Bench to bedside Flashcards
Aspergillus fumigatus
Inactive condida are inhaled into the respiratory tract, where they swell . usually blocked by macrophages but they then go to germinate into hyphae. usually blocked by neutrophils but they then go and invade tissues and ultimately the blood stream. PHAGOCYTOSIS IS VERY IMPORTANT AND COMPLEMENT AS WELL.
Aspergilloma and invasive pulmonary aspergillosis
casued by aspergillus fumigata in immunocompromised patients
AFMP1 and galactomannoprotein
present on the cell surface in abundance of a. fumigata. ELISA was highly specific however only 50% of aspergillosis patients gave positive results due to them not having an immune system and are unable to form antibodies.
galactomannan ELISA
rat mAb targets GM. sandwich with a conjugated rat mAb. however gave false positives.
False positivies due to:
- mAb cross-reacting with GM from other fungi
- GM from beta-lactam antibiotics produced from penicillium species
- GM in food products
Pan-fungal detection
Detect all fungi. (1,3)-beta-D-glucan. abundant polymer and maker. “Fungitell” test. However cryptococcus doesn’t have it and it also gave high rate of false positives.
Alternative IPA (invasive pulmonary aspergillosis) detection - what do we need?
appropriate targets are extracelular - found in blood stream. found in abundance. constitutively-activated antigen. can discriminate between active growth and quiescence.
JF5
mous mAb. IgG3. recognises extracellular, constitutive glycoprotein. produced only in active growth. high specificity. use a lateral flow devivce for detection.
Human serum - LFA preparation
Mix isolated serum with buffer, heat to 100C for 3 mins. Spin for 5mins to get a clear fluid that will be used on the lateral flow assay.
bronchoalveolar lavage.
use a bronchoscope and then inject fluid into the lungs. bring back anything that is present within the lesion. negative predicted value is 97-100%.
ISCA diagnostics
Commercialisation of JF5.
Patent the Vh and Vl variable domains of the mAb.
Aspergillus LFD - CE marked to OLM. Aspergillus ELISA - CE marked to Euroimmun
why not use radiology?
non specific. pulmonary lymphoma, primary pulmonary tuberculosis, pulmonary cryptococcosis, IPA –> all look the same. radiologists say that fungal infections are “air crescents”
Immuno PET/MRI (non-invasive molecular imaging)
knockout neutrophils using anti-GR1 antibody. intratracheal injection of A. fumigatus conidia. Inject with copper 64 labeled antibody tracers which will bind to any antigen present. Take a PET/MRI at 3h, 24h and 48h. Histology at 48h. Show specific uptake of tracers in lungs.
How to get into humans from mouse models ? Immuno PET/MRI
Humanise antibody. USing the mouse vaiable regions (JF5 complementarity determining regions), graft to human k constant light chain, human IgG constant regions and hinge.
how do you prove the antigen isn’t present in the human anyway?
mmutant of the pathogen deficient in the epitope (beta 1,5-galactofuranose) which is an enzyme that adds galactose to the terminals of carbohydrates. Immunoimaging shows that there is loss of reactivity with KO so proven that the Ab binds to that particular enzyme.
What mAb did they take into clinical trials?
64Cu-labelled humanised JF5.
