TAQ DNA polymerase hotstart antibodies and methods of use thereof

Abstract

This disclosure relates to Taq polymerase binding antibody compositions and methods of use for nucleic acid amplification.

Description

[0001] TAQ DNA POLYMERASE HOTSTART ANTIBODIES AND METHODS OF USE THEREOF

[0002] RELATED APPLICATIONS

[0003] This Application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 604,290, filed on November 30, 2023, the entire contents of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (W109470006WO00-SEQ-ACZ.xml; Size: 24,171 bytes; and Date of Creation: November 15, 2024) are herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] The sensitivity and specificity of nucleic acid amplification techniques, such as polymerase chain reaction (PCR), often suffer from the presence of non-specific products and the formation of primer dimers. This can be caused by several factors, including annealing temperature, primer concentration, and polymerase activity during the setup of the reaction.

[0008] SUMMARY

[0009] Antibodies that can bind to a polymerase and inhibit its activities at low temperatures may be useful for preventing nonspecific nucleic acid amplification. There is a need for antibody compositions that can inhibit 5 ’-3’ exonuclease and / or 5 ’-flap endonuclease activity and polymerase activity at reaction set-up temperatures.

[0010] Provided herein are antibodies that can enhance the yield and / or homogeneity of primer extension products made by Taq polymerases. Taq polymerase maintains residual exonuclease activity when bound by currently available hot-start antibodies, such as Platinum® Taq Monoclonal Antibody and JumpStart™ Taq antibody. Antibodies of the disclosure inhibit 5 ’-3’ exonuclease and / or 5 ’-flap endonuclease activity, reducing the likelihood of spurious amplification events such as primer-dimer formation or amplification of non-specific DNA sequences. Also provided herein are methods of amplifying a nucleic acid template using the antibody bound to a Taq polymerase.

[0011] Accordingly, in some aspects, the present disclosure provides an antibody that binds to Taq-polymerase, wherein the antibody comprises: a heavy chain variable domain (VH) comprising a heavy chain complementary determining region 1 (CDR-H1) comprising an amino acid sequence of SEQ ID NO: 4; a heavy chain complementary determining region 2 (CDR-H2) comprising an amino acid sequence of SEQ ID NO: 5; a heavy chain complementary determining region 3 (CDR-H3) comprising an amino acid sequence of SEQ ID NO: 6; and a light chain variable domain (VL) comprising a light chain complementary determining region 1 (CDR-L1) comprising an amino acid sequence of SEQ ID NO: 10; a light chain complementary determining region 2 (CDR-L2) comprising an amino acid sequence of SEQ ID NO: 11; and a light chain complementary determining region 3 (CDR-L3) comprising an amino acid sequence of SEQ ID NO: 12.

[0012] In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 14, and the VL comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0013] In some embodiments, the antibody is a full length IgG, a Fab fragment, a F(ab’) fragment, a F(ab’)2 fragment, an scFv, or an Fv. In some embodiments, the antibody is a full- length rabbit IgG.

[0014] In some embodiments, the present disclosure provides a nucleic acid sequence encoding an antibody provided herein. In some embodiments, the VH is encoded by a nucleic acid sequence comprising SEQ ID NO: 13, and the VL is encoded by a nucleic acid sequence comprising SEQ ID NO: 15.

[0015] In some embodiments, the present disclosure provides an expression vector comprising a nucleic acid sequence provided herein. In some embodiments, the presence disclosure provides a cell comprising a nucleic acid sequence provided herein or an expression vector provided herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary cell (CHO). In some embodiments, the cell is a bacterial cell.

[0016] In some embodiments, the present disclosure provides an antibody -polymerase complex, wherein the complex comprises: an antibody of the present disclosure; and a Taq-polymerase. In some embodiments, the Taq-polymerase is a wild-type Taq-polymerase. In some embodiments, the Taq-polymerase is a variant Taq-polymerase. In some embodiments, the antibody- polymerase complex binds to DNA. In some embodiments, activity of the Taq-polymerase is inhibited when bound to the antibody. In some embodiments, the inhibited Taq-polymerase activity is 5 ’-3’ exonuclease and / or 5 ’-flap endonuclease activity. In some embodiments, the antibody -polymerase complex has decreased 5 ’-3’ exonuclease and / or 5 ’-flap endonuclease activity of the Taq polymerase as compared to a PLATINUM and / or JUMPSTART antibody- polymerase complex. In some embodiments, the antibody-polymerase complex disassociates at a temperature greater than 80 °C.

[0017] In some embodiments, the present disclosure provides a method of amplifying a nucleic acid template, the method comprising: combining, in a solution for amplifying the nucleic acid template: (a) an antibody of any one of the present disclosure and a Taq polymerase, or an antibody-polymerase complex of the present disclosure, (b) a dNTP mixture, (c) a target nucleic acid template, and (d) an oligonucleotide primer comprising a polynucleotide that is complementary to the nucleic acid template. In some embodiments, the Taq-polymerase is a wild-type Taq-polymerase. In some embodiments, the Taq-polymerase is a variant Taq- polymerase.

[0018] In some embodiments, combining occurs at a first temperature. In some embodiments, the first temperature is below 30°C. In some embodiments, the antibody-polymerase complex remains intact at the first temperature. In some embodiments, the method further comprises bringing the solution to a denaturation temperature. In some embodiments, the antibody- polymerase complex dissociates at the denaturation temperature. In some embodiments, the denaturation temperature is at least 80°C. In some embodiments, the method further comprises bringing the solution to an annealing temperature. In some embodiments, the target nucleic acid template and the oligonucleotide primer anneal at the annealing temperature. In some embodiments, the method further comprises bringing the solution to an extension temperature. In some embodiments, the oligonucleotide primer is extended at the extension temperature.

[0019] In some embodiments, the method comprises, in consecutive order: (a) bringing the solution to the first temperature for a first predetermined amount of time; (b) bringing the solution to the denaturation temperature for a second predetermined amount of time; (c) bringing the solution to the denaturation temperature for a third predetermined amount of time; (d) bringing the solution to the annealing temperature for a fourth predetermined amount of time; and (e) bringing the solution to the extension temperature for a fifth predetermined amount of time.

[0020] In some embodiments, the method further comprises repeating a plurality of times: (a) bringing the solution to the denaturation temperature for the third predetermined amount of time; (b) bringing the solution to the annealing temperature for the fourth predetermined amount of time; and (c) bringing the solution to the extension temperature for the fifth predetermined amount of time. In some embodiments, the method further comprises bringing the solution to the extension temperature for a sixth predetermined amount of time.

[0021] In some embodiments, amplification of the nucleic acid template produces a plurality of amplicons. In some embodiments, amplification is carried out by PCR, qPCR, digital PCR, droplet digital PCR, linear amplification, or multiplex PCR.

[0022] In some embodiments, the present disclosure provides a kit comprising: (a) an antibody provided herein; (b) a dNTP mixture; (c) a reaction buffer; and (d) a Taq-polymerase. In some embodiments, the antibody, the dNTP mixture, the reaction buffer, and the Taq-polymerase are provided as a single solution. In some embodiments, the Taq-polymerase is a wild-type Taq- polymerase. In some embodiments, the Taq-polymerase is a variant Taq-polymerase.

[0023] In some embodiments, the present disclosure provides a kit comprising: (a) an antibody- polymerase complex provided herein; (b) a dNTP mixture; and (c) a reaction buffer. In some embodiments, the antibody-polymerase complex, the dNTP mixture, and the reaction buffer are provided as a single solution.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are not intended to be drawn to scale. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0026] FIG. 1 shows the percentage inhibition of a Taq polymerase in the presence of 11 unique antibody clones, along with a negative supernatant and a Platinum® Taq Monoclonal Antibody as a control, when added in excess to a wild-type Taq polymerase (SEQ ID NOs: 19-20). An additional negative control using wild-type Taq polymerase was also evaluated (enzyme control).

[0027] FIG. 2 shows a standard curve of the measured concentrations of the antibody 37H9-4 (SEQ ID NOs: 17-18) as a function of a serial dilution of input antibody 37H9-4. FIG. 3 shows a comparison of the percentage inhibition of wild-type Taq polymerase (SEQ ID NOs: 19-20) using antibody 37H9-4 (SEQ ID NOs: 17-18), a Platinum® Taq Monoclonal Antibody, and a JumpStart™ Taq Antibody at different polymerase: antibody molar concentrations.

[0028] FIG. 4 shows the FAM signal of Taq polymerase nuclease activity bound by heat- inactivated and functional 37H9-4 (SEQ ID NOs: 17-18), Platinum® Taq Monoclonal Antibody, JumpStart™ Taq Antibody, and no antibody.

[0029] DETAILED DESCRIPTION

[0030] Antibodies

[0031] This disclosure provides antibodies that bind to Taq polymerase. In some embodiments, an antibody that binds to Taq polymerase comprises a heavy chain variable domain (VH) comprising a heavy chain complementary determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 4; a heavy chain complementary determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 5; a heavy chain complementary determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable domain (VL) comprisinga light chain complementary determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 10; a light chain complementary determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 11 ; and a light chain complementary determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 14, and the VL comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18. Sequences of this disclosure are provided in Table 1.

[0032] Table 1. Sequences

[0033] In some embodiments, the antibody is a full-length IgG, a Fab fragment, a F(ab’) fragment, a F(ab’)2 fragment, an scFv, or an Fv. In some embodiments, the antibody is a full- length rabbit IgG. In some embodiments, the antibody is a full-length cow IgG, a full-length goat IgG, a full-length chicken IgG, a full-length mouse IgG, or a full-length goat IgG. In some embodiments, the full-length IgG comprises a heavy chain constant region of isotype IgGl, IgG2, IgG3, or IgG4.

[0034] In some embodiments, a nucleic acid sequence encoding an antibody disclosed herein is provided. In some embodiments, the VH is encoded by a nucleic acid sequence comprising SEQ ID NO: 13, and the VL is encoded by a nucleic acid sequence comprising SEQ ID NO: 15.

[0035] Expression vectors and cells

[0036] In some embodiments, this disclosure provides an expression vector comprising a polynucleotide encoding an antibody described herein. An expression vector, or an expression construct, is a plasmid or other DNA molecule designed to facilitate the expression of a specific gene or protein in a cell. Expression vectors typically contain several key elements that enable gene expression, including: 1) a promoter, which is a DNA sequence that initiates transcription allowing the gene of interest to be transcribed into messenger RNA (e.g., the antibody); 2) a gene of interest or insert, which is the DNA sequence that encodes the protein or RNA molecule to be produced; 3) a selectable marker, which is a gene that confers resistance to an antibiotic (e.g., ampicillin or kanamycin) or another selective agent; 4) an origin of replication, which is a sequence that allows the vector to replicate autonomously within a cell and ensures that the vector and its inserted gene are maintained as the host cells divide; and, 5) a terminator sequence, which is a sequence downstream of the gene of interest that signals the end of transcription and ensures that the mRNA transcript is correctly processed.

[0037] In some embodiments, this disclosure provides a cell comprising an antibody described herein, a nucleic acid sequence described herein or an expression vector described herein. In some embodiments, a cell comprises (e.g., has been transformed with) an expression vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody and an amino acid sequence comprising the VL of the antibody. In some embodiments, the VH comprises a heavy chain complementary determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 4; a heavy chain complementary determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 5; a heavy chain complementary determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 6; and the VL comprises a light chain complementary determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 10; a light chain complementary determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 11 ; and a light chain complementary determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 14 and the VL comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, a cell comprises (e.g., has been transformed with) an expression vector comprising a nucleic acid that encodes an amino acid sequence comprising SEQ ID NO: 17 or SEQ ID NO: 18.

[0038] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese Hamster Ovary cell (CHO). In some embodiments, the mammalian cell is a HEK293 cell, a NSO cell, or an SP2 / 0 cell. In some embodiments, the cell is a bacterial cell. Non-limiting examples of bacterial cells include Escherichia coli, Bacillus subtilis, Streptomyces species, and Pseudomonas putida. In some embodiments, the cell is an insect cell (e.g., an Sf9 cell or an Sf21 cell). In some embodiments, the cell is a yeast cell (e.g., a Saccharomyces cerevisiae cell).

[0039] Polymerases

[0040] A polymerase is an enzyme responsible for catalyzing the polymerization of nucleotides to synthesize nucleic acid polymers. DNA polymerases are crucial components of nucleic acid amplification techniques, such as polymerase chain reaction (PCR). Taq polymerase is a DNA polymerase enzyme derived from the bacterium Thermus aquaticus. It is a heat-resistant polymerase known for its thermal stability, with a half-life of about 40 minutes at 95°C. Taq polymerase catalyzes the synthesis of a complementary DNA strand using a single-stranded DNA template and DNA primers, following the standard Watson-Crick base-pairing rules to synthesize the new DNA strand. In some embodiments, the Taq polymerase is a wild-type Taq polymerase (e.g., SEQ ID NOs: 19 and 20). In some embodiments, the Taq-polymerase is a variant Taq-polymerase.

[0041] Antibody-polymerase complexes

[0042] In some embodiments, this disclosure provides an antibody-polymerase complex, wherein the complex comprises an antibody described herein and a Taq polymerase. As used herein, the term “antibody-polymerase complex” refers to an antibody that is bound to a Taq polymerase. In some embodiments, the complex comprises: 1) an antibody comprising a heavy chain variable domain (VH) comprising a heavy chain complementary determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 4; a heavy chain complementary determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 5; a heavy chain complementary determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable domain (VL) comprisinga light chain complementary determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 10; a light chain complementary determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 11; and a light chain complementary determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 12; and, 2) a Taq polymerase. In some embodiments, the complex comprises: 1) an antibody comprising a VH comprising an amino acid of SEQ ID NO: 14 and a VL comprising an amino acid of SEQ ID NO: 16; and, 2) a Taq polymerase. In some embodiments, the complex comprises: 1) an antibody comprising an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18; and, 2) a Taq polymerase. In some embodiments, the Taq polymerase is a wild-type Taq polymerase (SEQ ID NOs: 19 and 20).

[0043] In some embodiments, the antibody binds to Taq polymerase. As used herein, the term “an antibody that binds to Taq polymerase” refers to an antibody that is capable of binding Taq polymerase with a high degree of selectivity and binding affinity relative to binding to an unrelated, non-Taq DNA polymerase. In some embodiments, the extent of binding of an antibody of the present disclosure to an unrelated, non-Taq DNA polymerase is less than about 15%, less than about 10%, less than about 5%, or less than about 2.5% of the extent of binding of an antibody of the present disclosure to a Taq polymerase as measured, e.g., by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the antibody of the present disclosure does not detectably bind to an unrelated, non-Taq DNA polymerase as measured, e.g., by enzyme-linked immunosorbent assay (ELISA).

[0044] In some embodiments, when an antibody of the present disclosure is bound to the Taq- polymerase, the activity of the Taq-polymerase is inhibited. In some embodiments, the inhibited Taq-polymerase activity is polymerization. In some embodiments, the inhibited Taq polymerase activity is 5 ’-3’ exonuclease activity. In some embodiments, the 5 ’-3’ exonuclease activity of a Taq polymerase bound to an antibody of the present disclosure is decreased by at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a Taq polymerase that is not bound to an antibody of the present disclosure as measured by e.g., a probe degradation assay. In some embodiments, the 5’- 3’ exonuclease activity of a Taq polymerase bound to an antibody of the present disclosure is decreased by 5-fold to 10-fold, 5-fold to 25-fold, 5-fold to 50-fold, 5-fold to 75-fold, 5-fold to 100-fold, 10-fold to 25-fold, 10-fold to 50-fold, 10-fold to 75-fold, 10-fold to 100-fold, 25-fold to 50-fold, 25-fold to 75-fold, 25-fold to 100-fold, 50-fold to 75-fold, 50-fold to 100-fold, 75- fold to 100-fold, 100-fold to 150-fold, 100-fold to 200-fold, 100-fold to 250-fold, 100-fold to 500-fold, 100-fold to 750-fold, 100-fold to 1,000-fold, 150-fold to 200-fold, 150-fold to 250- fold, 150-fold to 500-fold, 150-fold to 750-fold, 150-fold to 1,000-fold, 200-fold to 250-fold, 200-fold to 500-fold, 200-fold to 750-fold, 200-fold to 1,000-fold, 250-fold to 500-fold, 250-fold to 750-fold, 250-fold to 1,000-fold ,500-fold to 750-fold, 500-fold to 1,000-fold, 750-fold to 1,000-fold, 1,000-fold to 2,000-fold, 1,000-fold to 3,000-fold, 1,000-fold to 4,000-fold, 1,000- fold to 5,000-fold, 2,000-fold to 3,000-fold, 2,000-fold to 4,000-fold, 2,000-fold to 5,000-fold, 3,000-fold to 4,000-fold, 3,000-fold to 5,000-fold, 4,000-fold to 5,000-fold, 5,000-fold to 10,000-fold, 5,000-fold to 15,000-fold, 5,000-fold to 20,000-fold, 5,000-fold to 25,000-fold, 5,000-fold to 30,000-fold, 5,000-fold to 35,000-fold, 5,000-fold to 40,000-fold, 10,000-fold to 15,000-fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000-fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000-fold, 15,000-fold to 30,000-fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000- fold, 20,000-fold to 25,000-fold, 20,000-fold to 30,000-fold, 20,000-fold to 35,000-fold, 20,000- fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000-fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000- fold as compared to a Taq polymerase that is not bound to an antibody of the present disclosure as measured by e.g., a probe degradation assay.

[0045] In some embodiments, the inhibited Taq polymerase activity is 5 ’-flap endonuclease activity. In some embodiments, the inhibited 5 ’-flap endonuclease activity of a Taq polymerase bound to an antibody of the present disclosure is decreased by at least 5 -fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200- fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 10,000-fold, at least 15,000- fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a Taq polymerase that is not bound to an antibody of the present disclosure as measured by e.g., a probe degradation assay. In some embodiments, the inhibited 5 ’-flap endonuclease activity of a Taq polymerase bound to an antibody of the present disclosure is decreased by 5-fold to 10-fold, 5-fold to 25-fold, 5-fold to 50-fold, 5-fold to 75-fold, 5-fold to 100-fold, 10-fold to 25-fold, 10-fold to 50-fold, 10-fold to 75-fold, 10-fold to 100-fold, 25-fold to 50-fold, 25-fold to 75-fold, 25-fold to 100-fold, 50-fold to 75-fold, 50-fold to 100-fold, 75- fold to 100-fold, 100-fold to 150-fold, 100-fold to 200-fold, 100-fold to 250-fold, 100-fold to 500-fold, 100-fold to 750-fold, 100-fold to 1,000-fold, 150-fold to 200-fold, 150-fold to 250- fold, 150-fold to 500-fold, 150-fold to 750-fold, 150-fold to 1,000-fold, 200-fold to 250-fold, 200-fold to 500-fold, 200-fold to 750-fold, 200-fold to 1,000-fold, 250-fold to 500-fold, 250-fold to 750-fold, 250-fold to 1,000-fold ,500-fold to 750-fold, 500-fold to 1,000-fold, 750-fold to 1,000-fold, 1,000-fold to 2,000-fold, 1,000-fold to 3,000-fold, 1,000-fold to 4,000-fold, 1,000- fold to 5,000-fold, 2,000-fold to 3,000-fold, 2,000-fold to 4,000-fold, 2,000-fold to 5,000-fold, 3,000-fold to 4,000-fold, 3,000-fold to 5,000-fold, 4,000-fold to 5,000-fold, 5,000-fold to 10,000-fold, 5,000-fold to 15,000-fold, 5,000-fold to 20,000-fold, 5,000-fold to 25,000-fold, 5,000-fold to 30,000-fold, 5,000-fold to 35,000-fold, 5,000-fold to 40,000-fold, 10,000-fold to 15,000-fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000-fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000-fold, 15,000-fold to 30,000-fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000- fold, 20,000-fold to 25,000-fold, 20,000-fold to 30,000-fold, 20,000-fold to 35,000-fold, 20,000- fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000-fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000- fold as compared to a Taq polymerase that is not bound to an antibody of the present disclosure as measured by e.g., a probe degradation assay.

[0046] In some embodiments, the inhibited Taq polymerase activity is 5 ’-3’ exonuclease activity and 5 ’-flap endonuclease activity. In some embodiments, the 5 ’-3’ exonuclease activity and 5’- flap endonuclease activity of a Taq polymerase bound to an antibody of the present disclosure are decreased by at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a Taq polymerase that is not bound to an antibody of the present disclosure as measured by e.g., a probe degradation assay. In some embodiments, the 5 ’-3’ exonuclease activity and 5 ’-flap endonuclease activity of a Taq polymerase bound to an antibody of the present disclosure are decreased by 5-fold to 10-fold, 5-fold to 25-fold, 5-fold to 50-fold, 5-fold to 75-fold, 5-fold to 100-fold, 10-fold to 25-fold, 10-fold to 50-fold, 10-fold to 75-fold, 10-fold to 100-fold, 25-fold to 50-fold, 25-fold to 75-fold, 25-fold to 100-fold, 50-fold to 75-fold, 50-fold to 100-fold, 75- fold to 100-fold, 100-fold to 150-fold, 100-fold to 200-fold, 100-fold to 250-fold, 100-fold to 500-fold, 100-fold to 750-fold, 100-fold to 1,000-fold, 150-fold to 200-fold, 150-fold to 250- fold, 150-fold to 500-fold, 150-fold to 750-fold, 150-fold to 1,000-fold, 200-fold to 250-fold, 200-fold to 500-fold, 200-fold to 750-fold, 200-fold to 1,000-fold, 250-fold to 500-fold, 250-fold to 750-fold, 250-fold to 1,000-fold ,500-fold to 750-fold, 500-fold to 1,000-fold, 750-fold to 1,000-fold, 1,000-fold to 2,000-fold, 1,000-fold to 3,000-fold, 1,000-fold to 4,000-fold, 1,000- fold to 5,000-fold, 2,000-fold to 3,000-fold, 2,000-fold to 4,000-fold, 2,000-fold to 5,000-fold, 3,000-fold to 4,000-fold, 3,000-fold to 5,000-fold, 4,000-fold to 5,000-fold, 5,000-fold to 10,000-fold, 5,000-fold to 15,000-fold, 5,000-fold to 20,000-fold, 5,000-fold to 25,000-fold, 5,000-fold to 30,000-fold, 5,000-fold to 35,000-fold, 5,000-fold to 40,000-fold, 10,000-fold to 15,000-fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000-fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000-fold, 15,000-fold to 30,000-fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000- fold, 20,000-fold to 25,000-fold, 20,000-fold to 30,000-fold, 20,000-fold to 35,000-fold, 20,000- fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000-fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000- fold as compared to a Taq polymerase that is not bound to an antibody of the present disclosure as measured by e.g., a probe degradation assay.

[0047] In some embodiments, an antibody-polymerase complex disclosed herein binds to DNA. In some embodiments, the antibody-polymerase complex has decreased 5 ’-3’ exonuclease activity of the polymerase as compared to a Platinum® antibody-polymerase complex. In some embodiments, the 5 ’-3’ exonuclease activity of an antibody-polymerase complex of the present disclosure is decreased by at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a Platinum® antibody-polymerase complex as measured by e.g., a probe degradation assay. In some embodiments, the 5 ’-3’ exonuclease activity of an antibody-polymerase complex of the present disclosure is decreased by 10,000-fold to 15,000-fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000-fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000-fold, 15,000-fold to 30,000- fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000-fold, 20,000-fold to 25,000-fold, 20,000- fold to 30,000-cold, 20,000-fold to 35,000-fold, 20,000-fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000-fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000-fold as compared to a Platinum® antibody- polymerase complex as measured by e.g., a probe degradation assay.

[0048] In some embodiments, the antibody-polymerase complex has decreased 5 ’-flap endonuclease activity of the polymerase as compared to a Platinum® antibody-polymerase complex. In some embodiments, the 5 ’-flap endonuclease activity of an antibody-polymerase complex of the present disclosure is decreased by at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a Platinum® antibody-polymerase complex as measured by e.g., a probe degradation assay. In some embodiments, the 5 ’-flap endonuclease activity of an antibody- polymerase complex of the present disclosure is decreased by 10,000-fold to 15,000-fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000-fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000- fold, 15,000-fold to 30,000-fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000-fold, 20,000- fold to 25,000-fold, 20,000-fold to 30,000-cold, 20,000-fold to 35,000-fold, 20,000-fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000-fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000-fold as compared to a Platinum® antibody-polymerase complex as measured by e.g., a probe degradation assay.

[0049] In some embodiments, the antibody-polymerase complex has decreased 5 ’-3’ exonuclease activity and decreased 5 ’-flap endonuclease activity of the polymerase as compared to a Platinum® antibody-polymerase complex. In some embodiments, the 5 ’-3’ exonuclease activity and the 5 ’-flap endonuclease activity of an antibody-polymerase complex of the present disclosure are decreased by at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a Platinum® antibody-polymerase complex as measured by e.g., a probe degradation assay. In some embodiments, the 5 ’-3’ exonuclease activity and the 5 ’-flap endonuclease activity of an antibody-polymerase complex of the present disclosure are decreased by 10,000-fold to 15,000- fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000- fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000-fold, 15,000-fold to 30,000-fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000-fold, 20,000-fold to 25,000-fold, 20,000-fold to 30,000-cold, 20,000-fold to 35,000-fold, 20,000-fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000- fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000-fold as compared to a Platinum® antibody-polymerase complex as measured by e.g., a probe degradation assay.

[0050] In some embodiments, an antibody-polymerase complex disclosed herein binds to DNA. In some embodiments, the antibody-polymerase complex has decreased 5 ’-3’ exonuclease activity of the polymerase as compared to a JumpStart™ antibody-polymerase complex. In some embodiments, the 5 ’-3’ exonuclease activity of an antibody-polymerase complex of the present disclosure is decreased by at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a JumpStart™ antibody-polymerase complex as measured by e.g., a probe degradation assay. In some embodiments, the 5 ’-3’ exonuclease activity of an antibody-polymerase complex of the present disclosure is decreased by 10,000-fold to 15,000-fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000-fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000-fold, 15,000-fold to 30,000- fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000-fold, 20,000-fold to 25,000-fold, 20,000- fold to 30,000-cold, 20,000-fold to 35,000-fold, 20,000-fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000-fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000-fold as compared to a JumpStart™ antibody-polymerase complex as measured by e.g., a probe degradation assay. In some embodiments, the antibody-polymerase complex has decreased 5 ’-flap endonuclease activity of the polymerase as compared to a JumpStart™ antibody-polymerase complex. In some embodiments, the 5 ’-flap endonuclease activity of an antibody-polymerase complex of the present disclosure is decreased by at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a JumpStart™ antibody-polymerase complex as measured by e.g., a probe degradation assay. In some embodiments, the 5 ’-flap endonuclease activity of an antibody- polymerase complex of the present disclosure is decreased by 10,000-fold to 15,000-fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000-fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000- fold, 15,000-fold to 30,000-fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000-fold, 20,000- fold to 25,000-fold, 20,000-fold to 30,000-cold, 20,000-fold to 35,000-fold, 20,000-fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000-fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000-fold as compared to a JumpStart™ antibody-polymerase complex as measured by e.g., a probe degradation assay.

[0051] In some embodiments, the antibody-polymerase complex has decreased 5 ’-3’ exonuclease activity and decreased 5 ’-flap endonuclease activity of the polymerase as compared to a JumpStart™ antibody-polymerase complex. In some embodiments, the 5 ’-3’ exonuclease activity and the 5 ’-flap endonuclease activity of an antibody-polymerase complex of the present disclosure are decreased by at least 10,000-fold, at least 15,000-fold, at least 20,000-fold, at least 25,000-fold, at least 30,000-fold, at least 35,000-fold, or at least 40,000-fold as compared to a JumpStart™ antibody-polymerase complex as measured by e.g., a probe degradation assay. In some embodiments, the 5 ’-3’ exonuclease activity and the 5 ’-flap endonuclease activity of an antibody-polymerase complex of the present disclosure are decreased by 10,000-fold to 15,000- fold, 10,000-fold to 20,000-fold, 10,000-fold to 25,000-fold, 10,000-fold to 30,000-fold, 10,000- fold to 35,000-fold, 10,000-fold to 40,000-fold, 15,000-fold to 20,000-fold, 15,000-fold to 25,000-fold, 15,000-fold to 30,000-fold, 15,000-fold to 35,000-fold, 15,000-fold to 40,000-fold, 20,000-fold to 25,000-fold, 20,000-fold to 30,000-cold, 20,000-fold to 35,000-fold, 20,000-fold to 40,000-fold, 25,000-fold to 30,000-fold, 25,000-fold to 35,000-fold, 25,000-fold to 40,000- fold, 30,000-fold to 35,000-fold, 30,000-fold to 40,000-fold, or 35,000-fold to 40,000-fold as compared to a JumpStart™ antibody-polymerase complex as measured by e.g., a probe degradation assay.

[0052] In some embodiments, the antibody-polymerase complex is stable at a temperature less than or equal to 75°C. In some embodiments, the antibody-polymerase is stable (e.g., at least 90%, at least 95%, at least 97.5%, or 100% of the Taq-polymerase in solution remains bound to an antibody) 40°C and 55°C for at least 60 minutes. As used herein, an antibody-polymerase complex is considered “stable at a temperature” if at least 75% of the Taq-polymerase in solution remains bound to an antibody at a temperature. In some embodiments, an antibody-polymerase complex is stable at a temperature if at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or 100% of the Taq-polymerase in solution remains bound to an antibody at a temperature.

[0053] Methods of use

[0054] This disclosure also provides methods of amplifying a nucleic acid template. In some embodiments, the method comprises combining, in a solution for amplifying the nucleic acid template: (a) an antibody of the present disclosure and a Taq polymerase or an antibody- polymerase complex of the present disclosure; (b) a dNTP mixture; (c) a target nucleic acid template; and, (d) an oligonucleotide primer comprising a polynucleotide that is complementary to the nucleic acid template. In some embodiments, the Taq polymerase is a wild-type Taq polymerase. In some embodiments, the Taq polymerase is a variant Taq polymerase. In some embodiments, the amplification of the nucleic acid template produces a plurality of amplicons. In some embodiments, the amplification is carried out by PCR, qPCR, digital PCR, droplet digital PCR, linear amplification, or multiplex PCR.

[0055] In some embodiments, combining occurs at a first temperature. As used herein, the term “combining” refers to placing two more substances together. In some embodiments, the antibody and polymerase are combined at a first temperature. In some embodiments, the first temperature is less than 30°C. In some embodiments, the first temperature is about 20°C, 22°C, or about 25°C. In some embodiments, the first temperature is about 22°C and about 25°C. In some embodiments, the antibody and polymerase are further incubated for a first predetermined amount of time. In some embodiments, the first predetermined amount of time is at least 5 minutes, at least 10 minutes, or at least 15 minutes. In some embodiments, an antibody- polymerase complex is formed after combining the antibody and polymerase at a first temperature (e.g., 25 °C) and incubating at said first temperature for a first predetermined amount of time (e.g., 10 minutes). In some embodiments, the antibody-polymerase remains intact at the first temperature.

[0056] In some embodiments, the method further comprises bringing the solution to a denaturation temperature. The denaturation temperature is the temperature at which most of double-stranded DNA molecules in a sample separate into two single-stranded DNA molecules. In some embodiments, the denaturation temperature is about 80°C, about 85°C, about 90°C, about 95°C, or about 98°C. In some embodiments, the denaturation temperature is at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 98°C. In some embodiments, the solution is held at a denaturation temperature (e.g., 95°C) for a second predetermined amount of time. In some embodiments, the second predetermined amount of time is at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 25 seconds, at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds, or at least 60 seconds. In some embodiments, the solution is held at a denaturation temperature (e.g., 95°C) for a third predetermined amount of time. In some embodiments, the third predetermined amount of time is at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 25 seconds, at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds, or at least 60 seconds. In some embodiments, the antibody-polymerase complex dissociates at the denaturation temperature (e.g., 95°C). In some embodiments, the antibody- polymerase complex dissociates at a denaturation temperature (e.g., 95 °C) after incubation at said denaturation temperature (e.g., 95°C) for a second predetermined amount of time (e.g., 60 seconds).

[0057] In some embodiments, the method further comprises bringing the solution to an annealing temperature. The annealing temperature is the temperature at which an oligonucleotide primer comprising a polynucleotide that is complementary to the nucleic acid template binds to the complementary sequences on the target nucleic acid template. In some embodiments, the annealing temperature is about 55°C, about 60°C, about 65°C, about 70°C or about 72°C. In some embodiments, the solution is held at an annealing temperature (e.g., 60°C) for a fourth predetermined amount of time. In some embodiments, the fourth predetermined amount of time is at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 25 seconds, at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds, or at least 60 seconds.

[0058] In some embodiments, the method further comprises bringing the solution to an extension temperature. The extension temperature is the temperature at which the DNA polymerase enzyme synthesizes new DNA strands by adding complementary nucleotides to the template strand. In some embodiments, the extension temperature is about 68°C, about 70°C or about 72°C. In some embodiments, the solution is held at an extension temperature (e.g., 72°C) for a fifth predetermined amount of time. In some embodiments, the fifth predetermined amount of time is at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 25 seconds, at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds, or at least 60 seconds. In some embodiments, the solution is held at an extension temperature (e.g., 72°C) for a sixth predetermined amount of time. In some embodiments, the sixth predetermined amount of time is at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 25 seconds, at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds, or at least 60 seconds.

[0059] In some embodiments, the method comprises, in consecutive order: (1) bringing the solution to the first temperature (e.g., 25 °C) for the first predetermined amount of time (e.g., 10 minutes), which is expected to result in the formation of an antibody: polymerase complex; (2) bringing the solution to the denaturation temperature (e.g., 95 °C) for the second predetermined amount of time (e.g., 60 seconds), which is expected to result in denaturation of the antibody and template DNA; (3) bringing the solution to the denaturation temperature (e.g., 95°C) for the third predetermined amount of time (e.g., 15 seconds); (4) bringing the solution to the annealing temperature (e.g., 60°C) for the fourth predetermined amount of time (e.g., 15 seconds); (5) bringing the solution to the extension temperature (e.g., 72°C) for the fifth predetermined amount of time (e.g., 30 seconds); and (6) bringing the solution to the extension temperature (e.g., 72°C) for a sixth predetermined amount of time (e.g., 60 seconds), to extend all amplicons produced. In some embodiments, the method further comprises repeated a plurality (e.g., 1 and 12, 6-18, 12- 24, 18-30, 24-36, or 30-40) of times: (a) bringing the solution to the denaturation temperature for the third predetermined amount of time; (b) bringing the solution to the annealing temperature for the fourth predetermined amount of time; and, (c) bringing the solution to the extension temperature for the fifth predetermined amount of time. Kits

[0060] In some embodiments, this disclosure provides kits comprising (a) an antibody of the present disclosure; (b) a dNTP mixture; (c) a reaction buffer; and, (d) a Taq polymerase. The reaction buffer is a solution that provides a sufficient chemical environment for a nucleic acid amplification reaction to occur. The reaction buffer may contain: buffering agents (e.g., Tris) to maintain a stable pH, salts or ions (e.g., K+, Na+, or Mg2+) as cofactors for the polymerase, detergents (e.g., Tween-20 or Triton X-100) to reduce nonspecific DNA binding to reaction tubes or surfaces, stabilizers (e.g., bovine serum albumin (BSA) or gelatin) to minimize PCR inhibition caused by impurities in the DNA template or reaction components, dyes (e.g., bromophenol blue or xylene cyanol FF) for visualizing sample loading during electrophoresis, and / or PCR enhances (e.g., betaine or DMSO) which can help reduce secondary structures or improve the amplification of challenging templates. In some embodiments, the antibody, the dNTP mixture, the reaction buffer, and the Taq polymerase are provided as a single solution. In some embodiments, the Taq polymerase is a wild-type Taq polymerase. In some embodiments, the Taq polymerase is a variant Taq polymerase. In some embodiments, a kit comprising (a) an antibody -polymerase complex of the present disclosure; (b) a dNTP mixture; and, (c) a reaction buffer are provided. In some embodiments, the antibody-poly merase complex, the dNTP mixture, and the reaction buffer are provided as a single solution.

[0061] EXAMPLES

[0062] Example 1. Hot-start antibody production.

[0063] New Zealand rabbits were immunized with a Taq DNA polymerase (aCat77) . Two more booster immunizations in the same conditions were administered. Following immunizations, total IgG were isolated. Specific activity of isolated IgG against the Taq-DNA polymerase was confirmed by indirect ELISA. Affinity purification of the Taq-DNA polymerase- specific IgG was performed using Protein A agarose. Eluate fractions were assayed for protein by absorbance at 280 nm. Eluate fractions positive for protein were pooled, neutralized, and dialyzed.

[0064] The genes of the antibody were then inserted into a mammalian expression vector, and the plasmid was linearized and transfected into suspended CHO-K1 cells. The antibody was then produced in CHO cells initially by transient expression and subsequently by the creation of a stable expression cell line.

[0065] Example 2. Hot-start antibody identification and characterization.

[0066] The Malachite green-based polymerization assay was first used to test antibody blocking activity on Taq DNA polymerase to identify a candidate antibody. A total of eleven antibodies that showed binding to Taq-DNA polymerase, as determined using indirect ELISA, were chosen for this study. An excess of these antibody clones and Platinum® Taq Monoclonal Antibody (Invitrogen, USA) were separately combined with a Taq DNA polymerase. A negative supernatant and wild- type Taq-DNA polymerase alone was included in both conditions as negative controls. The percentage inhibition was then determined using the malachite greenbased assay and quantified by dividing the polymerization activity in the presence of the active antibody by the polymerization in the sample where the antibody was heat-inactivated. Of the antibody clones tested, antibody 37H9-4 was identified as a clear candidate, with nearly 100% inhibition against Taq polymerase (FIG. 1).

[0067] In order to compare antibody 37H9-4 to existing hot-start antibodies, a dilution series of antibody 37H9-4, JumpStart™ Taq antibody (Sigma- Aldrich, USA), and Platinum® Taq antibody were loaded onto a Protein Expression LabChip (PerkinElmer) to separate and quantify the proteins. The dilution series was used to create a standard curve that was used to measure the concentrations of JumpStart™ Taq antibody and Platinum® Taq antibody (FIG. 2), which were determined to be 1,203 ng / pL and 717 ng / pL, respectively.

[0068] To determine the molar ratio of antibody to Taq DNA polymerase needed to inhibit the polymerase, different molar ratios of 37H9-4:Taq DNA polymerase were assessed using the EvaEZ Fluorometric Polymerase Activity Assay Kit (Biotum, USA). Molar ratios (polymerase: antibody) of 1:0, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:06, 1:0.7, 1:0.8, 1:0.9, 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, and 1: 1.4 were tested antibody 37H9-4, JumpStart™ antibody, and Platinum® antibody (FIG. 3). Results from the EvaEZ Fluorometric Polymerase Activity assay showed that antibody 37H9-4 only started to lose efficacy around a molar ratio of 1:0.5 (polymerase: antibody), indicating that antibody 37H9-4 is more effective at decreasing Taq DNA polymerase activity at lower concentrations than its competitors (JumpStart™ antibody and Platinum® antibody) which started to lose efficacy around a molar ratio of 1:0.8 (polymerase: antibody). Further, because the antibody is bivalent and should be capable of binding and inhibiting two polymerase molecules per antibody molecule meets the theoretical minimum molar ratio of 1:0.5 (polymerase: antibody) and is competitive as a hot-start Taq DNA polymerase antibody.

[0069] Methods

[0070] Malachite green-based polymerization assay: A reaction mixture containing 0.4 pg / pL activated calf thymus DNA (Sigma-Aldrich), 0.2 mM dNTPs, 8 mM MgC12, 20 mM Tris-HCl pH 8.3, 40 mM KC1 and 0.004% Tween-20 was prepared. Antibody-Taq DNA polymerase complexes were prepared with 4 times molar excess of antibody. These samples were split into two and one half was heat treated at 90°C for 2 minutes to denature the antibodies. The heat- treated and non-heat-treated complexes were then added to the reaction mixture such that each reaction contained 1 ng of Taq DNA polymerase. The reaction mixtures were incubated at 37 °C for 16 hours. The reaction mixtures were then incubated with an excess of inorganic pyrophosphatase to convert the pyrophosphate byproduct to monophosphate. The monophosphate was quantified by color reaction using the Malachite Green Phosphate Assay Kit (Sigma- Aldrich). The absorbance was measured at 600-660nm using a spectrophotometer. The percentage inhibition was calculated by dividing the non-heated sample’s absorbance by the heated sample’s absorbance, converting to percentage by multiplying with 100 and subtracting the result from the theoretical 100%.

[0071] EvaEZ fluorometric polymerase activity assay (Biotium, USA): A reaction mixture containing IX EvaEz master mix and antibody-Taq DNA polymerase complexes (at different molar ratios and as described above, heated and non-heated) such that the Taq DNA polymerase concentration was 0.75 ng / pL, was incubated at 37°C for 1 hour. A real-time qPCR instrument was used to measure the fluorescence in the FAM channel (emission 520 nm). The initial rate of fluorescence change (fluorescence unit / second) resulting from the polymerase activity was obtained by taking the slope of the fluorescence change during the initial velocity of the enzymatic reaction. The percentage inhibition was calculated as described above by using the slopes of the non-heated and heated samples. Example 3. Evaluating the impact of antibody 37H9-4 on Taq DNA polymerase activity.

[0072] First, the impact of antibody 37H9-4, Platinum® antibody and JumpStart® antibody on 5 ’-3’ exonuclease / 5’-flap endonuclease Taq polymerase activity was measured using a fluorescence-based assay. The experiment was run either without heat denaturation or with heat denaturation (FIG. 4). In the absence of dNTPs, polymerization does not occur, allowing for evaluation of 5 ’-3’ exonuclease / 5'-flap endonuclease activity.

[0073] The fluorescence in the FAM channel was measured using a spectrophotometer to evaluate the extent of exonuclease / 5’-flap endonuclease activity, where an increase in FAM signal corresponds to increased exonuclease / 5’-flap endonuclease activity. After heating, all antibody: enzyme combinations showed a similar fluorescence increase compared to wild-type Taq polymerase alone (-1000 dF / dT) indicating that all antibodies were deactivated and activity of the wild-type Taq polymerase 5 ’-3’ exonuclease / 5’-flap endonuclease activity was restored. Without heating, the antibody 37H9-4 showed the least amount of fluorescence indicating that it effectively inhibits the 5 ’-3’ exonuclease / 5’-flap endonuclease activity. In contrast, without heat denaturation the Platinum® and JumpStart™ antibodies both increased fluorescence by more than 2-fold, indicating that they stimulate 5 ’-3’ exonuclease / 5’-flap endonuclease activity.

[0074] Together, these results demonstrate that the antibody 37H9-4 inhibits 5 ’-3’ exonuclease / 5’-flap endonuclease activity of Taq DNA polymerase and provides advantages in PCR applications over existing hot-start antibodies.

Claims

CLAIMSWhat is claimed is:

1. An antibody that binds to Taq-polymerase, wherein the antibody comprises: a heavy chain variable domain (VH) comprising a heavy chain complementary determining region 1 (CDR-H1) comprising an amino acid sequence of SEQ ID NO: 4; a heavy chain complementary determining region 2 (CDR-H2) comprising an amino acid sequence of SEQ ID NO: 5; a heavy chain complementary determining region 3 (CDR-H3) comprising an amino acid sequence of SEQ ID NO: 6; and a light chain variable domain (VL) comprising of a light chain complementary determining region 1 (CDR-L1) comprising an amino acid sequence of SEQ ID NO: 10; a light chain complementary determining region 2 (CDR-L2) comprising an amino acid sequence of SEQ ID NO: 11; and a light chain complementary determining region 3 (CDR-L3) comprising an amino acid sequence of SEQ ID NO: 12.

2. The antibody of claim 1 , wherein the VH comprises an amino acid sequence of SEQ ID NO: 14, and the VL comprises an amino acid sequence of SEQ ID NO: 16.

3. The antibody of claim 1, wherein the antibody comprises an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

4. The antibody of any one of claims 1-3, wherein the antibody is a full length IgG, a Fab fragment, a F(ab’) fragment, a F(ab’)2 fragment, an scFv, or an Fv.

5. The antibody of any one of claims 1-4, wherein the antibody is a full-length rabbit IgG.

6. A nucleic acid sequence encoding the antibody of any one of claims 1-5.

7. The nucleic acid sequence of claim 6, wherein the VH is encoded by a nucleic acid sequence comprising SEQ ID NO: 13, and the VL is encoded by a nucleic acid sequence comprising SEQ ID NO: 15.

8. An expression vector comprising the nucleic acid sequence of claim 6 or claim 7.

9. A cell comprising the nucleic acid sequence of claim 7 or the expression vector of claim 8.

10. The cell of claim 9, wherein the cell is a mammalian cell.

11. The cell of claim 10, wherein the mammalian cell is a Chinese hamster ovary cell (CHO).

12. The cell of claim 9, wherein the cell is a bacterial cell.

13. An antibody-polymerase complex, wherein the complex comprises: the antibody of any one of claims 1-5; and a Taq-polymerase.

14. The antibody-polymerase complex of claim 13, wherein the Taq-polymerase is a wildtype Taq-polymerase.

15. The antibody-polymerase complex of claim 13, wherein the Taq-polymerase is a variant Taq-polymerase.

16. The antibody-polymerase complex of any one of claims 13-15, wherein the antibody- polymerase complex binds to DNA.

17. The antibody-polymerase complex of any one of claims 13-16, wherein activity of the Taq-polymerase is inhibited when bound to the antibody.

18. The antibody-polymerase complex of claim 17, wherein the inhibited Taq-polymerase activity is 5 ’-3’ exonuclease and / or 5 ’-flap endonuclease activity.

19. The antibody-polymerase complex of any one of claims 13-18, wherein the antibody- polymerase complex has decreased 5 ’-3’ exonuclease and / or 5 ’-flap endonuclease activity of the Taq polymerase as compared to a PLATINUM and / or JUMPSTART antibody-polymerase complex.

20. The antibody-polymerase complex of any one of claims 13-19, wherein the antibody- polymerase complex disassociates at a temperature greater than 80°C.

21. A method of amplifying a nucleic acid template, the method comprising: combining, in a solution for amplifying the nucleic acid template:(a) the antibody of any one of claims 1-5 and a Taq polymerase, or the antibody- polymerase complex of any one of claims 13-20,(b) a dNTP mixture,(c) a target nucleic acid template, and(d) an oligonucleotide primer comprising a polynucleotide that is complementary to the nucleic acid template.

22. The method of claim 21, wherein the Taq-polymerase is a wild-type Taq-polymerase.

23. The method of claim 21, wherein the Taq-polymerase is a variant Taq-polymerase.

24. The method of any one of claims 21-23, wherein combining occurs at a first temperature.

25. The method of claim 24, wherein the first temperature is below 30°C.

26. The method of claim 25, wherein the antibody-polymerase complex remains intact at the first temperature.

27. The method of any one of claims 21-26 further comprising bringing the solution to a denaturation temperature.

28. The method of claim 27, wherein the antibody-polymerase complex dissociates at the denaturation temperature.

29. The method of claim 27 or claim 28, wherein the denaturation temperature is at least 80°C.

30. The method of any one of claims 21-29, further comprising bringing the solution to an annealing temperature.

31. The method of claim 30, wherein the target nucleic acid template and the oligonucleotide primer anneal at the annealing temperature.

32. The method of any one of claims 21-31, further comprising bringing the solution to an extension temperature.

33. The method of claim 32, wherein the oligonucleotide primer is extended at the extension temperature.

34. The method of claim 32 or claim 33 comprising, in consecutive order:(a) bringing the solution to the first temperature for a first predetermined amount of time;(b) bringing the solution to the denaturation temperature for a second predetermined amount of time;(c) bringing the solution to the denaturation temperature for a third predetermined amount of time;(d) bringing the solution to the annealing temperature for a fourth predetermined amount of time; and(e) bringing the solution to the extension temperature for a fifth predetermined amount of time.

35. The method of claim 34, further comprising repeating a plurality of times:(a) bringing the solution to the denaturation temperature for the third predetermined amount of time;(b) bringing the solution to the annealing temperature for the fourth predetermined amount of time; and(c) bringing the solution to the extension temperature for the fifth predetermined amount of time.

36. The method of claim 35, further comprising bringing the solution to the extension temperature for a sixth predetermined amount of time.

37. The method of any one of claims 21-36, wherein amplification of the nucleic acid template produces a plurality of amplicons.

38. The method of any one of claims 21-37 wherein amplification is carried out by PCR, qPCR, digital PCR, droplet digital PCR, linear amplification, or multiplex PCR.

39. A kit comprising:(a) the antibody of any one of claims 1-5;(b) a dNTP mixture;(c) a reaction buffer; and(d) a Taq-polymerase.

40. The kit of claim 39, wherein the antibody, the dNTP mixture, the reaction buffer, and the Taq-polymerase are provided as a single solution.

41. The kit of claim 39 or claim 40, wherein the Taq-polymerase is a wild-type Taq- polymerase.

42. The kit of any one of claims 39-41, wherein the Taq-polymerase is a variant Taq- polymerase.

43. A kit comprising:(a) the antibody-poly merase complex of any one of claims 13-20:(b) a dNTP mixture; and(c) a reaction buffer.

44. The kit of claim 43, wherein the antibody-polymerase complex, the dNTP mixture, and the reaction buffer are provided as a single solution.

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