v v
PCRs : No. of Direct primer tested
No. of effective primer pairs 16
2 16
2 16
2 16
2
Sequence Analysis : No. of different candidate sequences
2
2
2
2

No. of positive candidates (1)
1
1 1 2
length of positive cloned fragment 365 bp
121 aa 479 bp
159 aa 365 bp
121 aa 429 bp
143 aa

Sequences (10G12.C2 and 10G12.G6) CDR denoted with underlined region

The original clone was TACP-G6 and when we amplified the gene from Mouse Ab- Library set, we used 11 degenerative primers which were numbered from A-M, hence G6 when amplified and given positive sequence with 1L primer then it was called G6-1L and if it gave a positive sequence from 1H then it is called G6-1H. Most of the primers amplified the same sequence. The primers used were from Mouse IgG Library Primer Set from Progen cat: F2010. The primer names are given below.
These primers are commercially supplied by Progen, and are PCR oligonucleotide primer sets consisting of 25 primers for amplification and 25 primers for cloning of mouse IgG heavy and light chain variable domain coding regions for generating universal or antibody-specific scFv phage-display libraries (Reference (2) Zhou et al 888-889 Nucleic Acids Research, 1994, Vol. 22, No. 5)
Table 3 gives the sequences of the CDRs and the variable heavy and light chain regions. The CDRs are underlined for each VH and VL domain.

Table 3
Chain Clone ID/ primer Nucleotide amino acid
G6-VH1 G6-1H

SEQ ID NO: 25 SEQ ID NO: 1: G6-VH1-CDR1
SEQ ID NO: 2: G6-VH1-CDR2
SEQ ID NO: 3: G6-VH1-CDR3
SEQ ID NO: 10: G6-VH1

G6-VH2 G6-1L
SEQ ID NO: 26
SEQ ID NO: 4: G6-VH2-CDR1
SEQ ID NO: 5: G6-VH2-CDR2
SEQ ID NO: 6: G6-VH1-CDR3
SEQ ID NO: 10: G6-VH1

G6-VL G6-1O
SEQ ID NO: 27

SEQ ID NO: 7: G6-VL-CDR1
SEQ ID NO: 8: G6-VL-CDR2
SEQ ID NO: 9: G6-VL-CDR3
SEQ ID NO: 12: G6-VL

C2-VH1 C2 -L SEQ ID NO: 28 SEQ ID NO: 13: C2-VH1-CDR1
SEQ ID NO: 14: C2-VH1-CDR2
SEQ ID NO: 15: C2-VH1-CDR3
SEQ ID NO: 22: C2-VH1

C2-VH2 C2-H SEQ ID NO: 29 SEQ ID NO: 16: C2-VH2-CDR1
SEQ ID NO: 17: C2-VH2-CDR2
SEQ ID NO: 18: C2-VH2-CDR3
SEQ ID NO: 22: C2-VH2

C2-VL C2-1W SEQ ID NO: 30
SEQ ID NO: 19: C2-VH2-CDR1
SEQ ID NO: 20: C2-VH2-CDR2
SEQ ID NO: 21: C2-VH2-CDR3
SEQ ID NO: 24: C2-VH2

Example 5: Development of Anti-TACP Polyclonal Antibody in Rabbit
Immunization :
Rabbit Polyclonal antibody was developed to standardize sandwich ELISA assay in order to detect antigen. One or more immunization schedules was used to generate the anti-TACP Rabbit polyclonal antibody. TACP protein was injected to primed Rabbit at the concentration of 400µg/dose. Primary Dose and four booster doses were given. At each booster, the titre was measured. Blood sample was withdrawn at the titre 1: 754000. Rabbit Polyclonal Anti-TACP Antibody was isolated using Protein A Affinity Chromatography. The Antibody titre was checked and used as detection antibody in Sandwich ELISA.

Example 6: Sandwich Assay
Development of Sandwich ELISA for detection of recombinant TACP.
The monoclonal Antibody C2 was purified and coated into 96 well (Maxisorp, Nunc) plate at the concentration of 5-10µg/ml in bicarbonate buffer and incubated at 4ºC overnight. The plate is washed the next day with PBS and blocked with 2% BSA prepared in 1XPBST. Blocking is done at 37ºC for 1 hr and then the plate is washed. Antigen (TACP) is prepared in dose dependent dilution from 3µg/ml-0.003µg/ml and added on the wells. The unbound antigen is washed and detection antibody which is Rabbit polyclonal Antibody s added to each well. The plate was then subjected to TMB substrate and test was read by adding stop solution (1N HCl) at 450nm.
Table 4: Detection of TACP protein in the sandwich ELISA developed using C2 Antibody and Rabbit Polyclonal Antibody as sandwich pair.
TACP in ng/ml Antigen Detection Negative control
3200 2 0.2806
320 1.819 0.1864
32 1.1892 0.2017
3.2 0.6529 0.1179
0.32 0.6716 0.1249
0.032 0.6026 0.1263
0.0032 0.603 0.1296
0 0.3596 0.0633

The coating antibody used is C2 clone purified antibody while detection antibody used is the rabbit polyclonal antibody against TACP and affinity purified against the Antigen. The Antigen used was in dose dependent manner starting from 3.2?g/ml to 0.0032 ng/ml. Negative control included the wells without coating of capture antibody. The negative control defines that the Sandwich ELISA developed using C2 monoclonal Antibody is able to detect the antigen as low as 3.2pg/ml of the antigen. ELISA test results were considered positive when the optical density was > 0.1 over the negative control.
The affinity was calculated based on titre of antibody which is around 3.2 ng/ml.
The antibodies disclosed herein can detect as less as 3.2ng/ml of protein in a sample. (the data as given in figures).
As can be seen from the cross-reactivity experiment, (western as well as cross reactivity experiment) the monoclonal antibodies disclosed herein are showing no reactivity to other protein. Hence, they are very high specificity.
Fig. 2A shows sandwich assay done for detection of TACP where X-axis denotes the concentration of antigen in ng/ml quantity while the Y-axis is the signal produced in the ELISA.
Example7: Animal sera assays
Bovine cattle blood samples were collected from across Vidarbha region as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines, Ministry of Fisheries, Animal Husbandry and Dairying (MoFAH&D) constituted under the Prevention of Cruelty to Animals (PCA) Act, 1960
(244/GO/ReBi-S/Re-L/2000/CPCSEA). Half of the sample poured in heparin containing tube and other half allowed to clot for separation of serum. Clinical symptoms, microscopic smear analysis and PCR using gene specific primers as a gold standard were used for to confirm the Theileriosis diseased animals. Blood sample was used for to isolate DNA and PCR. Same animal serum sample was used for sandwich ELISA (Fig. 2B: Sandwich assay for detection of TACP in serum samples). There were total 24 serum samples as equal number for positive and negative assayed by above confirmatory test. Optical density 0.4 at 450 nm was kept as a cutoff value. Only two positive samples shown to be negative in ELISA out of 12 samples. The sensitivity and specificity calculated as 83.3 % and 100 % respectively (Ref 1).

Fig. 2B shows Sandwich ELISA for detection of TACP in cattle serum samples. Total 12 positive and 12 negative serum sample from cow assayed on Sandwich ELISA developed using C2 as capture Antibody and Rabbit polyclonal as detection antibody. The cut off value for positive sample is >0.4. X-axis is the sample while Y-axis is the signal of the sample at 450nm in spectrophotometer.

TACP full length protein sequence was retrieved from NCBI Accession: AAA30135 (SEQ ID NO: 31).
The monoclonal antibodies produced by the method given in the current disclosure are novel and specifically recognized by the recombinant TACP (228-437 fragment, SEQ ID NO:33) as well as in blood/sera samples.
Fig. 3 shows western blot of animal (Theileria annulate +/-ve ) sera samples.
TACP detection of 2 representative positive samples and 3 representative negative samples. The purified r-TACP protein along with sera was immunoblotted onto a nitrocellulose membrane. C2 purified antibody was used as primary antibody, followed by a peroxidase-conjugated IgG secondary antibody. DAB was used as the horseradish peroxidase substrate, and the membrane was developed for 30 s. The arrow indicates the location of the TACP protein in sera sample.
Fig. 4 shows Monoclonal antibody GNG-TACP-C2 and GNG- TACP-G6 cross-reactivity analysis with different proteins.

Five recombinant proteins including TACP 23kDa Fragment protein ( SEQ ID NO: 33) were used for cross reactivity ELISA assay. MTB 51D (Mycobacterium tuberculosis PepA (protease), ESPS 46 (erythrose 4-phosphate and phosphoenolpyruvate of Agrobacterium ) and STAT 63 (STAT3 protein of Human) recombinant protein did not match substantial nucleotide or amino acid identity with TACP 23 protein. TACP 8 (8kDa fragment ; SEQ ID NO: 34) was the other domain of TACP full length protein. Clone C2 antibody was used as the primary antibody followed by anti-mouse antibody HRPO conjugate. The plate was developed using TMB substrate. There was no cross reactivity of antibody against the different target antigen.

Reference:
1) MedCalc Software Ltd. Diagnostic test evaluation calculator.(Version 20.009; accessed August 19, 2021)
2) Zhou et al Optimization of primer sequences for mouse scFv repertoire display library construction888-889 Nucleic Acids Research, 1994, Vol. 22, No. 5