US20050118569A1 - Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to venezuelan equine encephalitis virus - Google Patents

Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to venezuelan equine encephalitis virus Download PDF

Info

Publication number
US20050118569A1
US20050118569A1 US10/784,305 US78430504A US2005118569A1 US 20050118569 A1 US20050118569 A1 US 20050118569A1 US 78430504 A US78430504 A US 78430504A US 2005118569 A1 US2005118569 A1 US 2005118569A1
Authority
US
United States
Prior art keywords
scfv
sbp
fusion protein
tagged
vee
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
US10/784,305
Other versions
US7527796B2 (en
Inventor
R. Fulton
Leslie Nagata
Azhar Alvi
Weigang Hu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Minister of National Defence of Canada
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US10/784,305 priority Critical patent/US7527796B2/en
Assigned to HER MAJESTY THE QUEEN IN RIGHT OF CANADA, AS REPRESENTED BY THE MINSTER OF NATIONAL DEFENCE reassignment HER MAJESTY THE QUEEN IN RIGHT OF CANADA, AS REPRESENTED BY THE MINSTER OF NATIONAL DEFENCE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FULTON, R. ELAINE, HU, WEIGANG, NAGATA, LESLIE P., ALVI, AZHAR Z.
Publication of US20050118569A1 publication Critical patent/US20050118569A1/en
Application granted granted Critical
Publication of US7527796B2 publication Critical patent/US7527796B2/en
Expired - Fee Related legal-status Critical Current
Adjusted expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag

Definitions

  • This invention relates to the construction of a recombinant gene encoding a single-chain variable fragment antibody cloned into an expression vector and fused with a streptavidin-binding peptide sequence to produce a fusion protein.
  • the resultant fusion protein can be used as reagent for immunoassay of Venezuelan equine encephalitis virus when detected by horseradish peroxidase-conjugated streptavidin.
  • This invention is related to U.S. Provisional Patent Application No. 60/361,698 filed by Fulton et al., the same inventors and assignee, which is herein incorporated by reference.
  • VEE Venezuelan equine encephalitis virus
  • Togaviridae Venezuelan equine encephalitis virus
  • VEE causes a spectrum of human diseases ranging from inapparent infection to acute encephalitis (Franck et al; 1970; Johnson et al., 1968). Since the VEE genome is composed of positive sense RNA, its nucleic acid is infectious independent of the complete viral particle (Johnston et al., 1996). Furthermore, VEE is highly infectious by aerosol inhalation in humans (Johnston et al., 1996). Thus, VEE is a potential biological warfare and bioterrorist agent of concern. Therefore, simple, stable, and efficient immunoassays are required for rapid identification of VEE in environmental or clinical samples in order that immediate therapeutic and preventive counter measures can be taken to limit the epidemic spread of VEE infection.
  • the present inventors have previously cloned and characterized several single-chain variable fragment antibodies (scFv Abs) against VEE (Alvi et al., 1999; Alvi et al., 2002; Alvi et al., 2003). Among them, mA116 scFv Ab was well characterized, showing sensitivity and specificity in recognition of VEE by immunoassay (Alvi et al., 2003). In order to further explore the potentiality of mA116 scFv Ab as an immunodiagnostic reagent for detecting VEE, the present inventors successfully fused a streptavidin-binding peptide (SBP) to mA116 scFv Ab by DNA recombinant technique.
  • SBP streptavidin-binding peptide
  • SBP streptavidin-binding peptide
  • it provides a method for constructing a recombinant gene encoding a single-chain variable fragment antibody cloned into an expression vector and fused with a SBP gene sequence to produce a fusion protein, comprising: (a) encoding anti-VEE scFv Ab gene to a recombinant plasmid and inserting a SBP gene and a 6His tag downstream to develop a SBP tagged scFv Ab construct; (b) amplifying the resultant scFv/SBP/6His by polymerase chain reaction; (c) inserting the amplified PCR products into cloning vector to produce a SBP-plasmid; (d) constructing said SBP-plasmid with promote
  • the genetically recombinant SBP tagged mA116 scFv Ab can be used as an excellent reagent for detecting VEE by means of immunoassay.
  • it provides a method for using the SBP tagged recombinant scFv Ab fusion protein of claim 4 for detecting VEE, comprising: (a) reacting the SBP tagged scFv Ab with a sample containing VEE for observing antigen-binding activity; and (b) analyzing the reactant by enzyme-linked immunosorbent assay (ELISA).
  • FIG. 1 is a schematic diagram showing the construction of pCRT7mA116SBP.
  • Step 1 A single double-stranded oligonucleotide encoding SBP and 6His flanked by Not I and Sal I sticky ends was ligated into pPICZ ⁇ BmA116 digested with NotI/Sal I.
  • Step 2 scFv/SBP/6His tag sequence was amplified from pPICZ ⁇ BmA116SBP vector by PCR.
  • Step 3 PCR product was ligated into pCRT7 TA cloning vector as described under “Materials and Methods”.
  • FIG. 2 shows the nucleotide and deduced amino acid sequences (SEQ ID NOs. 1 and 2 respectively) of SBP tagged mA116 Ab. MA116 scFv followed by SBP and 6His tags.
  • FIG. 3 shows SDS-PAGE analysis of samples from the purification of the SBP tagged mA116 scFv Ab. Samples were resolved on 10% polyacrylamide gel and stained with Coomassie blue. Lane 1—molecular weight markers; Lane 2—bacterial lysate; Lane 3—solubilized protein fraction; Lane 4—column flow through fraction; Lane 5—purified fraction.
  • FIGS. 4A & 4B show Western blotting analysis of samples from the purification of the SBP tagged mA116 scFv Ab. Samples were resolved by SDS-PAGE, transferred to Immunobilon-P membranes, and probed with: FIG. 4A : HRP-conjugated Ni—NTA. FIG. 4B : HRP-conjugated streptavidin. Lane 1—bacterial lysate; Lane 2—solubilized protein fraction; Lane 3—column flow through fraction; Lane 4—purified fraction.
  • FIGS. 5A & 5B are graphs showing VEE antigen binding assay by ELISA.
  • FIG. 5A Various concentrations of Abs were added to 96-well plate coated with 10 ⁇ g/ml of VEE;
  • FIG. 5B 10 ⁇ g/ml of Abs were added to a 96-well plate coated with various concentrations of VEE. Binding was detected with HRP-conjugated streptavidin or HRP-conjugated anti-mouse Ig followed by ABTS solution. Each point represents the mean ⁇ the standard error of the mean (SEM) of the four determinations.
  • SEM standard error of the mean
  • the pPICZ ⁇ BmA116 recombinant plasmid containing anti-VEE mA116 scFv Ab gene, arranged in variable heavy (VH)-variable light (VL) chain orientation via (Gly 4 Ser) 3 linker, was constructed previously.
  • PCHPQFPRCYA SEQ ID NO. 3
  • 6His tag at the C-terminus of mA116 scFv Ab
  • two complementary oligonucleotides corresponding to the SBP sequence and 6His tag with flanking sequences for restriction enzymes Not I and Sal I were synthesized and purified by Life Technologies (Burlington, ON).
  • sequences were as follows: sense, 5′-ggccgcCCATTCTGGTGGTGGTGGCCCATGCCATCCGC AGTTCCCACGATGTTATGCGGGTGGTGGCGGTTCTCATCATCATCATCAT TGAg-3′ (SEQ ID NO. 4); anti-sense, tcgacTCAATGATGATGATGATGATGAGAAC CGCCACCACCCGCATAACATCGTGGGAACTGCGGATGGCATGGGCCACCACC ACCAGAATGGgc-3′ (SEQ ID NO. 5).
  • the two oligonucleotides were heated to denature, and then annealed to a single double-stranded oligonucleotide by slow cooling to room temperature.
  • the annealed dimer possessed a Not I sticky end on one side and Sal I on the other side, and was ligated to pPICZA ⁇ BmA116 that had been cut with Not I and Sal I.
  • the resulting plasmid was named pPICZ ⁇ BmA116SBP.
  • the PCR method was introduced to amplify the mA116 scFv/SBP/6His sequence in pPICZ ⁇ BmA116SBP vector and the PCR product was subcloned into a T7 RNA polymerase-regulated expression vector.
  • Two primers were synthesized on an Oligo 1000 DNA synthesizer (Beckman Instruments, Fullerton, Calif.). The sequence of the forward primer was 5′-ATGGCTAAAGAAGAAGGGGTATC-3′ (SEQ ID NO. 6) and the reverse was 5′-TCATGTCTAAGGCTACAAACTCAA-3′ (SEQ ID NO. 7).
  • PCR reaction in a 50 ⁇ l volume consisted typically of 200 ⁇ mol each dNTP, 0.6 ⁇ M primers, 0.1 ⁇ g template, and 1.25 unit of HotStarTaqTM DNA polymerase in buffer supplied by the manufacturer (Qiagen, Mississauga, ON). Initial activation (95° C. for 15 min) was carried out followed by cycling (94° C. for 1 min, 61° C. for 1 min, and 72° C. for 2 min), repeated 30 times, on a Peltier Thermal Cycler (DNA Engine PTC-200; MJ Research, Watertown, Mass.).
  • pCRT7mA116SBP contained the correct orientation of the insert, mA116 scFv/SBP/6His tag, as confirmed by restriction digestion fragment mapping and DNA sequencing.
  • the cell pellet was resuspended in 5 mM borate sodium, pH 9.3 and 4 M urea, and cell lysate was prepared by sonication (three cycles of 10 sec; amplitude 10 ⁇ m; 15 sec cooling on ice), using a MSE Soniprep 150-probe sonicator (Wolf Laboratories, Pocklington, UK).
  • the sonicates were centrifuged (13,000 g for 10 min) and pellets were resuspended in 5 mM borate sodium, pH 9.3, 8 mM urea, and 100 mM sodium chloride (solubilizing agent).
  • Proteins were separated by 10% SDS-PAGE gels by use of a Mini-PROTEAN II apparatus (Bio-Rad Laboratories). The bands were visualized by Coomassie blue staining. The molecular weights of the samples were estimated by comparison to the relative mobility values of standards of known molecular weights.
  • Blots were washed three times for 5 min with PBS containing 0.1% tween-20 (PBST) and then incubated directly with a 1:1000 dilution of HRP-conjugated streptavidin (Sigma) or a 1:2000 dilution of HRP-conjugated Ni-nitrilotriacetic acid (NTA) (Qiagen) at room temperature for 1 hr. After three washes for 5 min with PBST, and two washes for 2 min with deionized water, the specific binding was detected by an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Baie d'Urfe, QC).
  • HRP-conjugated streptavidin Sigma
  • NTA Ni-nitrilotriacetic acid
  • the antigen-binding activity of the purified SBP tagged mA116 scFv Ab to VEE antigen was determined by an ELISA.
  • Nunc maxisorpTM flat-bottomed 96-well plates (Life Technologies) were coated overnight at 4° C. with whole VEE (strain TC-83) at a fixed concentration of 10 ⁇ g/ml, or various concentrations of 0.2-60 ⁇ g/ml, in carbonate bicarbonate buffer, pH 9.6, containing 0.02% sodium azide.
  • the plates were washed five times with PBST and then blocked twice in 2% bovine serum albumin for 1 hr at 37° C. After five washes with PBST, plates were incubated for 1 hr at 37° C.
  • the pPICZ ⁇ BmA116 recombinant plasmid, encoding anti-VEE mA116 scFv Ab gene was used as a source material to create the SBP tagged mA116 scFv Ab construct.
  • a synthetic double-stranded oligonucleotide encoding a SBP and 6His tag was inserted downstream to the pPICZ ⁇ BmA116, mA116 scFv/SBP/6His was amplified using PCR method with appropriate primers.
  • the PCR product was inserted into pCRT7 TA cloning vector.
  • the resulting plasmid designated pCRT7mA116SBP, contained the mA116 scFv Ab gene, followed by the SBP sequence under the control of T7 promoter ( FIG. 1 ).
  • the SBP sequence under the control of T7 promoter ( FIG. 1 ).
  • the nucleotide and deduced amino acid sequences are showed in FIG. 2 .
  • the encoded whole recombinant fusion protein was 296 residues with a predicted molecular weight of 31.3 kDa.
  • the SBP tagged mA116 scFv Ab was expressed in E.coli BL-21 cells as inclusion bodies and purified by IMAC.
  • SDS-PAGE demonstrated that there was a relatively small amount of protein in the bacterial lysate of molecular weight ⁇ 32 kDa corresponding to the predicted size (31.3 kDa) of the SBP tagged mA116 scFv Ab, due to the presence of large amounts of contaminating proteins ( FIG. 3 , Lane 2).
  • FIG. 3 , Lane 3 shows that after centrifugation of the lysate, and dissolution of the pellet in solubilizing agent, many of the bacterial host proteins were removed from the lysate, making the ⁇ 32 kDa band more visible ( FIG. 3 , Lane 3).
  • the solubilized protein fraction was incubated with metal affinity resin and loaded to an empty column. After thoroughly washing with wash buffer, the bound fractions were eluted by 100 mM imidazole. Only one band of ⁇ 32 kDa was observed in the purified fraction ( FIG. 3 , lane 5). In this purification protocol, the expressed protein could be purified to over 90%.
  • the immunoreactivity of the SBP tagged mA116 scFv Ab to VEE antigen was examined by ELISA.
  • the SBP tagged mA116 scFv Ab bound to VEE in a dose-dependent manner, similar to the binding to VEE of its parental 1A4A1 MAb ( FIG. 5A ).
  • An additional ELISA was performed in which a concentration gradient of VEE was titrated against a fixed concentration of Abs (10 ⁇ g/ml). A similar dose-response relationship was observed ( FIG. 5B ).
  • streptavidin-biotin system has one of the highest affinities (10 ⁇ 15 M) among receptor-ligand interactions (Green, 1963).
  • the strong interaction between streptavidin and biotin has been applied in many immunoassays (Guesdon et al., 1979; Hsu et al., 1981).
  • chemical biotinylation of Ab is time-consuming and, as most of the biotin binds to amino groups of the protein, the degree of labeling can differ from batch to batch.
  • the biological activity of the Ab may be affected by the labeling procedure (Mirables et al., 1991). With advent of recombinant DNA technology, it is possible to fuse a short peptide to the target protein through gene fusion technique.
  • SBPs constituting around 10 amino acids have been selected from random peptide libraries (Devlin et al., 1990; Osterguard et al., 1995; Gissel et al., 1995). Some of them have been well characterized (Schmidt et al., 1996; Skewa et al., 1999). SBPs have successfully been fused to scFv Abs to antigen CA125, Bacillus cereus spores, and scorpion toxin for use in immunoassay (Luo et al., 1998 Kao et al., 1998 Aubrey et al., 2001).
  • the present inventors genetically incorporated a SBP sequence in mA116 scFv Ab gene in order to biotinylate mA116 scFv Ab.
  • DNA sequencing confirmed that DNA cloning was successful.
  • the SBP tagged mA I16 scFv Ab was expressed in E. coli to high levels in the form of insoluble inclusion bodies.
  • the insoluble recombinant fusion protein was solubilized by denaturing agent, 8M urea. Inclusion of 6His tag allowed the solubilized recombinant fusion protein to be purified via IMAC. In this way, greater than 90% purity of the SBP tagged recombinant mA116 scFv Ab could be obtained.
  • arginine was introduced to the recombinant protein solution to direct correct refolding.
  • the streptavidin-binding peptide confers reversible binding activity toward the streptavidin. Therefore, it can be employed for the one step purification of a corresponding fusion protein via streptavidin affinity chromatography (Schmidt et al., 1994; Zwicker et al., 1999).
  • streptavidin affinity chromatography Schowicker et al., 1999.
  • purification of the recombinant SBP tagged mA116 scFv Ab using streptavidin affinity column chromatography yielded only small amounts of product containing relatively large amounts of host proteins (data not shown).
  • a large amount of streptavidin-binding protein of around 20 kDa showed up in the bacterial lysate on Western blot analysis of bacterial lysate ( FIG. 4B ).
  • a SBP sequence was introduced downstream to the sequence for mA116 scFv Ab to VEE.
  • the fusion protein was expressed and purified. Fusion to the SBP did not affect the ability of mA116 scFv Ab to recognize VEE antigen with an affinity similar to that observed for the parental MAb. Similarly, the streptavidin-binding property of the fusion protein was not impaired.
  • Western blot and ELISA results suggest that SBP tagged mA116 scFv Ab could be used for simple, stable, and efficient detection of VEE when used in conjunction with HRP-conjugated streptavidin. This approach eliminates the need for chemical biotinylation of Abs with resultant possible impairment of the antigen-binding site of the Ab.

Abstract

A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) being cloned into a prokaryotic T7 RNA polymerase-regulated expression vector was disclosed. A streptavidin-binding peptide (SBP) sequence fused to a 6His tag is then attached downstream to the scFv gene. The recombinant fusion protein is expressed in bacteria and then purified by immobilized metal affinity chromatography. ELISA and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody, but also possessed streptavidin-binding activity. This discovery obviates the need for chemical biotinylation of antibodies and the risk associated with antibody denaturation and provides a stable and reproducible reagent for rapid and efficient immunoassay of VEE.

Description

  • This Application claims the benefit of U.S. Provisional Application No. 60/448,902, filed on Feb. 24, 2003, the entire content of which is incorporated by reference in this application.
    • FIELD OF THE INVENTION
  • This invention relates to the construction of a recombinant gene encoding a single-chain variable fragment antibody cloned into an expression vector and fused with a streptavidin-binding peptide sequence to produce a fusion protein. The resultant fusion protein can be used as reagent for immunoassay of Venezuelan equine encephalitis virus when detected by horseradish peroxidase-conjugated streptavidin. This invention is related to U.S. Provisional Patent Application No. 60/361,698 filed by Fulton et al., the same inventors and assignee, which is herein incorporated by reference.
    • BACKGROUND OF THE INVENTION
  • List of Prior Art Literatures
    • Johnston R E and Peters C J: Alphavirus. In: Fields virology, 3rd ed. Fields B N, Knipe D M and Howley P M (Eds.), Raven Publishers, Philadelphia, Pa., 1996, pp.843-898.
    • Franck P T and Johnson K M: An outbreak of Venezuelan encephalitis in man in the Panama Canal Zone. Am J Trop Med Hyg 1970;19:860-865.
    • Johnson K M, Shelokov A, Peralta P H, Dammin G J, and Young N A: Recovery of Venezuelan equine encephalomyelitis virus in Panama: a fatal case in man. Am J Trop Med Hyg 1968;17:432-440.
    • Alvi A Z, Stadnyk L L, Nagata L P, Fulton R E, Bader D E, Roehrig J T, and Suresh M R: Development of a functional monoclonal single-chain variable fragment antibody against Venezuelan equine encephalitis virus. Hybridoma 1999;18:413-421.
    • Alvi A Z, Fulton R E, Chau D, Suresh M R, and Nagata L P: Development of a second generation monoclonal single chain variable fragment antibody against Venezuelan equine encephalitis virus: expression and functional analysis. Hybridoma and Hybridomics 2002;21:169-178.
    • Alvi A Z, Hu W G, Fulton R E, Nagata L P, Coles J E, and Long M C: Functional enhancement of a partially active single chain variable fragment antibody to Venezuelan equine encephalitis virus. Viral Immunology 2003;16:213-222.
    • Luo D, Geng M, Schultes B, Ma J, Xu D Z, Hamza N, Qi W, Noujaim A A, and Madiyalakan R: Expression of a fusion protein of scFv-biotin mimetic peptide for immunoassay. J. Biotechnol 1998;65:225-228.
    • Long M C, Jager S, Mah D C, Jebailey L, Mah M A, Masri S A, and Nagata L P: Construction and characterization of a novel recombinant single-chain variable fragment antibody against Western equine encephalitis virus. Hybridoma 2000;19:1-13.
    • Nakamura R M: Monoclonal antibodies: methods and clinical laboratory applications. Clin Physiol Biochem 1983;1:160-172.
    • Huston J S, Levinson D, Mudgett-Hunter M, Tai M S, Novotny J, Margolies M N, Ridge R J, Bruccoleri R E, Haber E, and Crea R: Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA 1988;85:5879-5883.
    • Marin M, Brockly F, Noel D, Etienne-Julan M, Biard-Piechaczyk M, Hua T D, Gu Z J, and Piechaczyk M: Cloning and expression of a single-chain antibody fragment specific for a monomorphic determinant of class I molecules of the human major histocompatibility complex. Hybridoma 1995; 14:443-451.
    • Bruyns A M, De Jaeger G, De Neve M, De Wilde C, Van Montagu M, and Depicker A: Bacterial and plant-produced scFv proteins have similar antigen-binding properties. FEBS Lett 1996;386:5-10.
    • George A J, Jamar F, Tai M S, Heelan B T, Adams G P, McCartney J E, Houston L L, Weiner L M, Oppermann H, and Peters A M: Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide. Proc Natl Acad Sci USA 1995;92:8358-8362.
    • Boleti E, Deonarain M P, Spooner R A, Smith A J, Epenetos A A and George A J: Construction, expression and characterisation of a single chain anti-tumour antibody (scFv)-IL-2 fusion protein. Ann Oncol 1995;6:945-947.
    • Wels W, Harwerth I M, Mueller M, Groner B, and Hynes N E: Selective inhibition of tumor cell growth by a recombinant single-chain antibody-toxin specific for the erbB-2 receptor. Cancer Res 1992;52: 6310-6317.
    • Green N M: Avidin: 1. The use of 14C-biotin for kinetic studies and for assay. Biochem J 1963; 89:585-590.
    • Guesdon J L, Ternynck T, and Avrameas S: The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 1979;27:1131-1139.
    • Hsu S M, Raine L, and Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577-580.
    • Miralles F, Takeda Y, and Escribano M J: Comparison of carbohydrate and peptide biotinylation on the immunological activity of IgG1 murine monoclonal antibodies. J Immunol Methods 1991;140:191-196.
  • Devlin J J, Panganiban L C, and Devlin P E: Random peptide libraries: a source of specific protein binding molecules. Science 1990;249:404-406.
    • Ostergaard S, Hansen P H, Olsen M, and Holm A: Novel avidin and streptavidin binding sequences found in synthetic peptide libraries. FEBS Lett 1995;362:306-308.
    • Gissel B, Jensen M R, Gregorius K, Elsner H I, Svendsen I, and Mouritsen S: Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids. J Pept Sci 1995; 1:217-226.
    • Schmidt T G, Koepke J, Frank R, and Skerra A: Molecular interaction between the strep-tag affinity peptide and its cognate target, streptavidin. J Mol Biol 1996; 255:753-766.
    • Skerra A, and Schmidt T G: Applications of a peptide ligand for streptavidin: the strep-tag. Biomol Eng 1999;16:79-86.
    • Koo K, Foegeding P M, and Swaisgood H E: Construction and expression of a bifunctional single-chain antibody against Bacillus cereus spores. Appl Environ Microbiol 1998;64:2490-2496.
    • Aubrey N, Devaux C, di Luccio E, Goyffon M, Rochat H, and Billiald P: A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin Biol Chem 2001;382:1621-1628.
    • Schmidt T G and Skerra A: One-step affinity purification of bacterially produced proteins by means of the “strep tag” and immobilized recombinant core streptavidin. J Chromatogr A 1994;676:337-345.
    • Zwicker N, Adelhelm K, Thiericke R, Grabley S, and Hanel F: Strep-tag II for one-step affinity purification of active bHLHzip domain of humanc-myc. Biotechniques 1999;27:368-375.
    • Sutton M R, Fall R R, Nervi A M, and Alberts A W, Vagelos P R, and Bradshaw R A: Amino acid sequence of Escherichia coli biotin carboxyl carrier protein (9100). J Biol Chem 1977;252:3934-3940.
  • Venezuelan equine encephalitis virus (VEE), belonging to alphavirus genus of the family Togaviridae, is an important pathogen of epidemics in humans and of epizootics in some animals (Johnston et al., 1996). VEE causes a spectrum of human diseases ranging from inapparent infection to acute encephalitis (Franck et al; 1970; Johnson et al., 1968). Since the VEE genome is composed of positive sense RNA, its nucleic acid is infectious independent of the complete viral particle (Johnston et al., 1996). Furthermore, VEE is highly infectious by aerosol inhalation in humans (Johnston et al., 1996). Thus, VEE is a potential biological warfare and bioterrorist agent of concern. Therefore, simple, stable, and efficient immunoassays are required for rapid identification of VEE in environmental or clinical samples in order that immediate therapeutic and preventive counter measures can be taken to limit the epidemic spread of VEE infection.
  • The present inventors have previously cloned and characterized several single-chain variable fragment antibodies (scFv Abs) against VEE (Alvi et al., 1999; Alvi et al., 2002; Alvi et al., 2003). Among them, mA116 scFv Ab was well characterized, showing sensitivity and specificity in recognition of VEE by immunoassay (Alvi et al., 2003). In order to further explore the potentiality of mA116 scFv Ab as an immunodiagnostic reagent for detecting VEE, the present inventors successfully fused a streptavidin-binding peptide (SBP) to mA116 scFv Ab by DNA recombinant technique. This confers a streptavidin-binding function on the mA116 scFv Ab and therefore obviates the need for conventional chemical biotinylation. Chemical biotinylation is commonly associated with impairment of the antigen-binding site of the Ab and it is hence desirable to use the recombinant SBP tagged mA116 scFv of the present invention as reagent to develop a simple, stable and efficient immunoassay for VEE.
    • SUMMARY OF THE INVENTION
  • It is an object of the present invention to teach a method for constructing a streptavidin-binding peptide (SBP) to the sequence for mA I16 scFv Ab to VEE. According to one aspect of the present invention, it provides a method for constructing a recombinant gene encoding a single-chain variable fragment antibody cloned into an expression vector and fused with a SBP gene sequence to produce a fusion protein, comprising: (a) encoding anti-VEE scFv Ab gene to a recombinant plasmid and inserting a SBP gene and a 6His tag downstream to develop a SBP tagged scFv Ab construct; (b) amplifying the resultant scFv/SBP/6His by polymerase chain reaction; (c) inserting the amplified PCR products into cloning vector to produce a SBP-plasmid; (d) constructing said SBP-plasmid with promoter to produce a SBP tagged scFv Ab; and (e) expressing said SBP tagged scFv Ab in E. coli cells as inclusion bodies and purifying the expressed SBP tagged scFv Ab by immobilized metal affinity chromatography.
  • It is another object of the invention to demonstrate that the recombinant fusion protein retains antigen-binding affinity to VEE and possesses streptavidin-binding function. Hence, the genetically recombinant SBP tagged mA116 scFv Ab can be used as an excellent reagent for detecting VEE by means of immunoassay. According to another aspect of the present invention, it provides a method for using the SBP tagged recombinant scFv Ab fusion protein of claim 4 for detecting VEE, comprising: (a) reacting the SBP tagged scFv Ab with a sample containing VEE for observing antigen-binding activity; and (b) analyzing the reactant by enzyme-linked immunosorbent assay (ELISA).
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a schematic diagram showing the construction of pCRT7mA116SBP. Step 1—A single double-stranded oligonucleotide encoding SBP and 6His flanked by Not I and Sal I sticky ends was ligated into pPICZαBmA116 digested with NotI/Sal I. Step 2—scFv/SBP/6His tag sequence was amplified from pPICZαBmA116SBP vector by PCR. Step 3—PCR product was ligated into pCRT7 TA cloning vector as described under “Materials and Methods”.
  • FIG. 2 shows the nucleotide and deduced amino acid sequences (SEQ ID NOs. 1 and 2 respectively) of SBP tagged mA116 Ab. MA116 scFv followed by SBP and 6His tags.
  • FIG. 3 shows SDS-PAGE analysis of samples from the purification of the SBP tagged mA116 scFv Ab. Samples were resolved on 10% polyacrylamide gel and stained with Coomassie blue. Lane 1—molecular weight markers; Lane 2—bacterial lysate; Lane 3—solubilized protein fraction; Lane 4—column flow through fraction; Lane 5—purified fraction.
  • FIGS. 4A & 4B show Western blotting analysis of samples from the purification of the SBP tagged mA116 scFv Ab. Samples were resolved by SDS-PAGE, transferred to Immunobilon-P membranes, and probed with: FIG. 4A: HRP-conjugated Ni—NTA. FIG. 4B: HRP-conjugated streptavidin. Lane 1—bacterial lysate; Lane 2—solubilized protein fraction; Lane 3—column flow through fraction; Lane 4—purified fraction.
  • FIGS. 5A & 5B are graphs showing VEE antigen binding assay by ELISA. FIG. 5A: Various concentrations of Abs were added to 96-well plate coated with 10 μg/ml of VEE; FIG. 5B: 10 μg/ml of Abs were added to a 96-well plate coated with various concentrations of VEE. Binding was detected with HRP-conjugated streptavidin or HRP-conjugated anti-mouse Ig followed by ABTS solution. Each point represents the mean ± the standard error of the mean (SEM) of the four determinations.
    • DETAILED DESCRIPTION OF THE INVENTION
  • Materials and Methods
  • Construction of pCRT7mA116SBP
  • The pPICZαBmA116 recombinant plasmid, containing anti-VEE mA116 scFv Ab gene, arranged in variable heavy (VH)-variable light (VL) chain orientation via (Gly4Ser)3 linker, was constructed previously. In order to introduce a SBP sequence, PCHPQFPRCYA (SEQ ID NO. 3) (Lue et at., 1998) followed by a 6His tag at the C-terminus of mA116 scFv Ab, two complementary oligonucleotides corresponding to the SBP sequence and 6His tag with flanking sequences for restriction enzymes Not I and Sal I, were synthesized and purified by Life Technologies (Burlington, ON). The sequences were as follows: sense, 5′-ggccgcCCATTCTGGTGGTGGTGGCCCATGCCATCCGC AGTTCCCACGATGTTATGCGGGTGGTGGCGGTTCTCATCATCATCATCATCAT TGAg-3′ (SEQ ID NO. 4); anti-sense, tcgacTCAATGATGATGATGATGATGAGAAC CGCCACCACCCGCATAACATCGTGGGAACTGCGGATGGCATGGGCCACCACC ACCAGAATGGgc-3′ (SEQ ID NO. 5). The two oligonucleotides were heated to denature, and then annealed to a single double-stranded oligonucleotide by slow cooling to room temperature. The annealed dimer possessed a Not I sticky end on one side and Sal I on the other side, and was ligated to pPICZAαBmA116 that had been cut with Not I and Sal I. The resulting plasmid was named pPICZαBmA116SBP.
  • To obtain high expression of the recombinant fusion protein, the PCR method was introduced to amplify the mA116 scFv/SBP/6His sequence in pPICZαBmA116SBP vector and the PCR product was subcloned into a T7 RNA polymerase-regulated expression vector. Two primers were synthesized on an Oligo 1000 DNA synthesizer (Beckman Instruments, Fullerton, Calif.). The sequence of the forward primer was 5′-ATGGCTAAAGAAGAAGGGGTATC-3′ (SEQ ID NO. 6) and the reverse was 5′-TCATGTCTAAGGCTACAAACTCAA-3′ (SEQ ID NO. 7). PCR reaction in a 50 μl volume consisted typically of 200 μmol each dNTP, 0.6 μM primers, 0.1 μg template, and 1.25 unit of HotStarTaq™ DNA polymerase in buffer supplied by the manufacturer (Qiagen, Mississauga, ON). Initial activation (95° C. for 15 min) was carried out followed by cycling (94° C. for 1 min, 61° C. for 1 min, and 72° C. for 2 min), repeated 30 times, on a Peltier Thermal Cycler (DNA Engine PTC-200; MJ Research, Watertown, Mass.). After gel-purification, the PCR fragment was cloned into the pCRT7 vector by use of a pCRT7 TA cloning expression kit in accordance with the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). The recombinant plasmid, named pCRT7mA116SBP, contained the correct orientation of the insert, mA116 scFv/SBP/6His tag, as confirmed by restriction digestion fragment mapping and DNA sequencing.
  • Expression, Purification and Refolding of the SBP Tagged mA116 scFv Ab
  • Expression, purification and refolding of the SBP tagged mA116 scFv Ab were performed using minor modifications of previously described methodologies (Long et al., 2000). In brief, an overnight culture of E. coli BL-21 (DE3) pLys cells that had been transformed with pCRT7mA116SBP vector, was diluted 1:50 with LB-medium containing 100 μg/ml ampicillin and incubated with shaking at 37° C. to OD600 of 0.5. The promoter was then induced for 3 hr by isopropyl β-D-thiogalactoside (IPTG). The cell pellet was resuspended in 5 mM borate sodium, pH 9.3 and 4 M urea, and cell lysate was prepared by sonication (three cycles of 10 sec; amplitude 10 μm; 15 sec cooling on ice), using a MSE Soniprep 150-probe sonicator (Wolf Laboratories, Pocklington, UK). The sonicates were centrifuged (13,000 g for 10 min) and pellets were resuspended in 5 mM borate sodium, pH 9.3, 8 mM urea, and 100 mM sodium chloride (solubilizing agent). Purification of the recombinant protein was performed on Talon™ metal affinity resin (Clontech, Palo Alto, Calif.). A solution of 5 mM borate sodium, pH 9.3, 8 M urea, and 100 mM sodium chloride was used as wash buffer. Bound fractions were eluted with 100 mM imidazole and then 1 M arginine (final concentration) was added as cosolvent, to encourage the correct folding of the protein molecules. The recombinant protein was refolded by removal of 8 M urea, by dialyzing against 5 mM borate sodium, pH 9.3, and 1 M arginine; the cosolvent was then removed by dialyzing against 5 mM borate sodium, pH 9.3. The purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue R-250 (Bio-Rad Laboratories, Mississauga, ON) staining after samples had been concentrated in dialysis bags on a bed of polyethylene glycol compound, molecular weight (MW) 15,000-20,000 (Sigma, Oakville, ON).
  • SDS-PAGE and Western Blot Analysis
  • Proteins were separated by 10% SDS-PAGE gels by use of a Mini-PROTEAN II apparatus (Bio-Rad Laboratories). The bands were visualized by Coomassie blue staining. The molecular weights of the samples were estimated by comparison to the relative mobility values of standards of known molecular weights.
  • Gels were immunoblotted to Immunobilon-P membranes (Millipore Corp, Bedford, Mass.) using a western blot semi-dry transfer apparatus (Bio-Rad Laboratories) with Towbin buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, and 20% methanol). Blots were blocked with blocking buffer (2% bovine serum albumin in phosphate-buffered saline (PBS)). Blots were washed three times for 5 min with PBS containing 0.1% tween-20 (PBST) and then incubated directly with a 1:1000 dilution of HRP-conjugated streptavidin (Sigma) or a 1:2000 dilution of HRP-conjugated Ni-nitrilotriacetic acid (NTA) (Qiagen) at room temperature for 1 hr. After three washes for 5 min with PBST, and two washes for 2 min with deionized water, the specific binding was detected by an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Baie d'Urfe, QC).
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • The antigen-binding activity of the purified SBP tagged mA116 scFv Ab to VEE antigen was determined by an ELISA. Nunc maxisorp™ flat-bottomed 96-well plates (Life Technologies) were coated overnight at 4° C. with whole VEE (strain TC-83) at a fixed concentration of 10 μg/ml, or various concentrations of 0.2-60 μg/ml, in carbonate bicarbonate buffer, pH 9.6, containing 0.02% sodium azide. The plates were washed five times with PBST and then blocked twice in 2% bovine serum albumin for 1 hr at 37° C. After five washes with PBST, plates were incubated for 1 hr at 37° C. with various concentrations of 0.6-50 μg/ml, or a fixed concentration of 10 μg/ml of the SBP tagged mA116 scFv Ab or its parental monoclonal antibody (MAb) 1A4A1, diluted in PBST. Following five washes with PBST, plates were incubated for 1 hr at 37° C. with 1:1000 dilution of HRP-conjugated streptavidin in PBST for the SBP tagged mA116 scFv Ab and 1:2000 dilution of HRP-conjugated goat anti-mouse Ig for 1A4A1 MAb. Finally, the plates were washed five times with PBST and developed for 30 min at room temperature with a substrate consisting of 2,2′-azino-di-(3-ethyl-benzthiazoline-sulfonic acid) diammonium salt (ABTS) and hydrogen peroxidate (Kirkegaard and Perry Laboratories, Gathersburg, Md.). The reactions were read at an absorbance of 405 nm by a microplate autoreader (Molecular Devices, Sunnyvale, Calif.).
  • Results
  • Construction, Expression and Purification
  • The pPICZαBmA116 recombinant plasmid, encoding anti-VEE mA116 scFv Ab gene was used as a source material to create the SBP tagged mA116 scFv Ab construct. After a synthetic double-stranded oligonucleotide encoding a SBP and 6His tag was inserted downstream to the pPICZαBmA116, mA116 scFv/SBP/6His was amplified using PCR method with appropriate primers. The PCR product was inserted into pCRT7 TA cloning vector. The resulting plasmid, designated pCRT7mA116SBP, contained the mA116 scFv Ab gene, followed by the SBP sequence under the control of T7 promoter (FIG. 1). In addition, there was a 6His tag located downstream of the SBP for immobilized metal affinity chromatography (IMAC) purification. The nucleotide and deduced amino acid sequences are showed in FIG. 2. The encoded whole recombinant fusion protein was 296 residues with a predicted molecular weight of 31.3 kDa.
  • The SBP tagged mA116 scFv Ab was expressed in E.coli BL-21 cells as inclusion bodies and purified by IMAC. SDS-PAGE demonstrated that there was a relatively small amount of protein in the bacterial lysate of molecular weight ˜32 kDa corresponding to the predicted size (31.3 kDa) of the SBP tagged mA116 scFv Ab, due to the presence of large amounts of contaminating proteins (FIG. 3, Lane 2). However, after centrifugation of the lysate, and dissolution of the pellet in solubilizing agent, many of the bacterial host proteins were removed from the lysate, making the ˜32 kDa band more visible (FIG. 3, Lane 3). The solubilized protein fraction was incubated with metal affinity resin and loaded to an empty column. After thoroughly washing with wash buffer, the bound fractions were eluted by 100 mM imidazole. Only one band of ˜32 kDa was observed in the purified fraction (FIG. 3, lane 5). In this purification protocol, the expressed protein could be purified to over 90%.
  • Biochemical Characterization
  • To confirm the integrity of the expressed SBP tagged mA116 scFv Ab, a series of Western blotting experiments was performed, in which the 32-kDa protein was detected by both HRP-conjugated streptavidin and HRP-conjugated Ni—NTA (FIG. 4). With HRP-conjugated Ni—NTA, a 32 kDa band was observed in all purification fractions (FIG. 4A, Lanes 2-4). With HRP-conjugated streptavidin, bands were visible at 32 kDa in all fractions (FIG. 4B, Lanes 2-4). In addition, bands were observed at 20 kDa in the bacterial lysate, solubilized protein fraction, and column flow through fraction (FIG. 4B, Lanes 1-3). However, after purification, only the 32 kDa band was present in the purified fraction (FIG. 4B, Lane 4).
  • Binding Properties to VEE Antigen
  • The immunoreactivity of the SBP tagged mA116 scFv Ab to VEE antigen was examined by ELISA. When the plates were coated with a fixed concentration of VEE (10 μg/ml), the SBP tagged mA116 scFv Ab bound to VEE in a dose-dependent manner, similar to the binding to VEE of its parental 1A4A1 MAb (FIG. 5A). An additional ELISA was performed in which a concentration gradient of VEE was titrated against a fixed concentration of Abs (10 μg/ml). A similar dose-response relationship was observed (FIG. 5B).
  • Discussion
  • Since the introduction of MAb technology, a number of types of immunodiagnostic assays have been developed based on specific binding between antigens and their corresponding MAbs (Nakamura, 1983). However, the disadvantages of MAbs as immunodiagnostic reagents are numerous. The cost and time required for growth and maintenance of hybridoma cell lines, and production and purification of MAbs, coupled with the potential for occurrence of genetic mutation during repeated cycles of cell growth, makes routine production of MAbs from hybridoma cell lines difficult, expensive, and time consuming. ScFv Abs are comprised of immunoglobulin VH and VL chains, covalently connected by a peptide linker (Huston et al., 1988). These small proteins generally retain the specificity and affinity for antigen similar to their parental MAb and possibly bind to poorly accessible epitopes more efficiently due to their small size (Marin et al., 1995; Bruyns et al., 1996). The attractiveness of scFv Abs is that they can be produced economically in bacteria and manipulated via genetic engineering, for example to form fusion proteins with additional functions (George et al., 1995; Boleti et al., 1995; Wels et al., 1992).
  • The streptavidin-biotin system has one of the highest affinities (10−15 M) among receptor-ligand interactions (Green, 1963). The strong interaction between streptavidin and biotin has been applied in many immunoassays (Guesdon et al., 1979; Hsu et al., 1981). However, chemical biotinylation of Ab is time-consuming and, as most of the biotin binds to amino groups of the protein, the degree of labeling can differ from batch to batch. Furthermore, the possibility exists that the biological activity of the Ab may be affected by the labeling procedure (Mirables et al., 1991). With advent of recombinant DNA technology, it is possible to fuse a short peptide to the target protein through gene fusion technique. SBPs, constituting around 10 amino acids have been selected from random peptide libraries (Devlin et al., 1990; Osterguard et al., 1995; Gissel et al., 1995). Some of them have been well characterized (Schmidt et al., 1996; Skewa et al., 1999). SBPs have successfully been fused to scFv Abs to antigen CA125, Bacillus cereus spores, and scorpion toxin for use in immunoassay (Luo et al., 1998 Kao et al., 1998 Aubrey et al., 2001).
  • The present inventors genetically incorporated a SBP sequence in mA116 scFv Ab gene in order to biotinylate mA116 scFv Ab. DNA sequencing confirmed that DNA cloning was successful. The SBP tagged mA I16 scFv Ab was expressed in E. coli to high levels in the form of insoluble inclusion bodies. The insoluble recombinant fusion protein was solubilized by denaturing agent, 8M urea. Inclusion of 6His tag allowed the solubilized recombinant fusion protein to be purified via IMAC. In this way, greater than 90% purity of the SBP tagged recombinant mA116 scFv Ab could be obtained. After purification, arginine was introduced to the recombinant protein solution to direct correct refolding.
  • The streptavidin-binding peptide confers reversible binding activity toward the streptavidin. Therefore, it can be employed for the one step purification of a corresponding fusion protein via streptavidin affinity chromatography (Schmidt et al., 1994; Zwicker et al., 1999). However, in the present invention, purification of the recombinant SBP tagged mA116 scFv Ab using streptavidin affinity column chromatography yielded only small amounts of product containing relatively large amounts of host proteins (data not shown). In fact, a large amount of streptavidin-binding protein of around 20 kDa showed up in the bacterial lysate on Western blot analysis of bacterial lysate (FIG. 4B). This may be attributed to whole cell extracts of E.coli containing biotinylated proteins, such as biotin carboxyl carrier protein (22.5 kDa) that binds strongly to streptavidin (Sutton et al., 1977). Accordingly, a 6His tag was introduced into the gene construct in order that IMAC could be used to purify the Ab of the present invention.
  • The results of Western blot analysis confirmed that the refolded recombinant fusion protein was intact, with a molecular weight of ˜32kDa. The in vitro binding characteristics of the SBP tagged mA116 scFv Ab to VEE antigen were assayed by ELISA. The recombinant fusion protein exhibited strong binding activity to VEE, indicating that the SBP did not interfere with the conformation of antigen-binding site or the bioactivity of mA116 scFv Ab. The parental 1A4A1 MAb showed similar binding activity to VEE. However, a direct comparison of the binding affinities between both Abs was not possible by ELISA due to the use of different HRP-conjugates.
  • In summary, a SBP sequence was introduced downstream to the sequence for mA116 scFv Ab to VEE. The fusion protein was expressed and purified. Fusion to the SBP did not affect the ability of mA116 scFv Ab to recognize VEE antigen with an affinity similar to that observed for the parental MAb. Similarly, the streptavidin-binding property of the fusion protein was not impaired. Western blot and ELISA results suggest that SBP tagged mA116 scFv Ab could be used for simple, stable, and efficient detection of VEE when used in conjunction with HRP-conjugated streptavidin. This approach eliminates the need for chemical biotinylation of Abs with resultant possible impairment of the antigen-binding site of the Ab.
  • It is to be understood that the embodiments and variations shown and described herein are merely illustrative of the principles of this invention and that various modifications may be implemented by those skilled in the art without departing from the scope and spirit of the invention.
  • In addition, the List of Prior Art Literatures referred to in the Background of the Invention section is incorporated by reference herein.

Claims (15)

1. A method for constructing a recombinant gene encoding a single-chain variable fragment antibody cloned into an expression vector and fused with a streptavidin-binding peptide (SBP) gene sequence to produce a fusion protein, comprising:
(a) encoding anti-VEE single-chain variable fragment antibody (scFv Ab) gene to a recombinant plasmid and inserting a SBP gene and a 6His tag downstream to develop a SBP tagged scFv Ab construct;
(b) amplifying the resultant scFv/SBP/6His by polymerase chain reaction (PCR);
(c) inserting the amplified PCR products into cloning vector to produce a SBP-plasmid;
(d) constructing said SBP-plasmid with promoter to produce a SBP tagged scFv Ab; and
(e) expressing said SBP tagged scFv Ab in E. coli cells as inclusion bodies and purifying the expressed SBP tagged scFv Ab by immobilized metal affinity chromatography.
2. A method as in claim 1, wherein:
said recombinant plasmid in step (a) is a pPICZαBmA116 recombinant plasmid;
said cloning vector in step (c) is pCRT7 TA; and
said promoter in step (d) is a T7 promoter.
3. A method as in claim 1, wherein said anti-VEE scFv Ab is mA116 Ab.
4. A fusion protein, SBP tagged scFv Ab, comprising a single-chain variable fragment antibody (scFv Ab) fused with a streptavidin-binding peptide (SBP) sequence.
5. The SBP tagged recombinant scFv Ab fusion protein of claim 4, comprising an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.: 1.
6. The SBP tagged recombinant scFv Ab fusion protein of claim 4, comprising the amino acid sequence shown in SEQ ID NO.: 2.
7. The SBP tagged recombinant scFv Ab fusion protein of claim 4, wherein said fusion protein has a molecular weight of ˜32 kDa.
8. The SBP tagged recombinant scFv Ab fusion protein of claim 4, wherein said fusion protein displays high antigen-binding affinity to Venezuelan equine encephalitis virus (VEE).
9. The SBP tagged recombinant scFv Ab fusion protein of claim 4, wherein said fusion protein displays high streptavidin-binding activity.
10. The SBP tagged recombinant scFv Ab fusion protein of claim 4, wherein said scFv Ab is mA116 scFv Ab.
11. A method for using the SBP tagged recombinant scFv Ab fusion protein of claim 4 for detecting VEE, comprising:
(a) reacting the SBP tagged scFv Ab with a sample containing VEE for observing antigen-binding activity; and
(b) analyzing the reactant by enzyme-linked immunosorbent assay (ELISA).
12. The method of claim 11, wherein said ELISA immunoassay employs an indicator enzyme and substrate system to visually indicate presence of antigen-binding activity.
13. The method of claim 12, wherein horseradish peroxidase is used in said ELISA as the indicator enzyme.
14. The method of claim 12, wherein 2,2′-azino-di-(3-ethyl-benzthiazoline-sulfonic acid) diammonium salt (ABTS) is used in said ELISA as the substrate system.
15. The method of claim 11, wherein said scFv Ab is mA116 scFv Ab.
US10/784,305 2003-02-24 2004-02-24 Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus Expired - Fee Related US7527796B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/784,305 US7527796B2 (en) 2003-02-24 2004-02-24 Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US44890203P 2003-02-24 2003-02-24
US10/784,305 US7527796B2 (en) 2003-02-24 2004-02-24 Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus

Publications (2)

Publication Number Publication Date
US20050118569A1 true US20050118569A1 (en) 2005-06-02
US7527796B2 US7527796B2 (en) 2009-05-05

Family

ID=32908670

Family Applications (1)

Application Number Title Priority Date Filing Date
US10/784,305 Expired - Fee Related US7527796B2 (en) 2003-02-24 2004-02-24 Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus

Country Status (2)

Country Link
US (1) US7527796B2 (en)
CA (1) CA2458824A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103267841A (en) * 2013-05-15 2013-08-28 湖南农业大学 Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8501157B2 (en) 2007-11-01 2013-08-06 Her Majesty the Queen in right of Canada as represented by The Ministry of National Defence Humanized anti-Venezuelan equine encephalitis virus recombinant antibodies

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103267841A (en) * 2013-05-15 2013-08-28 湖南农业大学 Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle

Also Published As

Publication number Publication date
US7527796B2 (en) 2009-05-05
CA2458824A1 (en) 2004-08-24

Similar Documents

Publication Publication Date Title
CN112010965B (en) Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof
Kerschbaumer et al. Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay
Bach et al. Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies
Goodchild et al. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries
CN112794899B (en) Fully human monoclonal neutralizing antibody for resisting novel coronavirus and application thereof
Rosso et al. Expression and functional characterization of a single chain Fv antibody directed against secretions involved in plant nematode infection process
McElhiney et al. Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water
Suzuki et al. Open sandwich ELISA with VH-/VL-alkaline phosphatase fusion proteins
CN111378048A (en) Antibody-polypeptide bispecific immunotherapeutics against middle east respiratory syndrome coronavirus
Zhang et al. Cloning of scFv from hybridomas using a rational strategy: Application as a receptor to sensitive detection microcystin-LR in water
CA3109433A1 (en) Ns1-binding protein and uses thereof
KR20210035249A (en) NS1 protein binding protein
CN111303286B (en) anti-CD19 fully human antibody or antibody fragment, chimeric antigen receptor thereof and application thereof
KR20120002641A (en) Antibody against ompu of vibrio and method of detecting vibro using the same
Suzuki et al. Construction, bacterial expression, and characterization of hapten-specific single-chain Fv and alkaline phosphatase fusion protein
Velappan et al. Selection and characterization of scFv antibodies against the Sin Nombre hantavirus nucleocapsid protein
US7527796B2 (en) Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus
Furuta et al. Construction of mono-and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing.
Hu et al. Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus
WO2002008757A1 (en) Method for immuno-detection of epitopes
Mousli et al. Production and characterization of a bivalent single chain Fv/alkaline phosphatase conjugate specific for the hemocyanin of the scorpion Androctonus australis
Das et al. Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus: bacterial expression and refolding
EP4299745A1 (en) Anti-sars-cov-2 antibody
Pearce et al. Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents
Lah et al. Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector

Legal Events

Date Code Title Description
AS Assignment

Owner name: HER MAJESTY THE QUEEN IN RIGHT OF CANADA, AS REPRE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FULTON, R. ELAINE;NAGATA, LESLIE P.;ALVI, AZHAR Z.;AND OTHERS;REEL/FRAME:015055/0456;SIGNING DATES FROM 20040216 TO 20040220

REMI Maintenance fee reminder mailed
LAPS Lapse for failure to pay maintenance fees
STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 20130505