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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
  • G01N33/6857—Antibody fragments
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to a reagent for use in agglutination assays, and in particular whole blood agglutination assays.
• the invention also relates to a method for detecting an antigen, antibody or other analyte in a sample using the reagent, and to a kit containing the reagent.
• the invention describes the use of recombinant DNA methods in E. coli to produce the key reagents for this assay.
• Immunoassays and analogous specific binding assays are now very well-known and widely used in a variety of bio edical and other fields.
• the most commonly used immunoassays utilise complex detection systems involving radioisotopes or enzymes, and suffer from the disadvantage that the assay procedure is lengthy and involved, and requires expensive instrumentation.
• Radioimmunoassays further suffer from the disadvantage of the radioactive hazard presented by the isotopes.
• Non-specific agglutination is avoided if the erythrocyte binding molecule recognises an abundant, well-distributed erythrocyte membrane constituent such as glycophorin.
• WO 91/04492 describes an autologous agglutination assay of improved sensitivity. The entire disclosures of U.S. Patent No. 4,894,347 and International Patent Application No. WO 91/04492 are also incorporated herein by reference.
• test systems for use under field conditions should be stable, rapid, reliable and specific, and should provide a clear-cut demarcation between positive and negative results. In order to be cost effective, such a system should require the minimum number of reagents, which in themselves should be easy to produce.
• antigen-antibody constructs Two main types of reagent are desired for use in these immunoassays, namely antigen-antibody constructs and bispecific antibody constructs.
• the currently manufactured antigen-antibody reagents utilise, for example, an antigenic peptide from an immunodominant portion of HIV virus (HIV-1 or HIV-2), coupled chemically to the Fab fragment of antibody which is able to bind to glycophorin A on the red cell surface.
• HIV-1 or HIV-2 immunodominant portion of HIV virus
• Alternative reagents of the antigen-an ibody type utilise a larger protein, rather than an immunodominant peptide, for example hepatitis B surface antigen.
• Bispecific antibody and Ab-peptide conjugate reagents are currently manufactured by a series of steps involving chemical and enzymic manipulation of antibodies; they consist of two Fab molecules with differing specificities, linked by disulphide bonds at the hinge region.
• the resultant bispecific F(ab) 3 molecule reacts both with an indicator reagent, such as an erythrocyte, and a circulating antigen in a blood sample.
• Antibodies (Abs) and Ab fragments can be produced by recombinant DNA technology (Winter and Milstein, Nature, 1991 349 293; U.S. Patent No. 4,946,778 by Ladner et al; Australian Patent No.
• variable surface loops at the extremity of the molecule. These loops in the outer domain (Fv) are termed complementarity-determining-regions (CDRs), and provide the specificity of binding of the Ab to its antigenic target. Binding function is localised to the variable domains of the antibody molecule, which are located at the amino terminal end of both the heavy and light chains.
• the variable regions remain noncovalently associated (as V H V L dimers, termed Fv regions) even after proteolytic cleavage from the native antibody molecule, and retain much of their antigen recognition and binding capabilities (see, for example, Inbar et al, Proc. Natl. Acad. Sci.
• oligonucleotide synthesis can provide the gene fragments that encode the various C- terminal peptide tails that constitute analyte specificity.
• the red cell binding molecule providing it has sufficient affinity, may be an Fv fragment rather than a complete Fab.
• single chain scFv, or smaller domain structures can be engineered which may have advantages for product stability and yield. Further improvements to the reagents include the elimination of mouse constant domains, with resulting increased specificity, and improved solubility properties.
• Expression systems that are available for the production of antibody fragments include E. coli and alternative prokaryotes, yeast, baculoviral vectors and mammalian cells.
• E. coli secretion vectors which (Power et al, Gene 1992 113 95-99) now allow the expression, to exceptionally high levels, of the V E /V L /scFv domains of anti-neuraminidase Abs.
• Downstream processing has been addressed in the context of high-level Ab-domain production. A number of denaturation/ renaturation regimes have been tested, and molecular "flags" incorporated into the expressed antibody domains to aid in purification and conformational assessment.
• the synthesis of antibody variable region domains in recombinant organisms has the potential to enable the production of reagents which might otherwise be impossible to manufacture, such as constructs using large recombinant proteins, many of which are usually produced as insoluble molecules for solid phase assays.
• reagents which might otherwise be impossible to manufacture, such as constructs using large recombinant proteins, many of which are usually produced as insoluble molecules for solid phase assays.
• an antigen will not produce a single immunodominant response in an infected host, and several epitopes from an antigen are necessary to detect circulating antibodies.
• several peptides as a recombinant construct with Ab or fragments avoid the need for the multiple Fab-peptide conjugates.
• the use of multiple conjugates requires large amounts of blocking reagents to avoid non-specific agglutination resulting from interaction of the Fab constant regions.
• the ability to express the bifunctional molecules in a recombinant host dramatically decreases manufacturing costs for reagent
• the invention provides an assay, utilizing a series of reagents produced by recombinant DNA technology, that is useful for the detection of drugs, hormones, steroids, antibodies, and other molecules in a biological fluid, particularly in blood.
• Technology for this assay and these reagents is taught which provides a sensitive assay and a means to produce the key reagents as recombinant antibody molecules, including single chain antibody molecules, in E. coli, or in other expression systems known to the person skilled in the art.
• a bifunctional recombinant protein comprising a particle-binding antibody or antibody fragment (PBM), and an analyte-binding moiety or molecule (ABM) .
• PBM particle-binding antibody or antibody fragment
• ABSM an analyte-binding moiety or molecule
• the analyte may be an antigen or an antibody.
• the particle binding antibody or antibody fragment is an erythrocyte binding antibody or antibody fragment (EBM) .
• the ABM is selected from the group consisting of an antigenic peptide from an immunodominant region of the env gp41 protein of HIV-1 or HIV-2, and one of the gag proteins, and an immunodominant region from the surface antigen of Hepatitis B surface antigen.
• the specific ABMs may be produced by expression from gene fragments, for example, from synthesised oligonucleotides, that encode peptides which constitute the analyte specificity.
• the ABM is a single chain Fv region of an antibody directed against an antigen selected from the group consisting of Hepatitis B surface antigen, D-dimer and canine heartworm antigen.
• the EBM is a single chain Fv region of an anti-erythrocyte antibody, more preferably an anti- glycophorin antibody.
• single chain Fv region in the construct presents the advantage that the constant region of the antibody is almost completely removed, and consequently there is less opportunity for interference by heterophile antibody in the final assay, and the manufacture of a complete reagent is more efficient that would be the case if no blocker antibody were used.
• the EBM is the single chain Fv domain of the anti-glycophorin A monoclonal antibody produced by the hybrido a cell line G26.4.1C3/86, which is described in US-4,894,347, and WO91/04492.
• a sample of this cell line was deposited under the Budapest Treaty at the American Type Culture Collection (12301 Parklawn Drive, Rockville MD, 20852) on 7 September 1988, and received the ATCC accession number HB9893.
• a DNA sequence encoding as a single transcriptional unit a particle-binding moiety operatively linked to an analyte-binding moiety, as well as expression vectors and host cells comprising said sequence.
• a third aspect of the invention provides assay methods and kits utilising the recombinant protein of the invention.
• the host cell may be any of those currently used by those skilled in the art of expression in recombinant organisms, and is preferably E. coli . However, it will be clearly understood that other hosts, such as other bacteria, yeasts or insect, mammalian or plant cells may be used.
• the E. coli expression vectors described herein are novel, particularly with respect to the design of protease-resistant 'tails' with the unique features required by the diagnostic test. We have optimised the induction regime and fermentation conditions for high-yielding production.
• the DNA encoding both the erythrocyte binding activity and the specific analyte binding activity may be located on a DNA element capable of replication and the expression of the genes for the bifunctional reagents.
• This DNA element may be a plasmid or any equivalent DNA element capable of replication and expression in an appropriate host.
• the portion of the bifunctional reagent which has specific analyte binding activity may be encoded by DNA which has been produced from cells and tissues by any of the standard techniques known in the art for the amplification of DNA, such as the polymerase chain reaction, the ligase chain reaction, or isothermal amplification.
• the bifunctional fusion protein is made as a single polypeptide chain.
• Any two of a wide range of analyte-binding molecules can be incorporated into a bifunctional single polypeptide chain.
• a recombinant reagent which is derived from cloned DNA coding for the erythrocyte binding antibody, which as a result of genetic manipulation is fused to an analyte binding molecule encoded by the gene or gene fragment for the specific analyte binding molecule without substantially changing the binding characteristics of the binding portion.
• the reagent is non-agglutinating when incubated with endogenous erythrocytes in the absence of the analyte.
• Figure 1 illustrates the sequence of the IgG (1C3/86) gamma chain derived from clone gammal.1.la.
• the nucleotide and deduced amino acid sequence (mature sequence shown in bold type and single letter code) of 1C3/86 IgG gamma-l.l.la are shown;
• Figure 2 illustrates the sequence of the IgG (1C3/86) kappa chain derived by PCR amplification and clone 4AC1/C2.
• the nucleotide and deduced amino acid sequence (shown in bold type and single letter code) of mature 1C3/86 IgG kappa chain (sequence is a composite of that determined from clones K4AC1/C2 and the gene amplified directly from mRNA by polymerase chain reaction) are shown;
• Figure 3 illustrates the strategy for the amplification of 1C3/86 gamma and kappa gene variable domains and the construction of the scFv in expression vector pPOW. PCR primer-template combinations used to amplify various antibody fragments are shown.
• Figure 4 illustrates the strategy for the amplification and cloning of 1C3/86 scFv in expression vector pHFA. PCR primer-template combinations used to amplify various antibody fragments are shown.
• Figure 5 illustrates the strategies for amplification and cloning of scFv's with combined FLAG and HIV immunodominant peptide epitopes in the expression vector pHFA ⁇ c . PCR primer-template combinations used to amplify various antibody fragments are shown.
• Figure 6 illustrates the vectors pPOW, pHFA and pHFA S ⁇ C used for the construction and expression of the 1C3/86 scFv (described in figures 3,4 and 5) with pertinent cloning sites.
• Amp r ampicillin resistance gene, ColEl or Ori; E.
• M13 ORI M13 phage origin of replication
• Gene3 gene 3 phage surface protein gene, Amber; amber stop codon, (TAG) fD; transcription terminator, placZ; lacz promoter, cl857; lambda heat labile repressor gene, P r and V ; lambda phage right and left promoters, FLAG; gene for epitope recognised by M2 anti-flag IgG and pelB; gene for pectate lyase signal sequence.
• Figure 7 illustrates the protein sequences of peptide epitopes which may be generated by PCR reaction and linked in the reaction or added by recombinant DNA techniques.
• Figure 8 illustrates the activity of the recombinant protein in ELISA assays.
• the anti-glycophorin A monoclonal antibody 1C3/86 was selected as a model antibody.
• the gene encoding 1C3/86 IgG was cloned into an E ⁇ cherichia coli host, and the nucleotide sequence of the antibody was determined.
• Synthetic oligonucleotide primers were designed in order to enable the variable domains of the antibody to be cloned, linked together to form a single chain Fv domain (scFv), into various expression vectors.
• scFv single chain Fv domain
• Various peptide epitopes were added to the C-terminus of the scFv molecule.
• PCR to identify segments of the genes encoding the antibody and to add linkers and peptide epitopes to those segments to form single chain, antibody-based reagents was adopted.
• RNA messenger RNA
• ss- and ds-cDNA were synthesised.
• the ds-cDNA was cloned into lambda-gtlO arms and packaged into a phage library.
• the heavy chain clone gamma-M/1.1 (Tyler et al, Proc. Natl. Acad. Sci., 1982 21 2008-2012) and the light chain clone pH76-kappa-10 (Adams et al, Biochem., 1980 19.2711-2719) were used to source ds-DNA inserts for the screening of the gtlO library.
• the amplification reaction yielded a single product, which when cloned and sequenced showed a coding sequence consistent with a kappa light chain and identical at the 3' end with the overlapping kappa clone K4AC1.
• the sequences derived from PCR and gtlO library enabled the compilation of the sequence shown in Figure 2.
• a single chain antibody fragment was constructed from the 1C3/86 molecule as follows: i) Amplification and cloning of the heavy-chain variable domain.
• PCR polymerase chain reaction
• dNTP solution a mixed A,C,G and T deoxynucleotide (dNTP solution) with each base at a concentration of 2 mM, 5 1 of each terminal primer (10 pMolar each) and, where used, 1 1 of internal primers (0.05-0.1 pM) , Mg++ to a final concentration of 1-5 mM, a reaction buffer appropriate for the particular polymerase chosen (supplied by manufacturer), and water to 100 1.
• the reactants were mixed and overlayed with paraffin oil (Sigma biochemicals) and subjected to 25-30 cycles in a thermal cycler (Corbett Research, Australia) .
• Oligonucleotide primers (Table 1) were synthesized to amplify the variable domain (V h ) from the heavy chain cDNA clone gamma-l.l.la, , and to add a Thai restriction site at the 5' end (N907) and a Bst E2 - peptide epitope -Eco Rl sequence at the 3' end (N908/N909/N1296/N976), as described in Figure 3A.
• the product was digested with Tha 1 and Eco Rl, and cloned into the Use 1/ 'Eco Rl-digested expression vector pPOW (Power et al.
• scFv composite single-chain antibody
• V-. product described in Figure 3B was digested with Bst E2 and Eco Rl and cloned into the Bst E2/Eco Rl digested plasmid construct, pPOWlCSscV - ⁇ v . above ( Figure 3A) .
• TGI Transformed E. coli
• Oligonucleotide Sfil5 and NVKFO-RNOT (Table 1) were used to add Sfi 1 and Not 1 restriction sites (by PCR amplification) to the 5' and 3' ends respectively of the scFv gene construct in pPOWlC3scFv HIV1 ( see Figure 4) - in this amplification, the gp41 HIV1 epitope was removed.
• the PCR product was digested with these restriction enzymes and cloned into the likewise restricted vector pHFA (see Figure 6B) which contains the alternative octapeptide FLAG tag ( Figure 7A) - pHFA is the parent of the vector pHE ⁇ (Hoogenboom et al, 1991) . The construct was then transferred into the E.
• coli strain HB2151 a strain in which the nucleotide sequence TAG (amber mutation) is recognised as a stop codon .
• oligonucleotides N1294, N909, N1296, N976 and N1645 introduced a HIVl epitope, Eco Rl and Sac 2 sites.
• HIV2 epitope and restriction sites were added with 3' oligonucleotides N1297, N1311, N1310 and N1646 (Table 1).
• PCR products were restricted with Sfi 1 and Sac 2 were cloned into the likewise restricted vector pHFA ⁇ ( Figure 6C), a derivative of pHFA.
• Cultures of pPOW were induced by raising the temperature of the medium to 42°C for 15 minutes, after which the incubation was continued at 37°C for 2-4 hours.
• Periplasmic proteins were isolated by suspending the E. coli in 25% w/v sucrose/10 mM Tris-HCl (pH 7.5) and 16 mM EDTA. Cells were then collected by centrifugation and resuspended in ice-cold water. The particulate material and the soluble fractions were analysed by SDS-PAGE followed by Western blot. Active expression was assessed by the presence of a product of apparent M-.30 Kd.
• Citrate buffer 0.1M pH 4 2.58g citric acid 2.18g Na 2 HP0 4 make up to 200ml with distilled water and adjust pH to 4.
• Activated ABTS add 10 1 of 30% hydrogen peroxide to 10ml of diluted ABTS solution.
• Epitopes of the surface protein gp41 from HIVl and HIV2 virus types may be combined with epitopes from gpl20 surface protein or p24 core protein or substituted for the M2-FLAG epitope in scFv constructs or added to the scFv-M2 FLAG construct, thereby producing various bifunctional reagents capable of binding erythrocytes and serum antibodies which may be present in patient's serum.
• the sequences of the M2-FLAG, HIVl and HIV2 epitopes are shown in Figure 7.
• the host cells When cultured under the conditions described in Example 1, the host cells expressed scFv antibody protein, which was transported through the host cell membranes to the periplasmic space and culture supernatant.
• the recombinant 1C3/86 scFv-FLAG efficiently agglutinates erythrocytes in an assay which uses monoclonal antibody directed against the M2-FLAG epitope (IBI Corp, U.S.A.) as the cross-linking moiety, with activity comparable to that shown by the prior art SimpliRED assay, in which a synthetically produced conjugate of the HIV-1 gp41 immunodominant epitope and 1C3/86 Fab was used as the reagent, and antibody 1B1/114 was used as a known positive sample. Constructs with either HIV-1 or HIV-2 sequences were effective also in mediation of agglutination when respective monoclonal antibodies 1B1 or 2A6 and 2B4 (for HIVl and HIV2 respectively) were included in the assay.
• Example 8 A Hepatitis B surface antigen binding fragment may be substituted for the HIV-binding peptide, thereby producing a bifunctional reagent which has the capacity to bind a different analyte, in this case Hepatitis B and in so doing to agglutinate the erythrocytes.
• a bifunctional reagent which has the capacity to bind a different analyte, in this case Hepatitis B and in so doing to agglutinate the erythrocytes.

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