Rapid Diagnosis of Bacterial Infection

2017

"Developing a bacteriophage-Affimer antibody-scaffold diagnostic kit for Clostridium difficile infection"

Research for Masters degree

(University of Leeds, Michael McPherson Group)

I worked to develop a rapid diagnostic kit against Clostridium difficile bacterial infection, a common and expensive disease frequently acquired in hospitals.

This assay used bacteriophage viruses to present antibody-like Affimer (formerly adhiron) binding scaffolds. As part of a fast developing "ELISA" reaction, these scaffolds would detect toxin B from C. difficile.

Why is this work important?

Rapid detection of disease is often crucial for effective treatment.

This is certainly the case with C. difficile infection. This is a common hospital-acquired disease that is expensive to treat and can be particularly nasty for the patient.

By developing faster, more reliable and more cost effective solutions, we can begin to improve the quality of life for those infected by speeding up treatment, while also helping to reduce costs and increase bed-space for healthcare providers.

Laboratory Techniques

Below is a list of the type of work I carried out in the lab as part of this project.

Molecular Biology

This is the manipulation and analysis of genetic material. For me this was in the form of DNA, the genetic code for all living things.

I extracted and purified DNA from bacteria, sequenced the DNA to reveal the code within and made changes to the DNA.

I worked with things known as antibody-like structures called Affimers. These are protein structures (folded peptide-chains made out of things known as amino acids). In living things, DNA encodes for protein.

In order to work with the protein Affimers, I first had to work with the DNA!

With some molecular biology know-how, I was able to get bacteria to make my protein scaffolds for me. I could then purify these protein structures and put them to the test.

Phage Display

Bacteriophage are viruses which only infect bacteria. Some of these viruses are able to "present" a protein on their surface.

I was able to get bacteriophage viruses to present my protein scaffolds on their surface.

I was interested in seeing what variants of these protein scaffolds were able to bind to the toxin B protein produced by the C. difficile bacteria.

By having the scaffolds attached to the bacteriophage virus, I was able to detect scaffold binding by detecting the bacteriophage - a far easier and more reliable process.

ELISA Assay Development

ELISA stands for enzyme-linked immunosorbent assay.

ELISA assays are a common test used to detect binding. In this project, I developed and optimised ELISA assays to detect bacteriophage-Affimer scaffold binding to toxin B of C. difficile.

This allowed me to screen through a large number of Affimer scaffold variants to find which ones bound the best to toxin B.

The best of those could then be taken forward for further testing and eventual commercial product development.

Comparison Testing

With the bacteriophage-Affimer scaffolds which bound the best to toxin B of C. difficile, it was important to test they were better than what we already have available.

This is important as commercial product development is an expensive and risky undertaking. We need to know our product would actually be worth making.

For this, I evaluated the effectiveness of our best candidate scaffolds against other existing detection methodology to see how it fared. This was achieved using a variety of bench-top assays to measure speed and reliability. Production costs could then be factored in to see whether these scaffolds held any potential advantage.