The aim of the thesis was to develop an immunoassay for the detection of dangerous bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA). MRSA infections cause global problems in health facilities which provide acute and follow-up care in inpatient and outpatient parts. That is why early and rapid diagnosis and targeted thereapies are very important for the subjects. E. coli was purified using the QIAquick PCR kit Purificatin isolated bacterial constructs, which were subsequently purified and dialyzed. The recombinant protein PBP2a in different concentrations was applied to a nitrocellulose membrane in the form of lines. Furthermore was performed optimized blot-line method for the detection of specific antibodies against the recombinant antigen PBP2a in the classes IgG, IgA and IgM. Several different concentrations of the conjugate Goat Anti-human IgG-AP, Goat Anti-human IgA-AP or Goat anti-Human IgM-AP were used for the detection. The color intensity of each line of the strip was evaluated with Immunoblot software. The measured values were used to determine diagnostic sensitivity, specificity and overall diagnostic match. Further testing of precision was carried out under repeatability conditions (intra-assay) and reproducibility (inter-assay). Precision of the method was expressed by coefficient of variation. As the most suitable for the manufacture of a IgG kit was determined the concentration of the antigen PBP2a from 0.30 mg/ml to 0.45 mg/ml and the concentration of IgG conjugate from 1:1500 to 1:1800. For class IgA as the most appropriate antigen was determined concentration PBP2a from 0.40 mg/ml to 0.52 mg/ml and conjugate concentrations of IgA from 1:500 to 1:1000. The coefficient of variation under repeatability conditions for the entire range of the IgG class is 10.09 %, and for IgA is 8.91 %. Variation coefficient reproducibility conditions for the entire range of the IgG class is 9.23 % and for IgA is 9.60 %. Precision of the method under conditions of repeatability and reproducibility for classes IgG and IgA meets the criteria for the manufacture of a diagnostic kit. Titration results showed that particular batch of cards made of nitrocellulose membrane coated with antigen PBP2a must always be verified on the panel of reference samples and the values of concentrations (for both antigen and conjugate) should be set according to the needs, but differently for the class of immunoglobulins IgG and IgA.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:430449 |
| Date | January 2017 |
| Creators | Šmídová, Lada |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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