Method for reducing lysozyme enzymatic activity

Abstract

The present invention generally features therapeutic and diagnostic compositions and methods for increasing or decreasing the binding of a lysozyme polypeptide to a Treponema pallidum P17 polypeptide (Tp17) or a Tp17-like polypeptide. More particularly, the invention relates to compositions and methods for detecting, treating, or preventing a pathogen infection or a chronic disorder; and to binding assays using a Tp17-like polypeptide and a lysozyme polypeptide.

Description

FIELD OF THE INVENTION [0001] The present invention generally features diagnostic and therapeutic compositions and methods derived from the characterization of the binding of a lysozyme polypeptide to a Treponema pallidum P17 polypeptide (Tp17) or Tp17-like polypeptides. In addition, the invention provides methods and compositions for increasing or decreasing the binding between a lysozyme and a TP-17 or a TP-17 like polypeptide. BACKGROUND OF THE INVENTION [0002] Syphilis is a disease caused by Treponema pallidum (hereinafter also referred to as “Tp”) infection. The diagnosis of syphilis is generally made by an immunoassay of anti-Tp antibody in the blood using, for example, the Treponema pallidum Hemagglutination Test (TPHA), the Fluorescent Treponema Antibody Absorption Test (FTA-ABS) and / or the Treponema pallidum Immobilization Test (TPI), as well as enzyme-linked immuno sorbent assay (ELISA) and Western Blot systems. These tests detect antibodies that react with Tp or antigen p...

AI Technical Summary

Benefits of technology

[0085]FIG. 4 illustrates an advantage of the present invention since conventional Tp17 antibody detection kits do not detect antibodies to the Tp17-lysozyme complex. In one embodiment, the invention features a kit containing a Tp17-like polypeptide and a lysozyme polypeptide. Such kits are capable of detecting antibodies to Tp17 alone, lysozyme alone, or a Tp17-lysozyme complex, if present.

[0086]FIG. 5 illustrates a far western blot lysozyme-protein interaction assay comprising the following five steps: (1) immobilization of purified lysozyme onto a solid support; (2) probing of the solid support with a ligand for lysozyme (e.g., GST-Tp17); (3) binding of GST-Tp17 with goat anti-GST antibody; (4) binding of the goat antibody with an anti-goat alkaline phosphatase conjugate; and (5) staining with nitroblue tetra zolium / bromochloro indolyl phosphate alkaline phosphatase chromogenic substrate (NBT / BCIP).

[0087]FIG. 6 illustrates the amino acid (SEQ ID NO:29) and nucleotide (SEQ ID NO:30) sequence of GST-Tp17 fusion protein. The sequences corresponding to the GST moiety and to Tp17 are represented in italics and bold, respectively. Numbered lines, SEQ ID NO: 30, represent the sense DNA strand; the antisense or RNA strand is below the sense strand.

[0088]FIG. 7 illustrates the Tp17-HIS expression construct pET24a_Tpp17His. This vector derives from the pET24 expression vector (Novagen Inc., Madison, Wis.).

[0089]FIG. 8A is a Coomasie Brilliant Blue-stained gel of purified GST-Tp17 chromatographic fractions. Lane 1: purification process with added chicken lysozyme; lane 2: purification process without added chicken lysozyme; lane 3: MWM: molecular weight markers.

[0090]FIG. 8B is a Western Blot and Coomasie Brilliant Blue-stained gel of purified Tp17-HIS chromatographic fractions. Lane 1: Western blot using a syphilis positive human serum of proteins recovered from the purification process in the presence of added chicken lysozyme; lane 2: Coomasie Brilliant Blue staining of proteins recovered from the purification process in the presence of added chicken lysozyme. The peptide sequences obtained by N-terminal amino acid sequencing of the proteins recovered from the electrophoretic bands in lanes one and two are indicated.

Problems solved by technology

This test is not generally used as a diagnostic due to its high cost.

Although these assays are widely used, they lack the sensitivity required for detecting early stage or low level infection, when Tp antibody levels in the body fluids are very low.

In addition, the popular use of individual recombinant or purified Tp antigens in existing immunoassays for syphilis does not account for the binding by some antibodies to complexes formed between Tp antigens and other antigens (i.e., Tp antigen binding partners) normally present in the subject's body fluid.

Existing immunoassays thus fail to detect such antibodies in the absence of the Tp antigen binding partner, which results in some instances in low sensitivity or false negative assay results.

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