Inhibitory polypeptide against a thermostable DNA polymerase

Abstract

This disclosure relates polypeptides that bind to and inhibit thermostable DNA polymerases and related compositions, kits, and methods.

Description

[0001] Attorney Docket: ABBTT-43290.601

[0002] INHIBITORY POLYPEPTIDE AGAINST A THERMOSTABLE DNA POLYMERASE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority to United States Provisional Patent Application Serial Number 63 / 778,737, filed March 27, 2025, the disclosure of which is herein incorporated by reference in its entirety.

[0005] SEQUENCE LISTING

[0006] The text of the computer readable sequence listing filed herewith, titled “43290-601 SEQUENCE LISTING”, created March 27, 2026, having a file size of 25,434 bytes, is hereby incorporated by reference in its entirety.

[0007] FIELD

[0008] This disclosure relates to the field of polypeptides that bind to and inhibit thermostable DNA polymerases and related compositions, kits, and methods.

[0009] BACKGROUND

[0010] Thermostable DNA polymerases are commonly used in biotechnology and molecular biology applications, including nucleic acid synthesis techniques such as amplification (e.g., PCR), which involves cycles of alternating denaturation and primer annealing and extension. Thermostable DNA polymerases are resistant to inactivation by high temperatures and are compatible with thermal denaturation steps. Hot Start DNA polymerase complexes have been utilized to enhance sensitivity and specificity in PCR by reducing off-target amplification events and minimizing primer dimer formation. Kellogg DE, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert PD, Chenchik A: TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. Biotechniques 1994, 16(6): 1134-1137. There is a need for improved inhibitory polypeptides that confer improved performance of hot start DNA polymerase complexes in assays.Attorney Docket: ABBTT-43290.601

[0011] SUMMARY

[0012] Provided herein are polypeptides that bind to and inhibit thermostable polymerases as well as compositions, kits, and methods employing the same. The polypeptides find use as inhibitors for hot-start nucleic acid amplification reactions (e.g., polymerase chain reactions).

[0013] In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1.

[0014] In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2.

[0015] In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence with at least 70% sequence identity to SEQ 1D NO:3.

[0016] In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:4.

[0017] In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:5.

[0018] In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:6.

[0019] In some aspects of the polypeptides disclosed herein, the at least 70% sequence identity comprises at least 80% sequence identity. In some aspects of the polypeptidesAttorney Docket: ABBTT-43290.601

[0020] disclosed herein, the at least 70% sequence identity comprises at least 90% sequence identity. In some aspects of the polypeptides disclosed herein, the at least 70% sequence identity comprises at least 95% sequence identity.

[0021] In some aspects of the polypeptides disclosed herein, the antigen binding variable region is a light chain variable region that comprises a CDR1, CDR2, and CDR3. In some aspects, the light chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:3.

[0022] In some aspects of the polypeptides disclosed herein, the antigen binding variable region is a heavy chain variable region that comprises a CDR1, CDR2, and CDR3. In some aspects, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:5, and a CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:6.

[0023] In some aspects, the polypeptide disclosed herein has a light chain variable region that comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:3; and a heavy chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:4, a CDR2 comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:6.Attorney Docket: ABBTT-43290.601

[0024] Tn some aspects, the polypeptide disclosed herein has a light chain variable region that comprises a CDR1 comprising the amino acid sequence of SEQ IDNO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a heavy chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:4, a CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0025] In some aspects, the polypeptide disclosed herein comprises (i) a light chain amino acid sequence of SEQ ID NO: 7, and (ii) a heavy chain amino acid sequence of SEQ ID NO: 8.

[0026] In some aspects of the polypeptides disclosed herein, the polypeptide is an antibody. In some aspects, the antibody is an IgG antibody. In some aspects of the polypeptides disclosed herein, the polypeptide is an antibody fragment. In some aspect, the antibody fragment is an Fab or scFv. In some aspects, the polypeptide is a diabody.

[0027] In some aspects, the polypeptide binds and reversibly inhibits a DNA polymerase. In some aspects, the polypeptide is heat labile.

[0028] In some aspects, the presently disclosed subject matter provides a composition comprising a polymerase-polypeptide complex comprising a polypeptide disclosed herein that is bound to a thermostable DNA polymerase, wherein 5’ to 3’ polymerase activity of the thermostable DNA polymerase is inhibited. In some aspects the thermostable polymerase is a synthetic, non-natural polymerase. In some aspects, the thermostable polymerase is a polymerase as described in WO2024102861, herein incorporated by reference in its entirety. In some aspects of the composition disclosed herein, the thermostable DNA polymerase comprises a sequence selected from the group consisting of SEQ ID Nos: 12-19, with or without histidine tags, or sequences having at least 70% (e.g., 80%, 90%, 95%, 98%, 99%) identity thereto.

[0029] In some aspects, the presently disclosed subject matter provides a reaction mixture comprising a polypeptide disclosed herein and a thermostable DNA polymerase.

[0030] In some aspects, the presently disclosed subject matter provides a kit comprising a polypeptide disclosed herein. In some aspects, the kit disclosed herein further comprises a thermostable DNA polymerase. In some aspects, the thermostable DNA polymerase is bound to said polypeptide. In some aspects, the thermostable polymerase is a polymerase asAttorney Docket: ABBTT-43290.601

[0031] described in WO2024102861 , herein incorporated by reference in its entirety. In some aspects of the composition disclosed herein, the thermostable DNA polymerase comprises a sequence selected from the group consisting of SEQ ID Nos: 12-19, with or without histidine tags, or sequences having at least 70% (e.g., 80%, 90%, 95%, 98%, 99%) identity thereto.

[0032] In some aspects, the kit disclosed herein further comprises at least one reagent for polymerase chain reaction. In some aspects, the at least one reagent for polymerase chain reaction is selected from controls, buffers, dNTPs, primers, and stabilizers. In some aspect, the at least one reagent for polymerase chain reaction is a buffer comprising MgCh.

[0033] In some aspects, the presently disclosed subject matter provides a method comprising: contacting a sample containing a target nucleic acid with a polypeptide disclosed herein that is bound to a thermostable DNA polymerase. In some aspects, the method disclosed herein further comprises heating the sample to a temperature that disassociates the polypeptide from the thermostable DNA polymerase. In some aspects, the method disclosed herein further comprises amplifying the target nucleic acid. In some aspects, the amplifying comprises a polymerase chain reaction. In some aspects, the method disclosed herein further comprises detecting amplicons, or byproduct thereof, of the target nucleic acid. In some aspects, the target nucleic acid is DNA. In some aspects, the target nucleic acid is RNA. In some aspects, the RNA is converted into cDNA by reverse transcription prior to the contacting. In some aspects of the method disclosed herein, the target nucleic acid is from an infectious disease agent. In some aspects, the infectious disease agent is at least one of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG). In some aspects, the sample is, or is derived from, an endocervical swab specimen, a clinician-collected vaginal swab specimen, a self-collected vaginal swab specimen, a gynecological specimen, or urine.

[0034] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.

[0035] BRIEF DESCRIPTION OF THE FIGURESAttorney Docket: ABBTT-43290.601

[0036] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0037] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0038] FIG. 1 Hot Start polymerase principle. An antibody binds to and inhibits a DNA polymerase. Hot Start DNA polymerase complexes contain a DNA polymerase and an inhibitory antibody that prevents DNA amplification. This helps to prevent off-target amplification and primer-dimer formation. Upon heating the complex, the antibody becomes dissociated, and the DNA polymerase activity is restored. The antibody is heat labile such that, upon heating to 95°C, the enzyme activity is restored to a level comparable to the enzyme without the inhibitory antibody.

[0039] FIG. 2 illustrates the design of the 1874 rFab for rapid manufacture of homogeneous preparations of antibody.

[0040] FIG. 3 shows the SDS-PAGE of the 1874 rFab comprising a heavy chain without an interchain disulfide. O: original design, heavy chain (HC) with interchain disulfide; N: new design, HC without interchain disulfide.

[0041] FIG. 4 shows the SDS-PAGE of polymerase H7 (SEQ ID NO: 13), 1874 IgG, and 1874 rFab-His7 on 4-15% TGX gel (4pg of enzyme or 6pg of antibody).

[0042] FIG. 5 is graph comparing purified IgG or rFab inhibition of relative activity (%) of H7 (20 pg / rxn) DNA polymerase before heating and after heating.

[0043] FIG. 6 is a RFU graph of 1874 mlgGk inhibition of H5 DNA polymerase variant (SEQ ID NO: 17) with 1-1874 mlgG at 50-95-50°C and a RFU graph of 1874 mlgGk inhibition of H6 DNA polymerase variant (SEQ ID NO: 18) with 1-1874 mlgG at 50-95-50°C.

[0044] FIG. 7 is a graph showing 1-1874 mlgG inhibition of relative activity (%) of H5 DNA polymerase variant and H6 DNA polymerase variant before heating and after heating.

[0045] FIG. 8. shows Alinity m STI assay performance comparison of enzyme alone (No Ab), Hot Start (H3, H4, H5, H6, H7 with 1874 rFAB), Hot Start (H3, H4, H5, H6, H7 with 1874 IgG), and Kapa2G for six targets (TV, MG, CT, NG, IC, and CC).Attorney Docket: ABBTT-43290.601

[0046] FIG. 9 shows HIV amplification, HIV Ct, and HIV End RFU graphs comparing Kapa2G Hotstart, H7, H7-IgG, and H7-rFab.

[0047] FIG. 10 shows HPV39 (FAM) amplification, HPV39 (FAM) Ct, and HPV39 (FAM) End RFU graphs comparing Kapa2G Hotstart, H7, H7-IgG, and H7-rFab.

[0048] FIG. 11 shows HPV16 (VIC) amplification, HPV16 (VIC) Ct, and HPV16 (VIC) End RFU graphs comparing Kapa2G Hotstart, H7, H7-IgG, and H7-rFab.

[0049] FIG. 12 shows HPV18 (Cal Red 610) amplification, HPV18 (Cal Red 610) Ct, and HPV18 (Cal Red 610) End RFU graphs comparing Kapa2G Hotstart, H7, H7-IgG, and H7-rFab.

[0050] FIG. 13 shows HPV58 (Quasar 670) amplification, HPV58 (Quasar 670) Ct, and HPV58 (Quasar 670) End RFU graphs comparing Kapa2G Hotstart, H7, H7-IgG, and H7-rFab.

[0051] FIG. 14 shows HPV45 (Quasar 705) amplification, HPV45 (Quasar 705) Ct, and HPV45 (Quasar 705) End RFU graphs comparing Kapa2G Hotstart, H7, H7-IgG, and H7-rFab.

[0052] BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO: 1 represents a light chain CDR-L1: RPSENVYSYLA

[0053] SEQ ID NO: 2 represents a light chain CDR-L2: NAKTLTE

[0054] SEQ ID NO: 3 represents a light chain CDR-L3: QQHYGAPYT

[0055] SEQ ID NO: 4 represents a heavy chain CDR-H1: GYAFTNYLME

[0056] SEQ ID NO: 5 represents a heavy chain CDR-H2: VINPGSGGTNYNEKFKG

[0057] SEQ ID NO: 6 represents a heavy chain CDR-H3: SYYNNFYFDY

[0058] SEQ ID NO: 7 represents a 1874 light chain (kappa) sequence:

[0059] DIQMTQSPASLSVSVGETVTITCRPSENVYSYLAWYQQKQEKSPLLLVYNAKTLTE GVPSRF SGSGSGTQF SLKINSLQPEDFGS YYCQQHYGAPYTFGGGTKLEMKRADAA PTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 8 represents a 1874 heavy chain (gamma 1) sequence:

[0060] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLMEWVKQRPGQGLEWIGVINPG SGGTNYNEKFKGKATLTADRS S STAYMHLS SLTSDDS AVYFCARSYYNNFYFDYAttorney Docket: ABBTT-43290.601

[0061] WGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGS LSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVE VHTAQTQPREEQFNSTFRSVSELPLMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQP

[0062] 1MDTDGS YF V Y SKLN VQK SN WEAGN TFTCS VLHEGLHNHHTEK SL SHSPGK SEQ ID NO: 9 represents a 1874 Fab light chain sequence:

[0063] DIQMTQSPASLSVSVGETVTITCRPSENVYSYLAWYQQKQEKSPLLLVYNAKTLTE GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQQHYGAPYTFGGGTKLEMKRADAA PTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNES SEQ ID NO: 10 represents a 1874 Fab heavy chain sequence:

[0064] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLMEWVKQRPGQGLEWIGVINPGS GGTNYNEKFKGKATLTADRSSSTAYMHLSSLTSDDSAVYFCARSYYNNFYFDYWG QGTTLT VS S AKTTPP S VYPLAPGS AAQTNSMVTLGCLVKGYFPEP VTVTWNSG SL S S GVHTFP AVLQ SDLYTL S S S VTVP S STWPSETVTCNVAHP AS STK VDKKIVPRDC

[0065] SEQ ID NO: 11 represents a 1874 Fab heavy chain sequence with a Cys to Ser mutation: QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLMEWVKQRPGQGLEWIGVINPG SGGTNYNEKFKGKATLTADRSSSTAYMHLSSLTSDDSAVYFCARSYYNNFYFDY WGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGS LSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDS

[0066] SEQ ID NO: 12 represents an amino acid sequence of polymerase HO MLPLFDLEEPPKRVLLVDGHHLAYRTFYALSLTTSRGEPVQMVYGFARSLLKALKE DGQAVVVVFDAKAPSFRHEAYEAYKAGRAPTPEDFPRQLALVKRLVDLLGLVRLE APGYEADDVLGTLAKKAEREGMEVCILTGDRDFFQLLSEKVSVLLPDGTLVTPKDV QEKYGVPPERWVDFRALTGDRSDNIPGVAGIGEKTALRLLAEWGSVENLLKNLDR VKPDSLRRKLEAHLEDLRLSLDLARIRTDLPLEVDFKALRRRTPDLEGLRAFLEELEF GSLLHEFGLLGGEKPREEAPWPPPEGAFVGFLLSRKEPMWAELLALAAAAEGRVHR ATSPVEALADLKEARGFLAKDLAVLALREGVALDPTDDPLLVAYLLDPANTNPEGV ARRYGGEFTEDAAERALLSERLFQTLFPRLSEKLLWLYQEVERPLSRVLAHMEARG VRLDVPLLEALSFELEKEMERLEGEVFRLAGHPFNLNSRDQLEKVLFDELGLTPVGR TEKTGKRSTAQGALEALRGAHPIVELILQYRELSKLKSTYLDPLPRLVHPRTGRLHT RFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRKAFVAEEGWLLLAADYSQIELRVL AHLSGDENLI<RVFREGKDIHTETAAWMFGLDPALVDPI<MRRAAI<TVNFGVLYGM SAHRLSQELGIDYKEAEAFIERYFQSFPKVRAWIERTLEEGRTRGYVETLFGRRRYVAttorney Docket: ABBTT-43290.601

[0067] PDLASRVRSVREAAERMAFNMPVQGTAADLMKIAMVKLFPRLKPLGAHLLLQVHD ELVLEVPEDRAEEAKALVKEVMENAYPLDVPLEVEVGVGRDWLEAKQD SEQ ID NO: 13 represents an amino acid sequence of polymerase H7, containing a His-tag MHHHHHHGGSGMLPLFDLEEPPKRVLLVDGHHLAYRTFYALSLTTSRGEPVQMVY GFARSLLKALKEDGQAVVVVFDAKAPSFRHEAYEAYKAGRAPTPEDFPRQLALVK RLVDLLGLVRLEAPGYEADDVLGTLAI<I<AEREGMEVHILTGDRDFFQLLSEI<VSVL LPDGTLVTPKDVQEKYGVPPERWVDFRALTGDRSDNIPGVAGIGEKTALRLLAEWG S VENLLKNLDRVKPD SLRRKIE AHLEDLRL SLDL VRIRTDLPLE VDFKALRRRTPDL EGLRAFLEELELGSLLHEFGLLGGEKPREEAPWPPPEGAFVGFLLSRKEPMWAELLA LAAAAEGRVHRATSPVEALADLKEARGFLAKDLAVLALREGVALDPTDDPLLVAY LLDSANTNPERVARRYGGEFTEDAAERALLSERLFQTLFPRLSEKLLWLYQEVERPL SRVLAHMEARGVRLDVPLLEALSFEAAKELERLEGEVFRLAGHPFNLNSRDQLEKV LFDELGLTPVTRTEKTGKRSTSAAALEALRGVHPIVELILQYRELSKLKSTYLDRLPR LVHPRTGRLHTRFNQTATATGRLS S SDPNLQNIP VRTPLGQHIRKAFVAEEGWLLLA ADYSQIELRVLAHLSGDENLKRVFREGKDIHTETAAWMFGVDSALVDPKMRRAAK TVNFGVLYGMSAARLSQELGIDYKEAEAFIERYFQSFPKVRAWIERTLEEGRTRGYV ETLFGRRRYVPDLTSRVRSVREAAERMAWNMPVQGTAADLMKIAMVKLFPRLKPL GAHLLLQVHDELVLEVPEDRAEEAKALVKEVMENAYPLDVPLEVEVGVGRDWLE AKQD SEQ ID NO: 14 represents an amino acid sequence of polymerase H2, containing a His-tag MHHHHHHGGSGMLPLFDLEEPPKRVLLVDGHHLAYRTFYALSLTTSRGEPVQMVY GFARSLLKALKEDGQAVVVVFDAKAPSFRHEAYEAYKAGRAPTPEDFPRQLALVK RLVDLLGLVRLEAPGYEADDVLGTLAKKAEREGMEVCILTGDRDFFQLLSEKVSVL LPDGTLVTPKDVQEKYGVPPERWVDFRALTGDRSDNIPGVAGIGEKTALRLLAEWG S VENLLKNLDRVKPD SLRRKIE AHLEDLRL SLDL ARIRTDLPLE VDFKALRRRTPDL EGLRAFLEELEFGSLLHEFGLLGGEKPREEAPWPPPEGAFVGFLLSRKEPMWAELLA LAAAAEGRVHRATSPVEALADLKEARGFLAKDLAVLALREGVALDPTDDPLLVAY LLDPANTNPEGVARRYGGEFTEDAAERALLSERLFQTLFPRLSEKLLWLYQEVERPL SRVLAHMEARGVRLDVPLLEALSFELEKELERLEGEVFRLAGHPFNLNSRDQLEKV LFDELGLTPIGRTEKTGKRSTSAAALEALRGAHPIVELILQYRELSKLKSTYLDRLPR LVHPRTGRLHTRFNQTATATGRLS S SDPNLQNIP VRTPLGQRIRKAF VAEEGWLLLA ADYSQIELRVLAHLSGDENLKRVFREGKDIHTETAAWMFGLDPALVDPKMRRAAK TVNFGVLYGMSAHRLSQELGIDYKEAEAFIERYFQSFPKVRAWIERTLEEGRTRGYV ETLFGRRRYVPDLASRVRSVREAAERMAFNMPVQGTAADLMKIAMVKLFPRLKPL GAHLLLQVHDELVLEVPEDRAEEAKALVKEVMENAYPLDVPLEVEVGVGRDWLE AKQD SEQ ID NO: 15 represents an amino acid sequence of polymerase H4, containing a His-tag MHHHHHHGGSGMLPLFDLEEPPKRVLLVDGHHLAYRTFYALSLTTSRGEPVQMVY GFARSLLKALKEDGQAVVVVFDAKAPSFRHEAYEAYKAGRAPTPEDFPRQLALVK RLVDLLGLVRLEAPGYEADDVLGTLAKKAEREGMEVCILTGDRDFFQLLSEKVSVLAttorney Docket: ABBTT-43290.601

[0068] LPDGTLVTPKDVQEKYGVPPERWVDFRALTGDRSDNTPGVAGIGEKTALRLLAEWG S VENLLKNLDRVKPD S LR RK I E A H LE D LRL SLDL A R I RT DLPLE VDF K A LRRRT P D L EGLRAFLEELEFGSLLHEFGLLGGEKPREEAPWPPPEGAFVGFLLSRKEPMWAELLA LAAAAEGRVHRATSPVEALADLKEARGFLAKDLAVLALREGVALDPTDDPLLVAY LLDP ANTNPERVARRYGGEFTED AAERALL SERLFQTLFPRL SEKLLWL YQEVERPL SRVLAHMEARGVRLDVPLLEALSFELEKELERLEGEVFRLAGHPFNLNSRDQLEKV LFDELGLTPVTRTEKTGKRSTSAAALEALRGAHPIVELILQYRELSKLKSTYLDRLPR LVHPRTGRLHTRFNQTATATGRLS S SDPNLQNIP VRTPLGQHIRKAFVAEEGWLLLA ADYSQIELRVLAHLSGDENL1<RVFREGI<DIHTETAAWMFGVDPALVDPI<MRRAAI< TVNFGVLYGMSAHRLSQELGIDYKEAEAFIERYFQSFPKVRAWIERTLEEGRTRGYV ETLFGRRRYVPDLASRVRSVREAAERMAWNMPVQGTAADLMKIAMVKLFPRLKP LGAHLLLQVHDELVLEVPEDRAEEAKALVKEVMENAYPLDVPLEVEVGVGRDWL EAKQD SEQ ID NO: 16 represents an amino acid sequence of polymerase Hl, containing a His-tag MHHHHHHGGSGMLPLFDLEEPPKRVLLVDGHHLAYRTFYALSLTTSRGEPVQMVY GFARSLLKALKEDGQAVVVVFDAKAPSFRHEAYEAYKAGRAPTPEDFPRQLALVK RLVDLLGLVRLEAPGYEADDVLGTLAKKAEREGMEVCILTGDRDFFQLLSEKVSVL LPDGTLVTPKDVQEKYGVPPERWVDFRALTGDRSDNTPGVAGIGEKTALRLLAEWG S VENLLKNLDRVKPD S LR RK I E A H LE D LRL SLDL A R I RT DLPLE VDF K A LRRRT P D L EGLRAFLEELEFGSLLHEFGLLGGEKPREEAPWPPPEGAFVGFLLSRKEPMWAELLA LAAAAEGRVHRATSPVEALADLKEARGFLAKDLAVLALREGVALDPTDDPLLVAY LLDP ANTNPEGVARRYGGEFTED AAERALL SERLFQTLFPRL SEKLLWL YQEVERPL SRVLAHMEARGVRLDVPLLEALSFELEKELERLEGEVFRLAGHPFNLNSRDQLEKV LFDELGLTPIGRTEKTGKRSTSAAALEALRGAHPIVELILQYRELSKLKSTYLDPLPR LVHPRTGRLHTRFNQTATATGRLS S SDPNLQNIP VRTPLGQRIRKAF VAEEGWLLLA ADYSQIELRVLAHLSGDENLKRVFREGKDIHTETAAWMFGLDPALVDPKMRRAAK TVNFGVLYGMSAHRLSQELGIDYKEAEAFIERYFQSFPKVRAWIERTLEEGRTRGYV ETLFGRRRYVPDLASRVRSVREAAERMAFNMPVQGTAADLMI<1AMVI<LFPRLI<PL GAHLLLQVHDELVLEVPEDRAEEAKALVKEVMENAYPLDVPLEVEVGVGRDWLE AKQD SEQ ID NO: 17 represents an amino acid sequence of polymerase H5, containing a His-tag MHHHHHHGGSGMLPLFDLEEPPKRVLLVDGHHLAYRTFYALSLTTSRGEPVQMVY GFARSLLKALKEDGQAVVVVFDAKAPSFRHEAYEAYKAGRAPTPEDFPRQLALVK RLVDLLGLVRLEAPGYEADDVLGTLAKKAEREGMEVCILTGDRDFFQLLSEKVSVL LPDGTLVTPKDVQEKYGVPPERWVDFRALTGDRSDNTPGVAGIGEKTALRLLAEWG S VENLLKNLDRVKPD S LR RK I E A H LE D LRL SLDL A R I RT DLPLE VDF K A LRR RT P D L EGLRAFLEELELGSLLHEFGLLGGEKPREEAPWPPPEGAFVGFLLSRKEPMWAELLA LAAAAEGRVHRATSPVEALADLKEARGFLAKDLAVLALREGVALDPTDDPLLVAY LLDHANTNPERVARRYGGEFTEDAAERALLSERLFQTLFPRLSEKLLWLYQEVERP LSRVLAHMEARGVRLDVPLLEALSFELEKELERLEGEVFRLAGHPFNLNSRDQLEK VLFDELGLTPVTRTEKTGKRSTSAAALEALRGAHPIVELILQYRELSKLKSTYLDRLP RLVHPRTGRLHTRFNQTAT ATGRLS S SDPNLQNIP VRTPLGQHIRK AF VAEEGWLLLAttorney Docket: ABBTT-43290.601

[0069] AADYSQIELRVLAHLSGDENLKRVFREGKDIHTETAAWMFGVDPALVDPKMRRAA KTVNFGVLYGMSAARLSQELGIDYKEAEAFIERYFQSFPKVRAWIERTLEEGRTRGY VETLFGRRRYVPDLTSRVRSVREAAERMAWNMPVQGTAADLMKIAMVKLFPRLK PLGAHLLLQVHDELVLEVPEDRAEEAKALVKEVMENAYPLDVPLEVEVGVGRDW LEAKQD SEQ ID NO: 18 represents an amino acid sequence of polymerase H6, containing a His-tag MHHHHHHGGSGMLPLFDLEEPPKRVLLVDGHHLAYRTFYALSLTTSRGEPVQMVY GFARSLLKALKEDGQAVVVVFDAKAPSFRHEAYEAYKAGRAPTPEDFPRQLALVK RLVDLLGLVRLEAPGYEADDVLGTLAKKAEREGMEVCILTGDRDFFQLLSEKVSVL LPDGTLVTPKDVQEKYGVPPERWVDFRALTGDRSDNIPGVAGIGEKTALRLLAEWG SVENLLKNLDRVKPDSLRRKIEAHLEDLRLSLDLVRIRTDLPLEVDFKALRRRTPDL EGLRAFLEELELGSLLHEFGLLGGEKPREEAPWPPPEGAFVGFLLSRKEPMWAELLA LAAAAEGRVHRATSPVEALADLKEARGFLAKDLAVLALREGVALDPTDDPLLVAY LLD S ANTNPERVARRYGGEFTED AAERALL SERLFQTLFPRL SEKLLWL YQEVERPL SRVLAHMEARGVRLDVPLLEALSFEAAKELERLEGEVFRLAGHPFNLNSRDQLEKV LFDELGLTPVTRTEKTGKRSTSAAALEALRGVHPIVELILQYRELSKLKSTYLDRLPR LVHPRTGRLHTRFNQTATATGRLS S SDPNLQNIPVRTPLGQHIRKAFVAEEGWLLLA ADYSQIELRVLAHLSGDENLKRVFREGKDIHTETAAWMFGVDSALVDPKMRRAAK TVNFGVLYGMSAARLSQELGIDYKEAEAFIERYFQSFPKVRAWIERTLEEGRTRGYV ETLFGRRRYVPDLTSRVRSVREAAERMAWNMPVQGTAADLMKIAMVKLFPRLKPL GAHLLLQVHDELVLEVPEDRAEEAKALVKEVMENAYPLDVPLEVEVGVGRDWLE AKQD SEQ ID NO: 19 represents an amino acid sequence of polymerase H3, containing a His-tag MHHHHHHGGSGMLPLFDLEEPPKRVLLVDGHHLAYRTFYALSLTTSRGEPVQMVY GFARSLLKALKEDGQAVVVVFDAKAPSFRHEAYEAYKAGRAPTPEDFPRQLALVK RLVDLLGLVRLEAPGYEADDVLGTLAI<I<AEREGMEVC1LTGDRDFFQLLSEI<VSVL LPDGTLVTPKDVQEKYGVPPERWVDFRALTGDRSDNIPGVAGIGEKTALRLLAEWG SVENLLKNLDRVKPDSLRRKIEAHLEDLRLSLDLARIRTDLPLEVDFKALRRRTPDL EGLRAFLEELEFGSLLHEFGLLGGEKPREEAPWPPPEGAFVGFLLSRKEPMWAELLA LAAAAEGRVHRATSPVEALADLKEARGFLAKDLAVLALREGVALDPTDDPLLVAY LLDP ANTNPEGVARRYGGEFTED AAERALL SERLFQTLFPRL SEKLLWL YQEVERPL SRVLAHMEARGVRLDVPLLEALSFELEKELERLEGEVFRLAGHPFNLNSRDQLEKV LFDELGLTPVTRTEKTGKRSTSAAALEALRGAHPIVELILQYRELSKLKSTYLDRLPR LVHPRTGRLHTRFNQTATATGRLS S SDPNLQNIPVRTPLGQRIRKAF VAEEGWLLLA ADYSQIELRVLAHLSGDENLKRVFREGKDIHTETAAWMFGLDPALVDPKMRRAAK TVNFGVLYGMSAHRLSQELGIDYKEAEAFIERYFQSFPKVRAWIERTLEEGRTRGYV ETLFGRRRYVPDLASRVRSVREAAERMAWNMPVQGTAADLMKIAMVKLFPRLKP LGAHLLLQVHDELVLEVPEDRAEEAKALVKEVMENAYPLDVPLEVEVGVGRDWL EAKQDAttorney Docket: ABBTT-43290.601

[0070] DETAILED DESCRIPTION

[0071] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0072] The presently disclosed subject matter provides polypeptides, compositions, kits, and methods related to hot start DNA polymerases. In some aspects, the polypeptides disclosed herein have a paratope specific to a polymerase having, for example, SEQ ID NO: 12-19 and structurally related polymerases. The polypeptides disclosed herein inhibit enzymatic activity of thermostable DNA polymerases by forming a polymerase-polypeptide complex, which can be relieved upon heating the polymerase-polypeptide complex. The polypeptides disclosed herein confer improved performance of hot start DNA polymerase complexes in assays as shown in the non-limiting examples described herein. For example, polypeptides disclosed herein confer improved performance of a hot start DNA polymerase complex in ALINITY M assays and improve amplification at the limit of detection providing high sensitivity (as compared to the DNA polymerase alone and the Kapa2G hot start DNA polymerase complex).

[0073] A. Definitions

[0074] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs. Definitions set forth herein facilitate explanation of the presently disclosed subject matter.Attorney Docket: ABBTT-43290.601

[0075] These definitions are intended to supplement and illustrate, not preclude, the definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.

[0076] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to a polymeric form of amino acids of any length, which can include coded and noncoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0077] The term “antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0078] A whole immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2, and Cm) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda ( ), based upon the amino acid sequences of their constant domains. In a typical immunoglobulin, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the 0 sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

[0079] The term “monoclonal antibody” refers to an antibody of a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population areAttorney Docket: ABBTT-43290.601

[0080] identical except for possible naturally-occurring mutations that may be present in minor amounts. The term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be expressed by hybridomas, or may be made by recombinant DNA methods and expressed from another cell type. Monoclonal antibodies may also be isolated from phage libraries.

[0081] In some aspects, the polypeptide disclosed herein is an IgG antibody. In some aspects, the polypeptide disclosed herein is an IgGl, IgG2, IgG3, or IgG4 antibody. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, 8, 8, y, and p, respectively. The y and p classes are further divided into subclasses e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 41-7) any of which are suitable for use in some of the embodiments herein. Common allotypic variants in human populations are those designated by the letters a, f, n, z or combinations thereof. In any of the embodiments herein, a polypeptide, antibody, or antibody fragment may comprise a heavy chain Fc region comprising a human IgG Fc region. In further embodiments, the human IgG Fc region comprises a human IgG4.

[0082] The term “antibody fragment” refers to a fragment of an antibody. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. In some embodiments, the portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains, (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments chemically linked, e.g. by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab' fragment, which results from breaking the disulfide bridge of an F(ab')2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), (vi) a domain antibody (dAb), which is an antibody single variable region domain (Vuor VL) polypeptide that specifically binds antigen, (vii) Fab'-SH fragments, (viii) FdAttorney Docket: ABBTT-43290.601

[0083] fragments, (ix) diabodies, which are bispecific antibody fragments comprising two antigen binding Fv domains, (x) single-chain Fv (scFv) molecules, (xi) single-chain polypeptides containing only one light chain variable domain, (xii) single-chain polypeptides containing the three CDRs of the light-chain variable domain, (xii) single-chain polypeptides containing only one heavy chain variable region, and (xiii) single-chain polypeptides containing the three CDRs of the heavy chain variable region. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigenbinding site, and a residual “Fc” fragment. Pepsin treatment yields a F(ab')2 fragment that has two antigen-binding sites. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, goat, horse, sheep, chicken, etc. Furthermore, for all antibody constructs provided herein, variants having the sequences from other organisms are also contemplated, such as CDR-grafted antibodies or chimeric antibodies. Antibody fragments also include either orientation of single chain scFvs, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, etc. Antibody fragments also include nanobodies (sdAb, an antibody having a single, monomeric domain, such as a pair of variable domains of heavy chains, without a light chain). An antibody fragment can be referred to as being a specific species in some embodiments (for example, human scFv or a mouse scFv). This denotes the sequences of at least part of the non-CDR regions, rather than the source of the construct.

[0084] The term “antigen binding variable region” refers to a hypervariable region of an antibody or antibody fragment responsible for antigen binding and comprises at least one CDR. “Antigen” as used herein refers generally to targets to which the polypeptides, antibodies, or antibody fragments bind. As described in the Examples, the polypeptides, antibodies, or antibody fragments bind to DNA polymerases.

[0085] The term “CDR” (e.g. CDR1, CDR2, CDR3) denotes a complementarity determining region as defined by at least one method known in the art. In some embodiments, CDRs can be defined in accordance with any of the Chothia numbering schemes, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).Attorney Docket: ABBTT-43290.601

[0086] The term “heavy chain amino acid sequence” as used herein refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0087] The term “heavy chain variable region” as used herein refers to a region comprising at least three heavy chain CDRs. In some embodiments, the heavy chain variable region includes the three CDRs and at least FR2 and FR3. In some embodiments, the heavy chain variable region includes at least heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a heavy chain variable region also comprises at least a portion of a FR1 and / or at least a portion of a FR4.

[0088] The term “heavy chain constant region” as used herein refers to a region comprising at least three heavy chain constant domains, CHI, CH2, and CH3. Non-function-altering deletions and alterations within the domains are encompassed within the scope of the term “heavy chain constant region,” unless designated otherwise. Nonlimiting exemplary heavy chain constant regions include y, 8, and a, and variants thereof that do not alter the functions of the antibody needed for its intended use. Nonlimiting exemplary heavy chain constant regions also include s and p, and variants thereof that do not alter the functions of the antibody needed for its intended use. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a y constant region is an IgG antibody, an antibody comprising a 8 constant region is an IgD antibody, and an antibody comprising an a constant region is an IgA antibody. Further, an antibody comprising a p constant region is an IgM antibody, and an antibody comprising an e constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgGl (comprising a yl constant region), IgG2 (comprising a y2 constant region), IgG3 (comprising a y3 constant region), and IgG4 (comprising a y4 constant region) antibodies; IgA antibodies include, but are not limited to, IgAl (comprising an al constant region) and IgA2 (comprising an a2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgMl and IgM2.

[0089] The term “light chain amino acid sequence” as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In someAttorney Docket: ABBTT-43290.601

[0090] embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full-length light chain” as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0091] The term “light chain variable region” as used herein refers to a region comprising at least three light chain CDRs. In some embodiments, the light chain variable region includes the three CDRs and at least FR2 and FR3. In some embodiments, the light chain variable region includes at least light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. For example, a light chain variable region may comprise light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a light chain variable region also comprises at least a portion of a FR1 and / or at least a portion of a FR4.

[0092] The term “light chain constant region” as used herein refers to a region comprising a light chain constant domain, CL. Nonlimiting exemplary light chain constant regions include X and K, and variants thereof that do not alter the functions of the antibody needed for its intended use.

[0093] The “% sequence identity” of an amino acid sequence refers to the degree of identical amino acids when two or more amino acid sequences for comparison are optimally aligned. The identity of an amino acid sequence can be calculated using analysis tools that are commercially available or available through telecommunication lines (internet). For example, the identity may be calculated using the commercially available software GENETYX (Genetyx Corporation) or using default parameters in the homology algorithm BLAST (Basic Local Alignment Search Tool) (http: / / www.ncbi.nlm.nih.gov / BLAST / ) of the National Centre for Biotechnology Information (NCBI). As used herein, and throughout, “at least 70%” sequence identity comprises at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity, or values therein between.

[0094] “Sensitivity” as used herein, refers to the ability of a polymerase to amplify a target nucleic acid that is present at low copy number. In some embodiments, low copy number refers to a target nucleic acid that is present at fewer than 10,000 or fewer than 1,000 or fewer than 100 or fewer than 10 copies in the composition comprising the target nucleic acid and the polymerase.

[0095] “Specificity” as used herein, refers to the ability of a polymerase to amplify a target nucleic acid while producing fewer non-specific amplification byproducts, such as thoseAttorney Docket: ABBTT-43290.601

[0096] resulting from primer-dimers. As used herein, when an antibody or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (Ka) of at least IO7Al1(e.g., >107NT1, >10xM >IO9M >10'°M '. >1011M’1, >IO,2M >10l3M etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

[0097] Thermostable DNA polymerases are commonly used in biotechnology and molecular biology applications, including nucleic acid synthesis techniques such as amplification (e.g., PCR), which involves cycles of alternating denaturation and primer annealing and extension. Thermostable DNA polymerases are resistant to inactivation by high temperatures and so are compatible with thermal denaturation steps. DNA polymerases comprise a catalytic domain that extends a 3' terminus of a DNA strand in a template-dependent manner. DNA polymerases can also comprise an exonuclease domain, such as a 3' to 5' exonuclease domain. Such an exonuclease domain can reduce the frequency of misincorporation by removing mismatched nucleotides from the 3' end of a nascent DNA strand. Certain artificial DNA polymerases further comprise a sequence non-specific double-stranded DNA (dsDNA) binding domain. The presence of this domain can improve performance of the enzyme with respect to various parameters, including processivity, sensitivity, and yield.

[0098] Nucleic acid amplification can permit rapid detection of a target nucleic acid sequence and / or provide sufficient quantities of a sample for further analysis or manipulation, such as sequencing, cloning, restriction digestion, hybridization, ligation, mutagenesis, recombination, etc. Two key parameters of amplification are sensitivity and yield. Improving the sensitivity reduces the minimum amount of a target needed to produce a detectable product. Improving the yield increases the amount of product that results from a reaction, or reduces the amount of time and / or reagents necessary to obtain a given amount of product.Attorney Docket: ABBTT-43290.601

[0099] Hot start DNA polymerase complexes are used in nucleic acid synthesis (such as amplification) reactions to reduce non-specific nucleic acid synthesis at lower temperatures, improving specificity. In some embodiments, the present application provides polypeptides that bind a DNA polymerase and which may be used in hot start compositions. In some embodiments, hot start compositions are provided, comprising a polypeptide described herein bound to a DNA polymerase.

[0100] As used herein, the term “hot start” generally refers to an approach of limiting the availability of an essential reaction component (e.g., a polymerase) when the reaction mixture is maintained at a first condition (e.g., temperature (typically a lower temperature)) until a second condition (e.g., temperature (typically a higher temperature)) is reached which allows the essential component to participate in the reaction. Hot start reactions typically involve incubation at a first (e.g., lower) temperature and subsequent elevation to a second (e.g., higher) temperature which allows the desired reaction to take place. Activation of the hot start reaction is preferably achieved by an incubation at a temperature which is equal to or higher than the primer hybridization (annealing) temperature used in the amplification reaction to ensure primer binding specificity. The length of incubation required to recover enzyme activity depends on the temperature and pH of the reaction mixture and on the stability of the enzyme. A wide range of incubation conditions are usable; optimal conditions may be determined empirically for each reaction.

[0101] The terms “sample” and “biological sample” are used in their broadest sense and encompass samples or specimens obtained from any source that contains or may contain eukaryotic cells, including biological and environmental sources. As used herein, the term “sample” when used to refer to biological samples obtained from organisms, includes bodily fluids (e.g., blood or saliva), feces, biopsies, swabs (e.g., buccal swabs), isolated cells, exudates, and the like. The organisms include fungi, plants, animals, and humans. However, these examples are not to be construed as limiting the types of samples or organisms that find use with the present invention. In addition, in order to perform research or study the results related to use of a method or composition of the invention, in some embodiments, a “sample” or “biological sample” comprises fixed cells, treated cells, cell lysates, and the like. Samples also include environmental samples.Attorney Docket: ABBTT-43290.601

[0102] “Variant” is used herein to describe a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of “biological activity” include the ability to bind to another molecule. Variant is also used herein to describe a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change “Variant” also can be used to describe a polypeptide or a fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its antigen reactivity.

[0103] B. Polypeptides

[0104] In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1. In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2. In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:3.

[0105] In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:4. In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:5. In some aspects, the presently disclosed subject matter provides a polypeptide comprising an antigen binding variable region comprising a CDR3 comprising the amino acidAttorney Docket: ABBTT-43290.601

[0106] sequence of SEQ ID NO:6 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:6.

[0107] In some aspects of the polypeptides disclosed herein, the at least 70% sequence identity comprises at least 80% sequence identity. In some aspects of the polypeptides disclosed herein, the at least 70% sequence identity comprises at least 90% sequence identity. In some aspects of the polypeptides disclosed herein, the at least 70% sequence identity comprises at least 95% sequence identity. In some aspects of the polypeptides disclosed herein, the at least 70% sequence identity comprises at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity.

[0108] In some aspects of the polypeptides disclosed herein, the antigen binding variable region is a light chain variable region that comprises a CDR1, CDR2, and CDR3. In some aspects, the light chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:3.

[0109] In some aspects of the polypeptides disclosed herein, the antigen binding variable region is a heavy chain variable region that comprises a CDR1, CDR2, and CDR3. In some aspects, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:4, a CDR2 comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:5, and a CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:6.

[0110] In some aspects, the polypeptide disclosed herein has a light chain variable region that comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:3; and a heavy chainAttorney Docket: ABBTT-43290.601

[0111] variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:4, a CDR2 comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:6.

[0112] In some aspects, the polypeptide disclosed herein has a light chain variable region that comprises a CDR1 comprising the amino acid sequence of SEQ IDNO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a heavy chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:4, a CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0113] In some aspects, the polypeptide disclosed herein comprises (i) a light chain amino acid sequence of SEQ ID NO: 7, and (ii) a heavy chain amino acid sequence of SEQ ID NO: 8. In some aspects of the polypeptides disclosed herein, the polypeptide is an antibody. In some aspects, the antibody is an IgG antibody. In some aspects of the polypeptides disclosed herein, the polypeptide is an antibody fragment. In some aspect, the antibody fragment is Fab or scFv. In some aspects, the polypeptide is a diabody. In some aspects, the polypeptide binds and reversibly inhibits a DNA polymerase. In some aspects, the polypeptide is heat labile.

[0114] Methods for producing and screening for polypeptides, antibodies, and antibody fragments that are suitable for use in hot start complexes or compositions with the polymerases described herein are known in the art. In some aspects, a polypeptide disclosed herein inhibits the nucleic acid synthesis activity of the thermostable DNA polymerase disclosed herein. In some aspects, a polypeptide disclosed herein inhibits exonuclease activity of the thermostable DNA polymerase disclosed herein. In some aspects, a polypeptide disclosed herein inhibits both the nucleic acid synthesis activity and the exonuclease activity of the thermostable DNA polymerase disclosed herein.

[0115] In some aspects, a polypeptide disclosed herein increases the specificity of nucleic acid synthesis reactions, because they inactivate the DNA polymerase at room temperature, thus avoiding extension of nonspecifically hybridized primers. The functional activity of the DNA polymerase is restored by disassociating the antibody from the DNA polymerase, for example, by incubating the composition at a higher temperature. In some aspects, the “higherAttorney Docket: ABBTT-43290.601

[0116] temperature” is from about 65°C to about 99°C, from about 70°C to about 99°C, from about 75°C to about 99°C, or from about 80°C to about 99°C, or from about 85°C to about 99°C, or from about 90°C to about 99°C, for a time period of at least 15 seconds, or at least 30 seconds, or at least 1 minute, or at least 90 seconds, or at least 2 minutes; to about 3 minutes, or about 4 minutes, or about 5 minutes, or about 7 minutes, or about 10 minutes, or more. In some aspects, the higher temperature is at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, or at least 85°C. In some aspects, the temperature and duration of incubation to disassociate the antibody and activate the polymerase may be determined for the particular polymerase and antibody to be employed. One skilled in the art can determine the appropriate temperature and duration of incubation.

[0117] Methods for screening for polypeptides, antibodies, and antibody fragments of use in the present invention include methods known in the art, such as affinity -based ELISA assays, as well as functional assays for polymerase and / or exonuclease inhibition. For such functional assays, the amount of DNA produced or digested per unit of time can be correlated to the activity of the polymerase or exonuclease used, thus providing an estimate of the amount of inhibition a particular antibody can exert on either or both the polymerase and exonuclease activity of the polymerase.

[0118] Antibodies may be produced using any method known in the art. As a nonlimiting example, an antibody to a particular antigen (such as a polymerase described herein) may be produced by immunizing an animal (such as a mouse, rat, rabbit, goat, sheep, horse, etc.) with the antigen and isolating antibodies from the serum of the animal and / or immortalizing primary B cells from the animal to produce hybridomas that express the antibodies. Phage display technology may also be used to produce antibodies that bind to the polymerases described herein. Phage display libraries are commercially available and methods of selecting antibodies from such libraries are known in the art. See, e.g., Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581. In some embodiments, antibodies are produced by recombinant expression in a host organism (e.g., E. coli followed by purification.

[0119] C. CompositionsAttorney Docket: ABBTT-43290.601

[0120] Tn some aspects, the presently disclosed subject matter provides a polymerasepolypeptide complex comprising a polypeptide disclosed herein that is bound to a thermostable DNA polymerase, wherein 5 ’ to 3 ’ polymerase activity of the thermostable DNA polymerase is inhibited. In some aspects, the presently disclosed subject matter provides a composition comprising a polymerase-polypeptide complex comprising a polypeptide disclosed herein that is bound to a thermostable DNA polymerase, wherein 5’ to 3 ’ polymerase activity of the thermostable DNA polymerase is inhibited. In some aspects, the thermostable polymerase is a polymerase as described in WO2024102861, herein incorporated by reference in its entirety. In some aspects of the composition disclosed herein, the thermostable DNA polymerase comprises a sequence selected from the group consisting of SEQ ID Nos 12-19. In some aspects, the presently disclosed subject matter provides a reaction mixture comprising a polypeptide disclosed herein and a thermostable DNA polymerase. In some aspects, the thermostable polymerase is a polymerase as described in WO2024102861, herein incorporated by reference in its entirety. In some aspects of the reaction mixture disclosed herein, the thermostable DNA polymerase comprises a sequence selected from the group consisting of SEQ ID Nos 12-19. In some embodiments, the composition or reaction mixture disclosed herein comprising the polypeptide and thermostable DNA polymerase is a hot start composition.

[0121] In some aspects, the composition or reaction mixture comprises a 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1.5:1, 1.4:1, 1.3:1, 1.2:1, or 1.1:1 molar / mass ratio of the thermostable DNA polymerase to the polypeptide disclosed herein. In some aspects, the composition or reaction mixture comprises the thermostable DNA polymerase and the polypeptide disclosed herein at a mass ratio of 1:1.5, wherein the polypeptide is a monoclonal antibody. In some aspects, the composition or reaction mixture comprises the thermostable DNA polymerase and the polypeptide disclosed herein at a mass ratio of 1: 1, wherein the polypeptide is a rFab.

[0122] In some aspects, the thermostable DNA polymerase comprises a DNA polymerase catalytic domain, a 3' to 5' exonuclease domain, and / or a non-specific DNA-binding domain. In some aspects the thermostable DNA polymerase comprises a DNA polymerase catalytic domain, a 3' to 5' exonuclease domain, and a non-specific DNA-binding domain. In some aspects, all domains of the thermostable DNA polymerase are contained in a single polypeptide. In some aspects, the thermostable DNA polymerase comprises a plurality ofAttorney Docket: ABBTT-43290.601

[0123] polypeptide chains, which may be noncovalently associated or covalently associated. The plurality of polypeptide chains can include a first polypeptide comprising a polymerase catalytic domain and a second polypeptide comprising an additional domain, such as a sequence non-specific DNA-binding domain.

[0124] In some aspects, the reaction mixture is a dual hot start reaction mixture. The term “dual hot start reaction mixture” refers to the combination of reagents or reagent solutions which are used to block nucleic acid polymerase extension at low temperatures (e.g., ambient temperature) until the hot start conditions of the initial denaturation temperature in an amplification reaction (e.g., PCR) are reached. At the elevated amplification temperature, the nucleic acid polymerase is no longer inhibited and allows for primer extension. As used herein, the dual hot start reaction mixture is meant to include a reaction mixture that comprises at least two different mechanisms for hot start. Accordingly, “dual hot start reaction mixtures” may include more than two hot start mechanisms (e.g., “triple hot start reaction mixture”, “quadruple hot start reaction mixture”, “quintuple hot start reaction mixture”, and so on).

[0125] Nonlimiting exemplary hot start mechanisms include, but are not limited to, antibodies or combinations of antibodies that block nucleic acid polymerase activity at lower temperatures and which dissociate from the polymerase at elevated temperatures (see, e.g., Eastlund et al., LifeSci. Quarterly 2:2 (2001), Mizuguchi et al., J. Biochem. (Tokyo) 126:762 (1999)); affibodies (small synthetic protein molecules that have high binding affinity for a target protein) or combinations of affibodies, sometimes referred to as antibody mimetics; oligonucleotides that block nucleic acid polymerase activity at lower temperatures and which dissociate from the polymerase at elevated temperatures (see, e.g., Dang et al., J. Mol. Biol. 264:268 (1996)); reversibly chemical modification of the nucleic acid polymerase such that the nucleic acid polymerase activity is blocked at lower temperatures and the modifications reverse or dissociate at elevated temperatures (see, e.g., U.S. Pat. No. 5,773,258 and Moretti et al., Biotechniques 25:716 (1998)); amino acid mutations of the nucleic acid polymerase that provide reduced activity at lower temperatures (see, e.g., Kermekchiev et al., Nucl. Acids Res. 31:6139 (2003)); nucleic acid polymerase fusion proteins including hyperstable DNA binding domains and topoisomerases (see, e.g., Pavlov et al., Proc. Natl. Acad. Sci. USA 99:13510 (2002)); ligands that inhibit the nucleic acid polymerase in a temperature-dependent manner (for example, HotMaster™ Taq DNA polymerase fromAttorney Docket: ABBTT-43290.601

[0126] Eppendorf (Hauppauge, N.Y.) and 5 PRIME (Gaithersburg, Md.)); single-stranded binding proteins that sequester primers at low temperatures (see, e.g., U.S. Patent Application Publication No. 2008 / 0138878); thermostable pyrophosphatase which hydrolyzes inorganic pyrophosphate at elevated temperatures (see, e.g., U.S. Patent Application Publication No.

[0127] 2006 / 0057617); thermolabile blockers, such as a polymerase blocking protein (see, e.g., U.S. Patent Application Publication No. 2007 / 0009922); primer competitor sequences (see, e.g., Puskas et al., Genome Res. 5:309 (1995) and Vestheim et al., Front. Zool. 5:12 (2008)); modified primer constructs (see, e.g., Ailenberg et al., Biotechniques 29:22 (2000) and Kaboev et al., Nucl. Acids Res. 28:E94 (2000)); modified primers that improve hybridization selectivity (see, e.g., U.S. Pat. Nos. 6,794,142 and 6,001,611); primers with 3' modifications that are removable by 3'-5' exonuclease activity (see, e.g., U.S. Patent Application Publication No. 2003 / 0119150 and U.S. Pat. No. 6,482,590); primers with modified nucleobases that are removable by UV irradiation (see, e.g., Young et al., Chem. Commun. (Camb) 28:462 (2008)); primer modifications that are removable by thermal deprotection (see, e.g., U.S. Patent Application Publication No. 2003 / 0162199 and Lebedev et al., Nucl. Acids Res. 36:el31 (2008)); or modification of the dNTPs with thermolabile modification groups (see, e.g., U.S. Patent Application Publication No. 2003 / 0162199 andKoukharevaetal., Nucl. Acids Symp. Ser. (Oxford), 259 (2008)). Agents that are used as hot start mechanisms, such as, but not limited to, antibodies, oligonucleotides, Affibodies®, chemical modifications, etc., may be referred to as “hot start inhibitors.”

[0128] D. Kits

[0129] In some aspects, the presently disclosed subject matter provides a kit comprising a polypeptide disclosed herein. In some aspects, the kit disclosed herein further comprises a thermostable DNA polymerase. In some aspects, the thermostable DNA polymerase is bound to said polypeptide. In some aspects, the thermostable polymerase is a polymerase as described in WO2024102861, herein incorporated by reference in its entirety. In some aspect, the thermostable DNA polymerase comprises a sequence selected from the group consisting of SEQ ID Nos 12-19. In some aspects, the thermostable DNA polymerase is a Taq DNA polymerase or a variant thereof. In some aspects, the kit disclosed herein further comprises at least one reagent for a polymerase chain reaction. In some aspects, the at least one reagent for a polymerase chain reaction is selected from controls, buffers, dNTPs, primers, andAttorney Docket: ABBTT-43290.601

[0130] stabilizers. In some aspects, the at least one reagent for polymerase chain reaction is a buffer comprising MgCh. In some aspects, the at least one reagent for polymerase chain reaction is a reagent comprising MgCh, TMAC, or KC1.

[0131] In some aspects, the kit comprises the thermostable DNA polymerase and the polypeptide disclosed herein at a mass ratio of 1 : 1.5, wherein the polypeptide is a monoclonal antibody. In some aspects, the kit comprises the thermostable DNA polymerase and the polypeptide disclosed herein at a mass ratio of 1 : 1, wherein the polypeptide is a rFab. In some embodiments, the kit comprises 6mg / ml each of the thermostable DNA polymerase and the polypeptide disclosed herein, wherein the polypeptide is a rFab.

[0132] In some aspects, the kit further comprises a storage buffer. In some aspects, the storage buffer comprises a stabilizer. In some aspects, the storage buffer storage buffer comprises Tris-HCl, KC1, EDTA, Tween, and glycerol. In some aspects, the storage buffer comprises 10 mM Tris-HCl (pH 7.4), 100 mM KC1, 0.1 mM EDTA, 0.5% Tween 20, 22% glycerol. In some aspects, the kit can be stored in -20 °C storage conditions.

[0133] In some aspects, the kit comprises reagents for nucleic acid synthesis or polymerase chain reaction. In some aspects, reagents for nucleic acid synthesis include any one or any combination of target polynucleotides, particles attached with capture primers, solution-phase primers, fusion primers, other additional primers, enzymes (e.g., polymerases), accessory proteins (e.g., recombinase, recombinase loading protein, single-stranded binding protein, helicase or topoisomerase), nucleotides, divalent cations, binding partners, co-factors and / or buffer. In some aspects, reagents for nucleic acid synthesis include a dUTPase as an accessory protein.

[0134] In some aspects, the kit comprises compositions comprising one type, or a mixture of different types of nucleotides. A nucleotide comprises any compound that can bind selectively to, or can be polymerized by, a polymerase. Typically, but not necessarily, selective binding of the nucleotide to the polymerase is followed by polymerization of the nucleotide into a nucleic acid strand by the polymerase. Such nucleotides include not only naturally occurring nucleotides but also any analogs, regardless of their structure, that can bind selectively to, or can be polymerized by, a polymerase. While naturally occurring nucleotides typically comprise base, sugar and phosphate moieties, the nucleotides of the present disclosure can include compounds lacking any one, some or all of such moieties. In some embodiments, theAttorney Docket: ABBTT-43290.601

[0135] nucleotide can optionally include a chain of phosphorus atoms comprising three, four, five, six, seven, eight, nine, ten or more phosphorus atoms. In some embodiments, the phosphorus chain can be attached to any carbon of a sugar ring, such as the 5' carbon. The phosphorus chain can be linked to the sugar with an intervening O or S. In some embodiments, one or more phosphorus atoms in the chain can be part of a phosphate group having P and O. In some embodiments, the phosphorus atoms in the chain can be linked together with intervening O, NH, S, methylene, substituted methylene, ethylene, substituted ethylene, CNH2, C(O), C(CH2), CH2CH2, or C(OH)CH2R (where R can be a 4-pyridine or 1 -imidazole). In some embodiments, the phosphorus atoms in the chain can have side groups having O, BH3, or S. In the phosphorus chain, a phosphorus atom with a side group other than O can be a substituted phosphate group. In the phosphorus chain, phosphorus atoms with an intervening atom other than O can be a substituted phosphate group. Some examples of nucleotide analogs are described in Xu, U.S. Pat. No. 7,405,281.

[0136] Some examples of nucleotides that can be used in the disclosed kits or compositions (and related methods, systems, kits and apparatuses) include, but are not limited to, ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotide polyphosphates, modified ribonucleotide polyphosphates, modified deoxyribonucleotide polyphosphates, peptide nucleotides, modified peptide nucleotides, metallonucleosides, phosphonate nucleosides, and modified phosphate-sugar backbone nucleotides, analogs, derivatives, or variants of the foregoing compounds, and the like. In some embodiments, the nucleotide can comprise non-oxygen moieties such as, for example, thio- or borano-moieties, in place of the oxygen moiety bridging the alpha phosphate and the sugar of the nucleotide, or the alpha and beta phosphates of the nucleotide, or the beta and gamma phosphates of the nucleotide, or between any other two phosphates of the nucleotide, or any combination thereof. In some embodiments, a nucleotide can include a purine or pyrimidine base, including adenine, guanine, cytosine, thymine, uracil or inosine. In some embodiments, a nucleotide includes dATP, dGTP, dCTP, dTTP and / or dUTP.

[0137] In some aspects, the nucleotide is unlabeled. In some aspects, the nucleotide comprises a label and referred to herein as a “labeled nucleotide”. In some aspects, the label can be in the form of a fluorescent dye attached to any portion of a nucleotide including a base, sugarAttorney Docket: ABBTT-43290.601

[0138] or any intervening phosphate group or a terminal phosphate group, i.e., the phosphate group most distal from the sugar.

[0139] In some aspects, the kit comprises any one or any combination of capture primers, reverse solution-phase primers, fusion primers, target polynucleotides and / or nucleotides that are non-labeled or attached to at least one label. In some aspects, the label comprises a detectable moiety. In some aspects, the label can generate, or cause to generate, a detectable signal. In some aspects, the detectable signal can be generated from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). For example, a proximity event can include two reporter moieties approaching each other, or associating with each other, or binding each other. In some aspects, the detectable signal can be detected optically, electrically, chemically, enzymatically, thermally, or via mass spectroscopy or Raman spectroscopy. In some aspects, the label can include compounds that are luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent or electrochemical. In some aspects, the label can include compounds that are fluorophores, chromophores, radioisotopes, haptens, affinity tags, atoms or enzymes. In some aspects, the label comprises a moiety not typically present in naturally occurring nucleotides. For example, the label can include fluorescent, luminescent or radioactive moieties.

[0140] E. Methods

[0141] In some aspects, the presently disclosed subject matter provides a method comprising: contacting a sample containing a target nucleic acid with a polypeptide disclosed herein that is bound to a thermostable DNA polymerase. In some aspects, the method disclosed herein further comprises heating the sample to a temperature that disassociates the polypeptide from the thermostable DNA polymerase. In some aspects, the method disclosed herein further comprises amplifying the target nucleic acid. In some aspects, the amplifying comprises a polymerase chain reaction. In some aspects, the method disclosed herein further comprises detecting amplicons, or byproduct thereof, of the target nucleic acid. In some aspects, the target nucleic acid is DNA. In some aspects, the target nucleic acid is RNA. In some aspects, the RNA is converted into cDNA by reverse transcription prior to the contacting.Attorney Docket: ABBTT-43290.601

[0142] Tn some aspects of the method disclosed herein, the target nucleic acid is from an infectious disease agent. In some aspects, the infectious disease agent is at least one of a bacterium, a virus, a fungi, a protozoa, a helminth, or a prion. In some aspects, the infectious disease agent is at least one of monkeypox (MPXV), Epstein-Barr Virus (EBV), CMV (Cytomegalovirus), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG) Influenza virus (e.g., Flu A, FluB), Respiratory syncytial virus (RSV), SARS-CoV-2 virus, Hepatitis C virus (HCV), Hepatitis B virus (HBV), Human immunodeficiency virus (HIV), Human papillomavirus (HPV). In some aspects, the sample is, or is derived from, an endocervical swab specimen, a clinician-collected vaginal swab specimen, a self-collected vaginal swab specimen, a gynecological specimen, or urine, although any sample type may be used.

[0143] In some aspects, the presently disclosed subject matter provides a method of amplifying a target nucleic acid, the method comprising: (i) providing a solution comprising: (a) a DNA template, (b) a primer complementary to a target nucleic acid strand of the DNA template, (c) the polymerase-antibody complex disclosed herein, and (d) dNTPs, (ii) heating the solution wherein the antibody or fragment disassembles or disassociates from the thermostable DNA polymerase, and (iii) lowering the temperature of the solution wherein the primer is extended by the thermostable DNA polymerase to produce a target amplicon. In some aspects, the solution further comprises fluorescent dyes, colorimetric markers or radioactive markers. In some aspects, the method further comprises (iv) denaturing the target amplicon, (v) lowering the temperature of the solution wherein the primer is extended by the thermostable DNA polymerase to produce a new target amplicon, and repeating steps (iv)-(v) for 1-45 cycles. In some aspects, the method further comprises detecting target amplicons. In some aspects, the target amplicons are detected by sequencing, gel electrophoresis, or hybridization with labeled probes. Detection may be conducted in real-time or at one or more time points, including an “end point” after all cycles of amplification are completed.

[0144] In some aspects, the amplification reaction is a quantitative or semi-quantitative amplification reaction. In some aspects, the amplification is a multiplex reaction. In some aspects, the amplification reaction utilized nested primers. In some aspects, the amplification reaction utilized cDNA as a template, whereby the cDNA is generated from reverse transcription of RNA.Attorney Docket: ABBTT-43290.601

[0145] In some aspects the amplification reaction is an isothermal reaction. In some such embodiments, a first heating step is conducted to disrupt the polypeptide / polymerase inhibition, followed by amplification at a single temperature (or substantially single temperature), which may be the same or a different temperature than the temperature used to remove the hot start inhibition.

[0146] In some aspects, the presently disclosed subject matter provides a method of amplifying a target nucleic acid, the method comprising: (i) providing a solution comprising: (a) a DNA template, (b) a primer complementary to a target nucleic acid strand of the DNA template, (c) the polypeptide, antibody, or antibody fragment disclosed herein, (d) a thermostable DNA polymerase to which said antibody or fragment binds, and (e) dNTPs, (ii) heating the solution wherein the polypeptide, antibody or fragment disassembles or disassociates from the thermostable DNA polymerase, and (iii) lowering the temperature of the solution wherein the primer is extended by the thermostable DNA polymerase to produce a target amplicon. In some aspects, the solution further comprises fluorescent dyes, colorimetric markers or radioactive markers. In some aspects, the method further comprises (iv) denaturing the target amplicon, (v) lowering the temperature of the solution wherein the primer is extended by the thermostable DNA polymerase to produce a new target amplicon, and repeating steps (iv)-(v) for 1-45 cycles. In some aspects, the method further comprises detecting target amplicons. In some aspects, the target amplicons are detected by sequencing, gel electrophoresis, or hybridization with labeled probes.

[0147] In some aspects of the methods disclosed herein, the solution further comprises fluorescent dyes and the method further comprises: (iv) denaturing the amplicon, (v) lowering the temperature of the solution wherein the primer is extended by the thermostable DNA polymerase to produce a new target amplicon, (vi) repeating steps (iv)-(v) for 1-45 cycles, (vii) performing real-time fluorescent detection of target amplicons.

[0148] In some aspect of the methods disclosed herein, the method produces an amount of target amplicons greater than a second method that produces target amplicons with a KAPA2G Hotstart polymerase under the same amplification conditions and with the same reagents. In some aspects, the respective amounts are compared by Ct and / or RFU. In some aspect of the methods disclosed herein, the method produces an amount of target amplicons greater than a second method that produces target amplicons with a thermostable native TaqAttorney Docket: ABBTT-43290.601

[0149] DNA polymerase under the same amplification conditions and with the same reagents. In some aspects, the respective amounts are compared by Ct and / or RFU.

[0150] In some aspects, the presently disclosed subject matter provides a method for detecting at least one of Chlamydia trachomatis (CT), DNA from Neisseria gonorrhoeae (NG), RNA from Trichomonas vaginalis (TV), and RNA from Mycoplasma genitalium (MG), the method comprising the method disclosed herein, wherein the solution comprises: a primer complementary to a target CT RNA strand, a primer complementary to a target NG DNA strand, a primer complementary to a target MG RNA strand, and a primer complementary to a target TV RNA strand, and analyzing the corresponding target amplicons to detect at least one of CT, NG, TV, and MG. In some aspects, the sample is an endocervical swab specimen, a clinician-collected vaginal swab specimen, a self-collected vaginal swab specimen, a gynecological specimen, female urine, or male urine. In some aspects, the target amplicons can be detected at 3x or lOx Limit of Detection (LoD).

[0151] In some embodiments, conditions that are suitable for nucleic acid hybridization and / or nucleic acid synthesis include parameters such as salts, buffers, pH, temperature, % GC content of the polynucleotide and primers, and / or time. For example, conditions suitable for hybridizing nucleic acids (e.g., polynucleotides and primers) can include hybridization solutions having sodium salts, such as NaCl, sodium citrate and / or sodium phosphate. In some embodiments, a hybridization solution can be a stringent hybridization solution which can include any combination of formamide (e g., about 50%), 5*SSC (e g., about 0.75 M NaCl and about 0.075 M sodium citrate), sodium phosphate (e.g., about 50 mM at about pH 6.8), sodium pyrophosphate (e.g., about 0.1%), 5><Denhardfs solution, SDS (e.g., about 0.1%), and / or dextran sulfate (e.g., about 10%). In some embodiments, hybridization and / or nucleic acid synthesis can be conducted at a temperature range of about 45-55° C., or about 55-65° C., or about 65-75° C. In some embodiments, hybridization or nucleic acid synthesis conditions can be conducted at a pH range of about 5-10, or about pH 6-9, or about pH 6.5-8, or about pH 6.5-7.

[0152] Thermal melting temperature (Tm) for nucleic acids can be a temperature at which half of the nucleic acid strands are double-stranded and half are single-stranded under a defined condition. In some embodiments, a defined condition can include ionic strength and pH in an aqueous reaction condition. A defined condition can be modulated by altering theAttorney Docket: ABBTT-43290.601

[0153] concentration of salts (e g., sodium), temperature, pH, buffers, and / or formamide. Typically, the calculated thermal melting temperature can be at about 5-30° C. below the Tm, or about 5-25° C. below the Tm, or about 5-20° C. below the Tm, or about 5-15° C. below the Tm, or about 5-10° C. below the Tm. Methods for calculating a Tmare well known and can be found in Sambrook (1989 in “Molecular Cloning: A Laboratory Manual”, 2ndedition, volumes 1-3; Wetmur 1966, J. Mol. Biol., 31:349-370; Wetmur 1991 Critical Reviews in Biochemistry and Molecular Biology, 26:227-259). Other sources for calculating a Tmfor hybridizing or denaturing nucleic acids include OligoAnalyze (from Integrated DNA Technologies) and Primer3 (distributed by the Whitehead Institute for Biomedical Research).

[0154] EXAMPLES

[0155] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

[0156] 1874, 1874 IgG, purified IgG, 1874 mlgGk, Monoclonal antibody comprising SEQ ID NO: 7 light chain (kappa sequence) and 1-1874 mlgG

[0157] SEQ ID NO: 8 heavy chain (gamma 1) sequence

[0158] 1874 rFab, purified rFab Antibody fragment comprising SEQ ID NO: 9 Fab light chain sequence and SEQ 1874 rFab-His7

[0159] ID NO: 11 Fab heavy chain sequence with a C-terminal His7 tag

[0160] Heavy chain without interchain disulfide H7-rFab H7 polymerase - 1874 rFab complex H7-lgG H7 polymerase - 1874 IgG complex H3, H4, H5, H6, H7 DNA polymerase variants H3, H4, H5, H6,

[0161]

[0162] H7Attorney Docket: ABBTT-43290.601

[0163] Kapa2G, K2G, Commercial DNA polymerase

[0164] K2G Hotstart, Kapa2G Hotstart Commercial hotstart DNA polymerase HIV human immunodeficiency virus, sexually transmitted infection

[0165] HPV39 Human papillomavirus infection genotype, sexually transmitted infection

[0166] HPV16 Human papillomavirus infection genotype, sexually transmitted infection

[0167] HPV18 Human papillomavirus infection genotype, sexually transmitted infection

[0168] HPV58 Human papillomavirus infection genotype, sexually transmitted infection

[0169] HPV45 Human papillomavirus infection genotype,

[0170]

[0171] sexually transmitted infectionAttorney Docket: ABBTT-43290.601

[0172] Example 1 - Design of the 1874 rFab for rapid manufacture of homogenous preparations of antibody

[0173] The design of the 1874 rFab permits rapid manufacture of homogeneous preparations of antibody.

[0174] Plasmid used to express 1874 rFab. The bicistronic DNA sequence coding for 1874 rFab light chain (SEQ ID NO: 9) with a PelB signal sequence and for heavy chain (SEQ ID NO: 10) with a PhoA signal sequence and a C-terminal His7-tag was synthesized and inserted into the pD861 vector under the control of the rhaB promoter, resulting in the plasmid pD861 -1874_rFab for expressing the rFab by E.coli in shake flasks.

[0175] FIG. 2 illustrates the design of the 1874 rFab for rapid manufacture of homogeneous preparations of antibody by E.coli in shake flasks.

[0176] New design comprising heavy chain without an interchain disulfide. The similar above bicistronic sequence except a Ser mutation of the Cys of the rFab heavy chain (SEQ ID NO: 11) was cloned into a pBR322-derived vector driven by the araBAD promoter, generating the plasmid pBR322ara-1874_rFab for expressing the rFab by E.coli in fermenters. See FIG.

[0177] 3, A illustrates the design of the 1874 rFab for rapid manufacture of homogeneous preparations of antibody by fermentation. B shows the SDS-PAGE of the 1874 rFab comprising a heavy chain without an interchain disulfide (O: original design, heavy chain (HC) with interchain disulfide; N: new design, HC without interchain disulfide).

[0178] EXAMPLE 2 - Purification of enzyme and antibodies

[0179] Intact monoclonal antibody (IgGlk) was purified from the culture supernatant of the hybridoma cell line 1-1874 using Mab Select Xtra column (Cytiva) and then desalted using HiPrep 26 / 10 desalting column (Cytiva) on AKTA. The N-terminally His6-tagged DNA polymerase H7 was expressed in BL21 (NEB) under the control of the rhaB promoter and purified using prepacked cOmplete His-Tag purification column (Roche) and desalted by PD-10 column (Cytiva) with the storage buffer (10 mM Tris-HCl (pH 7.4), 100 mM KC1, 0.1 mM EDTA, 0.5% Tween 20, 22% glycerol). Recombinant Fab (rFab) with His7-tag fused to the C-terminus of the heavy chain was expressed in BL21 (NEB) under the control of theAttorney Docket: ABBTT-43290.601

[0180] rhamnose promoter or was expressed in 10-beta (NEB) driven by the arabinose promoter and purified using prepacked cOmplete His-Tag purification column (Roche) and desalted by PD-10 column (Cytiva) with the storage buffer.

[0181] FIG. 4 shows the SDS-PAGE of His6-H7, 1874 IgG, and 1874 rFab-His7 on 4-15% TGX gel (4pg of enzyme or 6 pg of antibody).

[0182] EXAMPLE 3 - Inhibition and reactivation of the DNA polymerase activity

[0183] EvaEZ fluorometric polymerase activity assay kit from BioTium was used to test the effect of the purified antibodies (IgG or rFab) on the enzymatic activity of the purified DNA polymerase. Briefly, 20 ng of the enzyme and certain amount (0 - 100 ng) of antibody were added into each well of a 96-well PCR plate and the volume was brought to 10 pl. 10 pl of EvaEZ assay master mix was then added into each well on ice. After short spin, the reactions were performed on a BioRad CFX96 Real-Time System using the following settings:

[0184] Cycling parameters for Biorad

[0185] CFX96

[0186] Cycle Temp Duration # of cycles Channel

[0187] I. Incubation 50 °C 15 sec 80 FAM

[0188] II. Denaturation 95 °C 15 sec 30 FAM

[0189]

[0190] III. Incubation 50 °C 15 sec 80 FAM

[0191] The initial rate of each reaction at step I and III (red and green in following figure) was calculated using the reference of that of the reaction without any antibody at step I as 100%.

[0192] FIG. 5 provides a graph comparing purified IgG or rFab inhibition of relative activity (%) of H7 (20 pg / rxn) DNA polymerase variant before heating and after heating.

[0193] The results demonstrated that both IgG from hybridoma and rFab from E.coli are able to inhibit the enzymatic activity of the DNA polymerase and the inhibition can be relieved by heat treatment.Attorney Docket: ABBTT-43290.601

[0194] EXAMPLE 4 -1874 mTgGk inhibitory activity of and activation post-heating

[0195] The 1874 mlgGk antibody inhibits a number of different polymerase variants. This antibody maintained inhibitory activity to eight polymerase variants suggesting the conserved nature of the epitope. See, for example, inhibition of H5 variant and H6 variant and activation post-heating. FIG. 6 and FIG. 7.

[0196] Inhibition of DNA polymerase activity

[0197] EvaEZ fluorometric polymerase activity assay kit from BioTium was used to test the effect of the purified antibodies (IgG) on the enzymatic activity of the purified variants H5 and H6. Briefly, 20 ng of either enzyme and certain amount (0 - 120 ng) of antibody were added into each well of a 96-well PCR plate and the volume was brought to 10 p.1. 10 pl of EvaEZ assay master mix was then added into each well on ice. After short spin, the reactions were performed on a BioRad CFX96 Real-Time System using the following settings:

[0198] Cycling parameters for Biorad

[0199] CFX96

[0200] Cycle Temp Duration # of cycles Channel

[0201] I. Incubation 50 °C 15 sec 45 FAM

[0202] IL Denaturation 95 °C 15 sec 15 FAM

[0203]

[0204] III. Incubation 50 °C 15 sec 60 FAM

[0205] The initial rate of each reaction at step I and III (red and green in following figure) was calculated using the reference of that of the reaction without any antibody at step I as 100%.

[0206] Results: FIG. 6 provides a RFU graph of 1874 mlgGk inhibition of H5 DNA polymerase variant with 1-1874 mlgG at 50-95-50°C and a RFU graph of 1874 mlgGk inhibition of H6 DNA polymerase variant with 1-1874 mlgG at 50-95-50°C.

[0207] Results: FIG. 7 provides a graph showing 1-1874 mlgG inhibition of relative activity (%) of H5 DNA polymerase variant and H6 DNA polymerase variant before heating and after heating. The 1874 mlgGk antibody has thermostability at 71 °C. It was found that a 1 : 1 molar ratio of 1874 mlgGk to DNA polymerase gave the desired performance. The antibody maintained inhibitory activity to eight polymerase variants suggesting the conserved nature of the epitope.Attorney Docket: ABBTT-43290.601

[0208] EXAMPLE 5 - ALTNITY M STI assay performance of 1874 IgG and 1874 rFab formulations

[0209] The 1874 mlgGk was reformatted into an rFab expressed in E. coli. This construct had a C-terminal His-tag added to the heavy chain to facilitate purification, and the interchain disulfide bond between the light and heavy chains was removed to eliminate heavy-heavy dimers that prevented a homogeneous rFab preparation. The protein could be isolated from the cell lysate and supernatant via IMAC.

[0210] The antibody is formulated with the DNA polymerase at an appropriate molar or mass ratio to abolish the enzyme activity as determined by a primer extension assay (e.g., EvaEZ™ Fluorometric Activity Assay - an EvaGreen-based DNA polymerase activity assay - Mao F, Leung WY, Xin X: Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol 2007, 7:76). The antibody is heat labile such that, upon heating to 95°C, the enzyme activity is restored to a level comparable to the enzyme without the inhibitory antibody (see FIG. 1).

[0211] Alinity m STI assay performance

[0212] The six targets of the STI assay were amplified at 3x or lOx Limit of Detection (LoD). The six targets were RNA from Trichomonas vaginalis (TV), RNA from Mycoplasma genitalium (MG), RNA from Chlamydia trachomatis (CT), DNA from Neisseria gonorrhoeae (NG). The on-market polymerase (Kapa2G) was compared to the enzyme alone (No Ab), Hot Start with 1874 rFAB, or Hot Start with 1874 IgG.

[0213] Results: FIG. 8 shows ALINITY M STI assay performance comparison of enzyme alone (No Ab), Hot Start (H3, H4, H5, H6, H7 with 1874 rFAB), Hot Start (H3, H4, H5, H6, H7 with 1874 IgG), and Kapa2G for six targets (TV, MG, CT, NG, IC, and CC).

[0214] Adding 1874 rFAB allows the enzyme to amplify the targets similarly to the on-market K2G enzyme. Formulation of a 1:1 ratio of DNA polymerase to rFab by mass was found to produce the most effective Hot Start formulation.Attorney Docket: ABBTT-43290.601

[0215] EXAMPLE 6 -Polymerase Formulation comprising antibody or antibody fragment and polymerase

[0216] The HotStart DNA polymerase complexes were formulated by mixing the purified enzyme and IgG at a mass ratio of 1 : 1.5 or by mixing equal mass amount of purified enzyme and rFab at room temperature for 5 minutes.

[0217] EXAMPLE 7 - HIV amplification assay

[0218] The HotStart formulations were tested in amplifying the HIV amplicon (0, 5, or 500 copies per reaction) along with the enzyme-only and a product from a commercial supplier (KAPA2G HotStart from Roche) by qPCR. The amplification was monitored by the signal in the FAM channel. The shape, quantification cycle (Ct), and the end RFU of the amplification curves were compared. The amplification reactions were performed on a BioRad CFX96 Real-Time System using the following settings:

[0219] Cycling parameters for Biorad

[0220] CFX96

[0221] Cycle Temp Duration # of cycles Channel Reverse

[0222] 52 °C 10 min 1

[0223] transcription

[0224] Hot start 95 °C 3 min 1

[0225] Amplification - melt 92 °C 3 sec

[0226] Amplification - 2

[0227] 50 °C 16 sec

[0228] anneal

[0229] Amplification - melt 92 °C 3 sec FAM Amplification - 8

[0230] 60 °C 16 sec

[0231] anneal

[0232] Amplification - melt 92 °C 3 sec

[0233] Amplification - 55 °C 16 sec 34

[0234] anneal

[0235]

[0236] Read 34 °C 16 sec

[0237] FIG. 9 shows HIV amplification, HIV Ct, and HIV End RFU graphs comparing Kapa2G Hotstart, H7, H7-IgG, and H7-rFab. Both formulations with either IgG from hybridoma or rFab from E.coli generated signal comparable to that from the control KAPA2G HotStart, stronger than that from the enzyme without antibody at 5 copies of HIV templates.Attorney Docket: ABBTT-43290.601

[0238] EXAMPLE 8 - HPV amplification assay

[0239] The HotStart formulations were also tested in amplifying the HPV (16, 18, 39, 45, and 58) amplicons (none, lx low, or lx standard concentration of HPV targets per reaction) along with the enzyme-only and a product from a commercial supplier (KAPA2G HotStart from Roche) by qPCR. The amplification was monitored by the signals in the FAM (HPV39), VIC (HPV16), Cal Red 610 (HPV18), Quasar 670 (HPV58), and Quasar 705 (HPV45) channels, respectively. The shape, quantification cycle (Ct), and the endRFU of the amplification curves were compared. The amplification reactions were performed on a BioRad CFX96 Real-Time System using the following settings:

[0240] Cycling parameters for Biorad

[0241] CFX96

[0242] Cycle Temp Duration # of cycles Channel UDG activity 46 °C 6 min 1

[0243] Hot start 97 °C 3.3 min 1

[0244] FAM

[0245] Amplification - melt 97 °C 3 sec

[0246] VIC

[0247] Amplification - 4

[0248] 51.6 °C 20 sec Cal Red anneal

[0249] 610 Quasar Amplification - melt 92 °C 3 sec

[0250] 670 Quasar Amplification - 51.6 °C 20 sec 36 705

[0251] anneal

[0252]

[0253] Read 54.7 °C 16 sec

[0254] See FIG. 10, which shows HPV39 (FAM) amplification, HPV39 (FAM) Ct, and HPV39 (FAM) End RFU graphs comparing Kapa2G HotStart, H7, H7-IgG, and H7-rFab.

[0255] See FIG. 11, which shows HPV16 (VIC) amplification, HPV16 (VIC) Ct, and HPV16 (VIC) End RFU graphs comparing Kapa2G HotStart, H7, H7-IgG, and H7-rFab.

[0256] See FIG. 12, which shows HPV 18 (Cal Red 610) amplification, HPV 18 (Cal Red 610) Ct, and HPV18 (Cal Red 610) End RFU graphs comparing Kapa2G HotStart, H7, H7-IgG, and H7-rFab.

[0257] See FIG. 13, which shows HPV58 (Quasar 670) amplification, HPV58 (Quasar 670) Ct, and HPV58 (Quasar 670) End RFU graphs comparing Kapa2G HotStart, H7, H7-IgG, and H7-rFab.

[0258] See FIG. 14, which shows HPV45 (Quasar 705) amplification, HPV45 (Quasar 705) Ct, and HPV45 (Quasar 705) End RFU graphs comparing Kapa2G HotStart, H7, H7-IgG, and H7-rFab.Attorney Docket: ABBTT-43290.601

[0259] The results indicate that both formulations with either IgG from hybridoma or rFab from E.coli generated signals with Ct delays of <3 cycles and similar or higher end RFU for all 5 HPV targets, compared to those from the control KAPA2G HotStart.

[0260] REFERENCES

[0261] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

[0262] 1. Kellogg DE, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert PD, Chenchik A: TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. Biotechniques 1994, 16(6): 1134- 1137.

[0263] 2. Mao F, Leung WY, Xin X: Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol 2007, 7:76.

[0264] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

Attorney Docket: ABBTT-43290.601CLAIMS:We claim:

1. A polypeptide comprising an antigen binding variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1.

2. A polypeptide comprising an antigen binding variable region comprising a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2.

3. A polypeptide comprising an antigen binding variable region comprising a CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 3.

4. A polypeptide comprising an antigen binding variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:4.

5. A polypeptide comprising an antigen binding variable region comprising a CDR2 comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 5.

6. A polypeptide comprising an antigen binding variable region comprising a CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:6.

7. The polypeptide of any of claims 1-6, wherein said at least 70% sequence identity comprises at least 80% sequence identity.

8. The polypeptide of any of claims 1-6, wherein said at least 70% sequence identity comprises at least 90% sequence identity.

9. The polypeptide of any of claims 1-6, wherein said at least 70% sequence identity comprises at least 95% sequence identity.Attorney Docket: ABBTT-43290.60110. The polypeptide of any of claims 1-6, wherein the antigen binding variable region is a light chain variable region that comprises a CDR1, CDR2, and CDR3.

11. The polypeptide of claim 10, wherein the light chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO:3.

12. The polypeptide of any of claims 1-6, wherein the antigen binding variable region is a heavy chain variable region that comprises a CDR1, CDR2, and CDR3.

13. The polypeptide of claim 12, wherein the heavy chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NON or an amino acid sequence with at least 70% sequence identity to SEQ ID NON, a CDR2 comprising the amino acid sequence of SEQ ID NON or an amino acid sequence with at least 70% sequence identity to SEQ ID NON, and a CDR3 comprising the amino acid sequence of SEQ ID NON or an amino acid sequence with at least 70% sequence identity to SEQ ID NON.

14. The polypeptide of any of claims 1-13, wherein said polypeptide has a light chain variable region that comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NON, and a CDR3 comprising the amino acid sequence of SEQ ID NON or an amino acid sequence with at least 70% sequence identity to SEQ ID NON; and a heavy chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NON or an amino acid sequence with at least 70% sequence identity to SEQ ID NON, a CDR2 comprising the amino acid sequence of SEQ ID NON or an amino acid sequence with at least 70% sequence identity to SEQ ID NON, and a CDR3 comprising the amino acid sequence of SEQ ID NON or an amino acid sequence with at least 70% sequence identity to SEQ ID NON.

15. The polypeptide of any of claims 1-14, wherein said polypeptide has a light chain variable region that comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2Attorney Docket: ABBTT-43290.601comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a heavy chain variable region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:4, a CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR3 comprising the amino acid sequence of SEQ ID NO:6.

16. The polypeptide of claim 15, comprising:(i) a light chain amino acid sequence of SEQ ID NO: 7 or 9, and(ii) a heavy chain amino acid sequence of SEQ ID NO: 8, 10, or 11.

17. The polypeptide of any of claims 1-16, wherein the polypeptide is an antibody.

18. The polypeptide of claim 17, wherein said antibody is an IgG antibody.

19. The polypeptide of any of claims 1-16, wherein the polypeptide is an antibody fragment.

20. The polypeptide of claim 19, wherein said antibody fragment is Fab or scFv.

21. The polypeptide of any of claims 1-16, wherein the polypeptide is a diabody.

22. The polypeptide of any of claims 1-21, wherein the polypeptide binds and reversibly inhibits a DNA polymerase.

23. The polypeptide of any of claims 1-22, wherein polypeptide is heat labile.

24. A composition comprising a polymerase-polypeptide complex comprising a polypeptide of any of claims 1-23 bound to a thermostable DNA polymerase, wherein 5’ to 3’ polymerase activity of the thermostable DNA polymerase is inhibited.

25. The composition of claim 24, wherein the thermostable DNA polymerase comprises a sequence selected from the group consisting of SEQ ID Nos 12-19.

26. A reaction mixture comprising a polypeptide of any of claims 1-23 and a thermostable DNA polymerase.

27. A kit comprising a polypeptide of any of claims 1-23.

28. The kit of claim 27 further comprising a thermostable DNA polymerase.Attorney Docket: ABBTT-43290.60129. The kit of claim 28, wherein said thermostable DNA polymerase is bound to said polypeptide.

30. The kit of claim 28 or 29, wherein the thermostable DNA polymerase comprises a sequence selected from the group consisting of SEQ ID Nos 12-19.

31. The kit of any of claims 28-30 further comprising at least one reagent for polymerase chain reaction.

32. The kit of claim 31, wherein the at least one reagent for polymerase chain reaction is selected from controls, buffers, dNTPs, primers, and stabilizers.

33. The kit of claim 32, wherein the at least one reagent for polymerase chain reaction is a buffer comprising MgCh34. A method comprising: contacting a sample containing a target nucleic acid with a polypeptide of any of claims 1-23 bound to a thermostable DNA polymerase.

35. The method of claim 34, further comprising heating the sample to a temperature that disassociates the polypeptide from the thermostable DNA polymerase.

36. The method of claim 35, further comprising amplifying the target nucleic acid.

37. The method of claim 36, wherein the amplifying comprises a polymerase chain reaction.

38. The method of claim 37, further comprising detecting amplicons, or byproduct thereof, of the target nucleic acid.

39. The method of any of claims 34-38, wherein the target nucleic acid is DNA.

40. The method of any of claims 34-38, wherein the target nucleic acid is RNA.

41. The method of claim 40, wherein the RNA is converted into cDNA by reverse transcription prior to the contacting.

42. The method of any of claims 34-41, wherein the target nucleic acid is from an infectious disease agent.Attorney Docket: ABBTT-43290.60143. The method of claim 42, wherein the infectious disease agent is at least one of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG).

44. The method of any of claims 34-43, wherein the sample is, or is derived from, an endocervical swab specimen, a clinician-collected vaginal swab specimen, a self-collected vaginal swab specimen, a gynecological specimen, or urine.

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