Diagnostic Assays : Pathogens


Enhanced Endotoxin Detection: New Advantages in Liquid Crystal Assays for Gram-Negative Pathogens

UW–Madison researchers have now demonstrated enhanced endotoxin detection in the presence of masking agents in their previous liquid crystal system.

Unlike the LAL assay, the LC-based method does not suffer from LER or any loss of sensitivity due to the presence of cations (e.g., Ca2+ or Mg2+), buffers (e.g., citrate), surfactants (e.g., SDS), chelating agents (e.g., EDTA), proteins or nucleic acids (e.g., DNA or RNA). Thus, the LC-based method provides faster and cheaper detection of endotoxin when compared to existing methods, such as the LAL assay.

Field Test for Mycobacterial Infections

A UW–Madison researcher has identified a set of biomarkers that can indicate whether a mammal is vaccinated or infected, as well as the type of infection (bovine tuberculosis or Johne’s disease).

Diagnostic Kit for Blastomycosis

UW–Madison researchers have developed a method for obtaining highly pure native BAD-1 protein that could be used to detect B. dermatitidis infection.

A solution containing native BAD-1 protein or fragments is collected from cultured fungus strains. The solution is combined with nickel-chelating resin, washed and eluted to obtain a highly pure form without the need for more expensive recombinant methods. This can be mixed and analyzed with a patient’s sample to determine if the fungus is present.

Paratuberculosis Test for Bovines and Other Animals

UW–Madison researchers have developed a new kit for diagnosing paratuberculosis infection in animals. The kit contains antigens from a novel M. avium paratuberculosis strain designated JTC303.

A sample such as serum or milk taken from an animal is tested. If the animal is infected, the sample will contain antibodies that bind to the antigens in the kit. This binding can be identified using a detecting molecule. The kit is best used in the widely used standard enzyme-linked immunosorbent assay (ELISA) format.

Simpler Culture Screening for Mycobacterium Avium

UW–Madison researchers have developed novel antigen and antibody preparations and kits that can be used in assays to detect MAC bacteria in liquid cultures.

The system uses capture and detection antibodies obtained from animal subjects immunized with Mycobacterium-secreted antigens. During incubation with the sample, capture antibodies will bind to MAC antigens, allowing others to be washed away. Detection antibodies then are added. They may be directly labeled or used with a conjugate enzyme to trigger a detection signal such as a color change.

Sensitive Assay for Detecting Botulinum Neurotoxin, Neutralizing Antibodies or Inhibitors

UW-Madison researchers have developed a sensitive and specific method of detecting the presence or activity of botulinum neurotoxin, neutralizing antibodies to the toxin or inhibitors of botulinum toxin.  This method may provide a viable alternative to the mouse bioassay. 

To detect neutralizing antibodies or other inhibitors of botulinum toxin, the method involves exposing cultured neuronal cells to the toxin and a test sample.  The cells are incubated to allow active toxin to cleave an endogenous substrate.  Then the cells are harvested and probed to determine how much substrate was cleaved.  The more neutralizing antibodies or other inhibitors there are, the less cleavage is observed as compared to the control. 

To detect the presence of active neurotoxin, the cultured cells are exposed to a test sample and control samples with known amounts of toxin.  The amount of substrate cleavage can be evaluated to determine how much botulinum neurotoxin is present in the test sample as compared to the control samples.

Quantitative Comet Assay for Measuring Viral Growth and Resistance to Anti-Viral Compounds

UW-Madison researchers have developed a sensitive means of measuring viral infectivity and replication activity by monitoring flow-induced viral comet formation. A layer of host cells is contacted with a viral sample and cultured in a thin layer of liquid culture medium. Preferably, the host cells are cultured with the virus particles in a microfluidic channel. The liquid medium flows controllably through the channel to enhance the spread of the viral progeny to uninfected host cells. Infected host cells develop an observable indication of viral gene expression, like cell death. The resulting comet-like infections can be digitally imaged and computer-processed for automated quantification of the spread of viral infection.

Method and Compositions for Detecting Botulinum Neurotoxin

UW-Madison researchers have developed a fluorescence resonance energy transfer (FRET) method for the sensitive detection of botulinum neurotoxin. The assay uses two fluorescent proteins, such as cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), which are linked together by a molecule that can be recognized and cleaved by botulinum neurotoxin. The emission spectrum of CFP partially overlaps with that of YFP. As a result, when CFP and YFP are very close together, excitation of CFP results in FRET-YFP emission and partial quenching of CFP emission. When botulinum neurotoxin cleaves the linker molecule separating these two fluorescent proteins, FRET is eliminated, i.e., excitation of CFP no longer results in YFP emission and partial quenching of CFP emission.

To detect botulinum neurotoxin, a sample is exposed to the CFP and YFP construct. The FRET signals are measured and compared before and after exposure, with a decrease in FRET after exposure indicating the toxin’s presence. This method is useful for detecting botulinum toxin both in vitro and in living cells.

Novel Sequences of E. coli 0157:H7

UW-Madison researchers have identified the entire sequence of E. coli O157:H7, allowing detection, diagnosis, prophylaxis and therapeutic tools for combating this pathogen. The researchers identified nearly all the DNA sequences unique to E. coli O157:H7. When combined with the genome sequence of E. coli strain K12, which has already been published in the field, these data provide virtually the entire genome of E. coli O157:H7.

Plasmid DNA from Yersinia pestis, the Causative Agent for Bubonic Plague

UW-Madison researchers have developed the complete DNA sequence of three virulence plasmids, pPCP1, pMT1 and pCD1, from Y. pestis. The open reading frames (ORFs), or protein coding regions, of the plasmids have been determined. The plasmid pPCP1 contains genes encoding plasminogen activator/coagulase and pesticin, a toxin that inhibits the growth of closely related bacteria. In the plasmid pMT1, 115 of the potential ORFs likely encode proteins, including seven new potential virulence factors. The plasmid pCD1 encodes a complex virulence property called the low calcium response (LCR). The sequence information will enable diagnostic, prophylactic, and therapeutic tools to be developed for use in combating Y. pestis.

Eight Hybridoma Cell Lines Producing Monoclonal Antibodies Against La Crosse Encephalitis Virus

UW-Madison researchers have now produced eight lines of hybridomas, one of which is specific for G1, five of which are specific for G2, and two of which recognize both G1 and G2. Frozen stocks are available at UW-Madison.

Monoclonal Antibodies That Recognize Recombinant Mengovirus 3C Proteinase

UW-Madison researchers have created hybridoma cell lines producing monoclonal antibodies raised to recombinant Mengo 3C protease; these antibodies are designated 6D10, 8F10 and 10C6. This material may be used in the specific molecular recognition and detection of protein 3C (protease), which is encoded within the genome of Mengovirus of the family Picornaviradae.

Monoclonal and Polyclonal Antibodies Raised to Recombinant Mengovirus 3D Polymerase

UW-Madison researchers have created polyclonal antibodies and various hybridoma cell lines producing monoclonal antibodies raised to recombinant Mengo 3D polymerase. These antibodies are designated 1B6, 3C5, 10D3, 2F11, 3C6, 2F6, 3B7, 10B3, 1D6, 5F6, and 8D10. This material can be used in the specific molecular recognition and detection of the protein 3D polymerase that is encoded within the genome of Mengovirus in the family Picornavirae.