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Bacterial vaginosis diagnostic

An activity and indicator technology, applied in disease diagnosis, instruments, peptides, etc., can solve problems such as lack of sensitivity and lack of accuracy of tests

Pending Publication Date: 2021-03-16
MOLOGIC LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] pH-based tests lack accuracy because various conditions can cause changes in the local pH of the vagina (eg, candidiasis and trichomoniasis and BV)
Known tests for detection of sialidase activity such as BV Blue lack sensitivity

Method used

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  • Bacterial vaginosis diagnostic
  • Bacterial vaginosis diagnostic
  • Bacterial vaginosis diagnostic

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0330] Example 1: Assay Chemistry Principles and Experimental Design

[0331] In summary, the principle works on the basis that antibodies recognize the chemical product of a sialidase reaction, where the chemical substrate is a peptide specifically designed to react with the sialidase, and that the reaction product is then recognized by an antibody raised against said synthetic product. As shown in Figures 1 and 2, glycopeptides containing sialic acid and having a biotin tag are provided. When contacted with a test sample containing sialidase activity, the sialylated group is cleaved from the glycopeptide by the sialidase to expose the pendant galactosyl group on the peptide. Antibodies raised against the desialylated product then bind specifically to the cleaved product. By using an antibody-gold conjugate and a lateral flow strip with a streptavidin test line, the presence of desialylated product can be detected as a red line, as well as sialidase activity measured directl...

example 2

[0333] Example 2: Peptide Design

[0334] In the design of candidate peptide sequences, many factors are considered:

[0335] size

[0336] In general, molecules with a molecular weight (MW) of less than 5000 Daltons are less likely to stimulate a good immune response in the host organism. The peptides were planned as relatively short sequences designed to be conjugated to KLH (keyhole limpet hemocyanin), resulting in more and more potent immunogens. A range of different lengths (9-20 amino acids) is proposed to cover any potential performance variation.

[0337] conjugation chemistry

[0338] Two different conjugation chemistries were used. For two of the peptides, a cysteine ​​tag was incorporated into the structure, allowing it to be conjugated to a carrier protein using standard maleimide-based chemistry. For the other three peptides, a relatively new hydrazine-based chemistry was used as a more controllable process than cysteine ​​chemistry because there is less ...

example 3

[0346] Example 3: Peptide Synthesis

[0347] Galactose-tagged peptides were synthesized using solid-phase chemistry on an automated microwave synthesizer. All peptides were purified using reverse phase HPLC and characterized by electrospray LCMS. To label the galactose moiety with sialic acid, an enzymatic approach using transsialic acid (TcTS) from T. cruzi and fetuin as sialic acid donor was employed. Figure 7 and 8 The synthetic methods used are summarized.

[0348] and related peptide immunogens conjugated to carrier proteins, two biotinylated derivatives of each sequence, one of which was labeled with sialic acid, were also synthesized. These biotinylated derivatives will form the basis for a lateral flow assay format. Figure 9 shows the peptides synthesized for this work based on various immunizations. Peptides labeled with sialic acid are designated with a "c"; eg, MOL136c. Peptides lacking a sialylation group do not have this designation (eg, MOL136).

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Abstract

The invention provides a sialidase enzyme activity detection kit or device comprising: (i) an indicator molecule comprising a sialylated peptide and a capture site; (ii) a capture zone comprising capture molecules; and (iii) binding molecules capable of binding to the de-sialylated derivative of the indicator molecule. Also provided are methods of using the kits or devices, as well as specific indicator molecules and specific binding molecules.

Description

technical field [0001] The present invention relates to detection of cleavage activity of sialidase and its use in detection of bacterial vaginosis. In particular, the present invention provides specially designed peptides and indicator molecules that can be used to detect the cleavage activity of sialidases. Various other aspects of the invention comprise enzyme detection devices, kits, methods and uses for detecting or measuring the presence in a test sample of the activity of a sialidase capable of cleaving an indicator molecule of the invention. Background technique [0002] Sialidases (otherwise known as neuraminidases) are glycoside hydrolases that split into two main classes (EC 3.2.1.18 and EC 3.2.1.129, respectively) that cleave exo- or endo-(poly)sialic acids . Sialic acid is an N- or O-substituted derivative of neuraminic acid (shown below): [0003] [0004] Sialic acid is commonly found in nature in glycoproteins and glycolipids (particularly gangliosides)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/34C12Q1/689G01N33/50G01N33/569G01N33/68
CPCC12Q1/34G01N33/5091G01N33/56911G01N33/6893G01N2800/36C07K7/06C07K16/00
Inventor 保尔·戴维斯詹姆斯·斯豪滕
Owner MOLOGIC LTD
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