Proximity ligation assay for the sensitive, specific and early diagnosis of invasive fungal disease

Ref ID: 19363

Author:

G. L. Johnson, M. Shannon, C. R. Thornton, S. G. Agrawal
and S. A. Bustin

Author address:

Queen Mary University of London, London, United Kingdom;
Life Technologies Corporation, Foster City, USA; School of
Biosciences, University of Exeter, Exeter, United Kingdom and
Postgraduate Medical Institute, Anglia Ruskin University,
Chelmsford

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

Objective The Proximity Ligation Assay (PLA) uses real-time PCR
(qPCR) to detect the binding of oligonucleotide-labelled antibodies to
their antigens. Our aim was to demonstrate the utility of this method
as a diagnostic assay for the early detection of fungal infections.
This first assay targets invasive aspergillosis by detecting Aspergillus-
specific proteins secreted only during active fungal growth.
Methods Generation of PLA-probes:
1. Polyclonal antibodies (pAb) were raised against an Aspergillus-specific
glycoprotein antigen that is secreted constitutively at the hyphal apex.
2. pAbs were biotinylated, and tested to ensure efficient biotinyla-
tion in a “œforced proximity test”.
3. Biotinylated pAbs were split into 2 pools and streptavidin-linked
oligonucleotides A or B were added to one or other of the pAb pools.
This generates probe pools attached to different DNA oligonucleo-
tides, one with a free 3’- (A), the other a free 5’-end (B).
The PLA assay:
4. Both PLA probes were combined and incubated with dilutions
of purified glycoprotein antigen or Aspergillus lysate. The reaction
mixture also contained a third oligonucleotide that is complementary
to the end of oligonucleotide A and the beginning of oligonucleotide
B. If antigen is present, it connects the two oligonucleotides, allowing
a ligase to seal the free ends. Control reactions contained antibodies
but no antigen.
5. Primers complementary to oligonucleotides A and B were used
to amplify ligated amplification products, which were detected with a
hydrolysis probe.

Analysis:
6. Serial dilutions of purified glycoprotein antigen in phosphate-
buffered saline (PBS) were used to determine the quantitative poten-
tial of this method. The Mann-Whitney U-test was used to calculate
the statistical significance between the quantification cycles (Cq) of
samples with and without A. fumigatus lysate.
Results The amplification plot of the forced proximity test is shown
in Figure 1 and indicated a 2,100-fold difference between the bioti-
nylated antibody (blue replicates) and the negative control (pink rep-
licates), which is significantly above the 360-fold quality control
threshold. Figure 2 shows increasing Cqs with serial 10-fold dilution
of purified glycoprotein antigen in PBS (A to D), with an 8-fold differ-
ence between the highest dilution (D) and the antigen-negative sam-
ple (Negative C). Serial dilutions of Aspergillus lysate were tested and
showed a statistically significant difference between Aspergillus-posi-
tive and Aspergillus-negative samples (n = 14, median positive Cq:
26.15, median negative Cq: 28.19; P < 0.05). Unlike standard qPCR, the negative control in a PLA generates aCq due to ligation of PLA probes being brought into proximity by chance. Conclusion This report describes the first ever use of PLA to detect proteins secreted by actively growing fungi. Results demonstrate the successful biotinylation and oligonucleotide attachment of the pAb, the ability of the PLA to detect its target antigen and a statistically significant difference between the Cq of an Aspergillus lysate contain- ing target antigen and an antigen-negative sample. Assay optimisa- tion will increase the Cq of the negative control to a value of >30.
Normalisation against chromosomal DNA present in the sample
should permit translation of qPCR quantification cycles into a quanti-
tative assay that determines proliferating fungal load within a patient
sample.

Figure 1 Amplification plot of the pAb Forced Proximity Probe. The
blue lines are the biotinylated antibody. The pink lines are the nega-
tive control.

Abstract Number: o2.6

Conference Year: 2013

Link to conference website: NULL

New link: NULL

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