Diagnostics and techniques

Question 1

Ouchterlony immunodiffusion (OID)

Answer 1

A qualitative technique for detecting antibody-antigen interacts. Can be done in a sold or liquid. It measures the interations by measuring the size of precipitation zone formed by aggultionation between antibody and antigen

Question 2

Immunofluorescence

Answer 2

A qualitative technique used to localise antibodies or antigens on cell surfaces or identification of pathogenic orgnaisms. It tags antibodies with fluorescent . Can be direct or indirect

Question 3

Immunoelectron gold microscopy (EM-gold)

Answer 3

used to visualise intracellular antigens and organelles. IS a qaulitative technique in which antibodies tagged with gold particles are used to detect molecules of interest

Question 4

Western blotting

Answer 4

A technique to identify and locate specific proteins in a sample based on their ability to bind to specific antibodies. Proteins are separated by SDS PAGE. Radiolabelled or enzyme linked antibodies can be used

Question 5

PTA-ELISA

Answer 5

plate trapped antigen ELISA

Question 6

DAS-ELISA

Answer 6

double antibody sandwich ELISA

Question 7

ELISA

Answer 7

quantitative technique used for detecting antigens or antibodies using enzyme-substrate reactions. Enzymes are usually coupled to antibodes (directly) or to anti-immunoglobilins (indirectly)

Question 8

IPA

Answer 8

Invasive pulmonary aspergillosis

Question 9

How can IPA be diagnosed?

Answer 9

Usually a probable diagnosis rather than a proven diagnosis. Serologically, microbilogical sources (galactomannan), using lateral flow assay, pan-fungal detection of (1-3)-b-glucans (high rates of false positives)

Question 10

Serological diagnosis of IPA

Answer 10

Involves removing sera from patients. Antibody detection of cell wall biomarkers like galactans and mannans, using ELISA- enzyme linked immunosorbant assay

Question 11

galactomannan detection of IPA

Answer 11

galactomannan is a carbohydrate cell wall component. Rat MAb E2-A2 used in detection with lab based DAS-ELISA. E2-A2 binds to (1-5)-b-galactofuranoside (an immunodominant epitope in GM). Can cause false-positive results

Question 12

disadvantages of pan-fungal detection

Answer 12

only detects (1-3)-b-D-glucans in cell walls which is not present in mucormycetes and cryptococcus.
Has higher rates of false positives

Question 13

Reasons for galactomannan detection false postives in IPA

Answer 13

cross-reactivity of EB-A2 antibody with galactomannan(GM) from other fungi present in patients (not infectious). cross reactivity of mAb with galactoxylomannan from crytococcus neoformans. Detection of GM in cross contaminated b-lactam antibiotics derived from penicillium species. GM common in food products and can gross the gut into the bloodstream. Cross-reactivity of mAb with lipteichoic acis from bifidobacteria in the gastrointestinal microbiota of neonates. Cross-reactivity with anti-cancer drug cyclophosphamide

Question 14

Lateral flow assay

Answer 14

allows quick detection of IPA, by using mouse monoclonal antibody JF5 that recognises glycoprotein antigen only secreted by active spores or active growth of hyphae. higher specificity than rat mAb EB-A2. Can be used with serum or haemorrhagic BAL

Question 15

Brochoalveolar lavage (BAL)

Answer 15

involves taking a tube and finding a lesion in the lung. Another tube is used to inject fluid into the lung which brings out anything that was in the lesion