Method for detecting anti-Streptococcus pyogenes DNase B antibody in a test sample

A method for detecting or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, wherein said antibody inhibits Streptococcus pyogenes DNase B enzyme activity, comprising the steps of:

• (a) providing a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• (b) adding a quantity of a Streptococcus pyogenes DNase B to the test sample, the quantity being sufficient to produce a detectable level of enzymatic activity in the absence of inhibition of the enzymatic activity by anti-DNase B antibody in the test sample, the DNase B added to the test sample produced by a process comprising:
• (i) culturing a bacterial host cell transformed with an expression vector comprising a DNA sequence including therein the nucleotide sequence of Streptococcus pyogenes DNase B enzyme as shown in FIG. 3 (SEQ ID NO:
• 7);
• (ii) using the cultured bacterial host cell to express the DNase B enzyme;
• and
• (iii) purifying the enzyme from the cultured bacterial host cell, the purified enzyme being substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNase B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• and
• (c) determining a level of DNase B enzyme in the test sample by performing a DNase B enzyme assay to detect or determine the anti-Streptococcus pyogenes antibody in the test sample.

A method for detecting or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, wherein said antibody inhibits Streptococcus pyogenes DNase B enzyme activity, comprising the steps of:

• (a) providing a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• (b) adding a quantity of substantially purified Streptococcus pyogenes DNase B enzyme to the test sample, the quantity being sufficient to produce a detectable level of enzymatic activity in the absence of inhibition of the enzymatic activity by anti-DNase B antibody in the test sample, the DNase B enzyme being substantially free of proteins other than (1) the S. pyogenes DNase B enzyme and (2) S. pyogenes DNase B enzyme fused at its amino terminus with a leader peptide, the substantially purified enzyme being substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNase B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• and
• (c) determining a level of DNase B enzyme in the test sample by performing a DNase B enzyme assay to detect or determine the anti-Streptococcus pyogenes antibody in the test sample.

A method for detecting or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, wherein said antibody inhibits Streptococcus pyogenes DNase B enzyme activity, comprising the steps of:

• (a) providing a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• (b) adding a quantity of substantially purified Streptococcus pyogenes DNase B enzyme to the test sample, the quantity being sufficient to produce a detectable level of enzymatic activity in the absence of inhibition of the enzymatic activity by anti-DNase B antibody in the test sample, the DNase B enzyme being produced by a process comprising:
• (i) absorption to and elution from diethylaminoethyl cellulose to produce a first eluate;
• (ii) chromatography of the first eluate on phenyl agarose to produce a second eluate;
• (iii) chromatography of the second eluate on heparin agarose to produce a third eluate;
• and
• (iv) chromatofocusing of the third eluate to produce substantially purified DNase B enzyme substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNase B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• and
• (c) determining a level of DNase B enzyme in the test sample by performing a DNase B enzyme assay to detect or determine the anti-Streptococcus pyogenes antibody in the test sample.

A method for detecting or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, comprising the steps of:

• (a) preparing a buffered solution of a DNase B produced by a process comprising:
• (i) culturing a bacterial host cell transformed with an expression vector comprising a DNA sequence comprising DNA encoding the amino acid sequence of Streptococcus pyogenes DNase B enzyme as shown in FIG. 4 (SEQ ID NO:
• 9);
• (ii) using the cultured bacterial host cell to express the DNase B enzyme;
• and
• (iii) purifying the enzyme from the cultured bacterial host cell, the purified enzyme being substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNase B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• (b) reacting the buffered DNase B solution with a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• and
• (c) detecting a reaction between the DNase B and the anti-DNase B antibody by observing or measuring a change in light absorption or light scattering in the solution.

A method for detecting or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, comprising the steps of:

• (a) preparing a buffered solution of a DNase B produced by a process comprising:
• (i) culturing a bacterial host cell transformed with an expression vector comprising a DNA sequence including therein the nucleotide sequence of Streptococcus pyogenes DNase B enzyme as shown in FIG. 3 (SEQ ID NO:
• 7);
• (ii) using the cultured bacterial host cell to express the DNase B enzyme;
• and
• (iii) purifying the enzyme from the cultured bacterial host cell, the purified enzyme being substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNase B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• (b) reacting the buffered DNase B solution with a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• and
• (c) detecting a reaction between the DNase B and the anti-DNase B antibody by observing or measuring a change in light absorption or light scattering in the solution.

A method for detecting or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, comprising the steps of:

• (a) preparing a buffered solution of a substantially purified DNase B enzyme, the DNase B enzyme being substantially free of proteins other than (1) the S. pyogenes DNase B enzyme and (2) S. pyogenes DNase B enzyme fused at its amino terminus with a leader peptide, the substantially purified enzyme being substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNase B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• (b) reacting the buffered DNase B solution with a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• and
• (c) detecting a reaction between the DNase B and the anti-DNase B antibody by observing or measuring a change in light absorption or light scattering in the solution.

A method for detecting or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, comprising the steps of:

• (a) preparing a buffered solution of substantially purified DNase B enzyme, the DNase B enzyme being produced by a process comprising:
• (i) absorption to and elution from diethylaminoethyl cellulose to produce a first eluate;
• (ii) chromatography of the first eluate on phenyl agarose to produce a second eluate;
• (iii) chromatography of the second eluate on heparin agarose to produce a third eluate;
• and
• (iv) chromatofocusing of the third eluate to produce substantially purified DNase B enzyme substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNase B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• (b) reacting the buffered DNase B solution with a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• and
• (c) detecting a reaction between the DNase B and the anti-DNase B antibody by observing or measuring a change in light absorption or light scattering in the solution.

A method for detecting and/or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, wherein said antibody inhibits Streptococcus pyogenes DNase B enzyme activity, comprising the steps of:

• (a) providing a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• (b) adding a detectable quantity of a Streptococcus pyogenes DNase B enzyme to the test sample, the quantity being sufficient to produce a detectable level of enzymatic activity in the absence of inhibition of the enzymatic activity by anti-DNase B antibody in the test sample, the DNase B enzyme produced by the process of:
• (i) culturing a bacterial host cell transformed with an expression vector comprising a DNA sequence that is substantially purified DNA comprising DNA encoding a Streptococcus pyogenes DNase B amino acid sequence selected from the group consisting of the amino acid sequence of:
• (1) Streptococcus pyogenes DNase B as shown in FIG. 4 (SEQ ID NO:
• 9);
• and (2) a Streptococcus pyogenes DNase B variant comprising the amino acid sequence shown in FIG. 4 (SEQ ID NO:
• 9) and having a deletion, insertion or substitution of one or more amino acids shown in FIG. 4 (SEQ ID NO:
• 9), wherein the variant is capable of being recognized and bound by anti-Streptococcus pyogenes DNase B antibody;
• (ii) using the cultured bacterial host cell to express the DNase B enzyme;
• and
• (iii) purifying the enzyme from the cultured bacterial host cell;
• and
• (c) determining a level of activity of DNase B enzyme in the test sample by performing a DNase B enzyme assay to detect and/or determine the anti-Streptococcus pyogenes antibody in the test sample.

A method for detecting and/or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, wherein said antibody inhibits Streptococcus pyogenes DNase B enzyme activity, comprising the steps of:

• (a) providing a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• (b) adding a detectable quantity of a Streptococcus pyogenes DNase B enzyme to the test sample, the quantity being sufficient to produce a detectable level of enzymatic activity in the absence of inhibition of the enzymatic activity by anti-DNase B antibody in the test sample, the DNase B enzyme produced by the process of:
• (i) culturing a bacterial host cell transformed with an expression vector comprising a DNA sequence that is the DNA having the nucleotide sequence of FIG. 3 (SEQ ID NO:
• 7);
• (ii) using the cultured bacterial host cell to express the DNase B enzyme;
• and
• (iii) purifying the enzyme from the cultured bacterial host cell;
• and
• (c) determining a level of activity of DNase B enzyme in the test sample by performing a DNase B enzyme assay to detect and/or determine the anti-Streptococcus pyogenes antibody in the test sample.

A method for detecting or determining anti-Streptococcus pyogenes DNase B antibody in a test sample, comprising the steps of:

• (a) preparing a buffered solution of a DNase B produced by a process comprising:
• (i) culturing a bacterial host cell transformed with an expression vector comprising a DNA sequence that is substantially purified DNA comprising DNA encoding a Streptococcus pyogenes DNase B amino acid sequence selected from the group consisting of the amino acid sequence of:
• (1) Streptococcus pyogenes DNase B as shown in FIG. 4 (SEQ ID NO:
• 9);
• and (2) a Streptococcus pyogenes DNase B variant comprising the amino acid sequence shown in FIG. 4 (SEQ ID NO:
• 9) and having a deletion, insertion or substitution of one or more amino acids shown in FIG. 4 (SEQ ID NO:
• 9), wherein the variant is capable of being recognized and bound by anti-Streptococcus pyogenes DNase B antibody;
• (ii) using the cultured bacterial host cell to express the DNase B enzyme;
• and
• (iii) purifying the enzyme from the cultured bacterial host cell, the purified enzyme being substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNase B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• (b) reacting the buffered DNase B solution with a test sample suspected of containing anti-Streptococcus pyogenes DNase B antibody;
• and
• (c) detecting a reaction between the DNase B and the anti-DNase B antibody by observing or measuring a change in light absorption or light scattering in the solution.

A method for detecting or determining anti-Streptococcus pyogenes DNAse B antibody in a test sample, comprising the steps of:

• (a) preparing a buffered solution of a DNAse B produced by a process comprising:
• (i) culturing a bacterial host cell transformed with an expression vector comprising a DNA sequence that is selected from the group consisting of:
• (1) the DNA having the nucleotide sequence of FIG. 3 (SEQ ID NO:
• 7), and (2) a Streptococcus pyogenes DNAse B variant comprising the polynucleotide sequence shown in FIG. 3 (SEQ ID NO:
• 7), the variant having a deletion, insertion or substitution of one or more nucleic acids shown in FIG. 3 (SEQ ID NO:
• 7), and wherein the variant encodes a Streptococcus pyogenes DNAse B capable of being recognized and bound by anti-Streptococcus pyogenes DNAse B antibody;
• (ii) using the cultured bacterial host cell to express the DNAse B enzyme;
• and
• (iii) purifying the enzyme from the cultured bacterial host cell, the purified enzyme being substantially free of mitogenic activity such that when added to a mitogenic assay with mammalian lymphocytes, the level of mitogenic activity observed in the purified DNAse B enzyme is equivalent to the level of mitogenic activity observed in a control sample to which no DNAse B enzyme has been added;
• (b) reacting the buffered DNAse B solution with a test sample suspected of containing anti-Streptococcus pyogenes DNAse B antibody;
• and
• (c) detecting a reaction between the DNAse B and the anti-DNAse B antibody by observing or measuring a change in light absorption or light scattering in the solution.