DEVELOPMENT OF NOVEL DIP STRIP BASED IMMUNOASSAYS

Trigg, Jay DEVELOPMENT OF NOVEL DIP STRIP BASED IMMUNOASSAYS. Doctoral thesis, University of Sunderland.

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Abstract

The introduction of enzymes into the detergent industry during the early 1960’s revolutionised the cleaning process for everyone. However shortly after this introduction high numbers of workers were reporting incidences of asthma. This
correlation was linked directly to the inhalation of the powdered enzymes,indicating steps had to be made to reduce the amount of enzyme in the atmosphere and the need for regular monitoring of the working environment. Current methods for the detection of atmospheric enzymes are often lengthy since they require specialist equipment and trained operators to perform the assays. There is still a need within the industry for a quick, simple assay that
can be performed on site, which is reproducible without the need for any specialist equipment or training. For the purpose of this research into development of an assay the enzyme Savinase was chosen. Savinase is a small monometric protease with a molecular weight of ~27 kDa which is part of the cocktail of enzymes used in modern detergents.

A novel type of dip strip assay has been developed termed Surface Layer Immuno-Chromatography (SLIC); this is an adaptation of the current ELISA method, which employs a novel combination of immuno-chromatography and enzyme-linked immuno sorbent assay. The assay is based on a phenomenon
which occurs when a dense bio-polymer is pulled by gravity over the surface membrane of a dip strip. The dip strip has a spot of immobilised antibody on its surface and 5-bromo-4-chloro-3-indolyl phosphate, nitro blue tetrazolium (BCIP/NBT) substrate deposited along with the bio-polymer on the top of the dip strip. At the bottom of the strip there is a deposit of enzyme-labelled antibody plus bio-polymer.
When the strip is placed into a sample, the analyte molecules bind to the immobilised spot, at the same time the lower reagent is released from the filter and flows to the bottom of the cuvette. After ten minutes the solution is stirred using the dip strip, thereby releasing the labelled antibody into the bulk of the solution and thus allowing the labelled antibody to cap the immobilised analyte-antibody spot. The sample is then topped up with substrate buffer, wetting the substrate which flows down the dip strip passing over the spot where the captured enzyme-antibody complex is located. The enzyme reacts with the substrate to produce an insoluble blue precipitate on the surface of the spot. The bound analyte can thus be observed visually and colorimetrically as a blue/purple spot.

The SLIC assay provides an analytical technique which can be used on site by the factory workers, providing quick results with a limit of detection at 5 ng mL-1. If sampling takes place over a complete 8 hr shift this is equivalent to an
atmosphereic concentration of 5 ng m-3 which is well below the maximum exsposure limit of 40 ng mL-1. In order to demonstrate the diversity of the SLIC assay a very different analyte which has a more complex structure was chosen. The yeast Malassezia furfur has been associated with the causes of dandruff. M. furfur is predominantly found around the face and scalp regions of the body. Current methods for the
investigation of yeast counts on the skin are very lengthy taking days to grow colonies taken from skin washes. After using the same washing technique as the current methods, the wash is used as the sample for the SLIC assay. An approximate cell count is produced within half an hour. The SLIC assay provides a quick result which clearly differentiates between what is considered a normal
cell count of 5 x 105 cells per cm2 and that which is considered an abnormal cell count of 5 x 106 cells per cm2.

Item Type: Thesis (Doctoral)
Depositing User: Klaire Purvis-Shepherd
Date Deposited: 30 Sep 2020 14:10
Last Modified: 30 Sep 2020 14:15
URI: http://sure.sunderland.ac.uk/id/eprint/12650

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