Baylor College of Medicine

David Y Graham

Mary K Estes

Xi Jiang

Saleh Naser

Fouad A El-Zaatari

Dolores G Evans

Doyle J Evans

Double-stranded cDNA was synthesized from nucleic acid extracted from Norwalk virus purified from stool specimens of volunteers. One clone was isolated from a cDNA library constructed in a pUC-13 vector after amplification of the cDNA. The specificity of this cDNA (pUCNV-953) was shown by hybridization assays. The cDNA reacted with post- (but not pre-) infection stool samples from Norwalk volunteers and with highly purified Norwalk virus, but not with other common enteric viruses such as hepatitis A virus and rotavirus. Finally, the probe detected virus in the same fractions of CsCl gradients in which viral antigen was detected using a specific Norwalk virus radioimmunoassay, and particles were detected by immune electron microscopy. Single-stranded RNA probes derived from the DNA clone after subcloning into an in vitro transcription vector were also used to show that the Norwalk virus contains a ssRNA genome of about 8 kb in size. The original clone was also used to detect additional cDNAs which represent at least 7 kb of nucleic acid of the Norwalk genome. The availability of a Norwalk-specific cDNA and the first partial genome sequence information allow rapid cloning of the entire genome and of establishment of sensitive diagnostic assays. Such assays can be based on detection of Norwalk virus nucleic acid or Norwalk viral antigen using polyclonal or monoclonal antibodies to proteins expressed from the cDNA or to synthetic peptides made based on the knowledge of the genome sequence. Vaccines made by recombinant DNA technology are now feasible.

Polyclonal and monoclonal antibodies to Norwalk virus and methods for

Recombinant clone pMptb #48 expressing a 36k M. paratuberculosis antigen is provided that provides a more sensitive and discriminating test than those in which crude antigens have been used. A mycobacterial genus-specific DNA probe corresponding to a 1.4 kb BamH1-DNA insert in pMptb #48 is also provided. The 1.4 kb insert includes the entire DNA coding sequence for an M. paratuberculosis 36k protein. There is also provided methods for serologically testing specimens which include the use of recombinant clone pMptb #48 and/or the p36k protein and/or fragments of the p36k protein. Also provided are methods for identifying mycobacterial infections using a probe corresponding to the 1.4 kb DNA sequence shown in FIG. 4, the coding portion of the 1.4 kb DNA sequence, or fragments thereof.

Mycobacterial genus-specific DNA probe and its expressed product

Double-stranded cDNA was synthesized from nucleic acid extracted from Norwalk virus purified from stool specimens of volunteers. One clone was isolated from a cDNA library constructed in a pUC-13 vector after amplification of the cDNA. The specificity of this cDNA (pACNV-953) was shown by hybridization assays. The cDNA reacted with post- (but not pre-) infection stool samples from Norwalk volunteers and with highly purified Norwalk virus, but not with other common enteric viruses such as hepatitis A virus and rotavirus. Finally, the probe detected virus in the same fractions of CsCl gradients in which viral antigen was detected using a specific Norwalk virus radioimmunoassay, and particles were detected by immune electron microscopy. Single-stranded RNA probes derived from the DNA clone after subcloning into an in vitro transcription vector were also used to show that the Norwalk virus contains a ssRNA genome of about 8 kb in size. The availability of a Norwalk-specific cDNA and the first partial genome sequence information allow rapid cloning of the entire genome and of establishment of sensitive diagnostic assays. Such assays can be based on detection of Norwalk virus nucleic acid or Norwalk vital antigen using polyclonal or monoclonal antibodies to proteins expressed from the cDNA or to synthetic peptides made based on the knowledge of the genome sequence. Vaccines made by recombinant DNA technology can also now be feasible.

Methods and reagents to detect and characterize Norwalk and related

An antigen for the detection of Compylobacter pylori infections and an assay for the serological detection of Campylobacter pylori. The antigen includes high molecular weight cell-associated proteins purified from Campylobacter pylori. The antigen can be used in a variety of assays including radioimmunoassay, ELISA, latex agglutination, complement fixation, and indirect hemagglutination. Furthermore, the antigens can be combined with a solid support in kit form.

Growing community of inventors

Houston, TX, United States of America

David Y Graham