H - DIRECCIÓN GENERAL DE CATASTRO E INFORMACIÓN TERRITORIAL

Figure 46: Format for the NP-based immunosorbant assay

Assays with confocal read out

Dengue immunoassays were performed in 96 well glass bottom plates. These plates had low fluorescence background and gave favourable results for the adsorption of antibodies. For confocal imaging using conventional fluorescent dyes, polyclonal rabbit anti-dengue type 1 - 4 antibody dissolved in carbonate/bicarbonate buffer (0.05 M, pH 9.2) at a concentration of 20 μg mL-1 was incubated overnight on the surface of the plate at 2 - 8o_{C. The experiment} was structured to allow each sample to be analysed in triplicate (sample of raw data shown in Appendix 3, page 196). The plate was washed three times with PBS and tapped dry. Dengue antigen type 3 or 4 was then added for 2 hours at a concentration of 10 μg mL-1_{. The plate was washed three times, tapped dry and} sera containing mouse anti-dengue IgG and IgM antibodies were added at a

Serum containing mouse anti- dengue IgM and IgG Surface coating of rabbit anti- dengue types 1-4 Type specific antigen Biotinylated goat anti-mouse IgG or IgM Avidin-linked SiO2 NPs doped with Eu, Tb or Sm probe

and tapped dry, after which goat anti-mouse IgG labelled with FITC and goat anti-mouse IgM labelled with TRITC were added at an antibody concentration of 5 μg mL-1 _{and left for 1 hour. A final wash step was carried out and the sample} was measured confocally. Confocal imaging was carried out with a confocal scanner head (TCS NT; Leica Microsystems, Germany) installed on an inverted microscope (DMIRIB, Leica Germany). The 488 nm line of an Ar ion laser was used for excitation of FITC and the intensity of fluorescence emitted was detected using a 530/30 nm bandpass filter. The 568 nm line of a Kr ion laser was used for excitation of TRITC and the intensity of fluorescence emitted was detected using a 570 nm long pass filter. The objective used was 40x NA 0.75 air objective. The thin film resulting from the immunoassays was measured in a 512 x 512 pixel area; which translates to ~ 159 μm2_{. The scan speed was set at 400 Hz, corresponding} to a dwell time of 4.9 µs. The axial response was measured in 0.4 μm intervals along the z-axis for a total of 50 images, using a 74 μm pinhole. The intensity as the sum of each x-y image was plotted against the z position, allowing the maximum intensity to be identified and used. Samples of the confocal images and resulting intensities are shown in Appendix 3 (page 196).

As a result of reagents constraints, the results from single well assays for dengue antibodies were compared to multi-welled assays performed using ELISA methods. The correlation with the ELISA method was used to determine the success of the multiplexed methods.

Assays using nanoparticles and QDs

For NP-based assays, (Figure 46), 100 μg mL-1 _{polyclonal rabbit anti-dengue type} 1 - 4 antibody dissolved in carbonate/bicarbonate buffer (pH 9.5) was coated on the surface of 384-well glass bottom plates overnight at 2 – 8o_{C. The plate was}

then added for 2 hours at a concentration of 10 μg mL-1_{. The plate was washed} three times, tapped dry and sera containing mouse anti-dengue IgG and IgM antibodies were added at a dilution of 1:500 in PBS for 2 hours. The plate was washed three times with PBS and tapped dry, after which each well was filled with a mixture of goat anti-mouse IgG biotin attached to an avidin conjugated Eu- doped NP and goat anti-mouse IgM biotin attached to an avidin conjugated Tb- doped NP. This mixture was left shaking overnight, after which the plate was washed with PBS and tapped dry in preparation for epi-fluorescence microscope measurement by a UV filter cube consisting of a 330 – 385 nm band pass excitation filter, 400 nm dichroic mirror and 420 nm long pass emission filter (U- MWU2; Olympus UK Ltd, Southall, UK). The biotinylated antibodies were mixed with the avidin conjugated NPs in PBS (pH 7.4) for more than 8 hours before their use. A 50-fold dilution of the antibody was dissolved in the required amount of avidin conjugated NP solution and left to shake. The mixture was then centrifuged at 8000 rpm for 10 minutes, after which it was washed twice with PBS. The pellet was redissolved in PBS and mixed with the second secondary antibody for use in multiplexed immunoassays.

Quantum dot-based assays were carried out in similar fashion to the NP-based assays. Streptavidin-linked quantum dots 655 nm and 525 nm were incubated with anti-mouse IgM-biotin and anti-mouse IgG-biotin respectively. Since these probes cannot be centrifuged, an excess of the QD was added to the antibodies to ensure that unlabelled anti-mouse IgG/M did not bind to mouse anti-dengue IgG/M. Excess streptavidin-QD label was established by observing the fluorescence of the tryptophan groups on streptavidin. When biotin is bound to streptavidin, the

nM solution of QD-streptavidin was prepared and the tryptophan fluorescence measured at 333 nm with 300 nm excitation with a fluorescence spectrophotometer (Carey Eclipse Fluorescence Spectrophotometer, Varian). Anti-mouse IgG-biotin was added in 2 μL increments until the tryptophan fluorescence peak decreased by 40 %. The volume and concentration of biotinylated antibody added was used to determine optimal concentrations of antibody and streptavidin-linked QD. These optimal concentrations were 80 nM 655 nm QD incubated with a 1 in 10 000 dilution of anti-mouse IgM-biotin and 100 nM 525 nm QD incubated with a 1 in 100 000 dilution of anti-mouse IgG-biotin. For the assays utilising the biotin- avidin/streptavidin interaction, there is the possibility of exchange between the two labels as a result of their dissociation equilibrium. This equilibrium has been investigated previously 10 _{using avidin, biotin and radioactively labelled biotin. The} authors began with the statement that thermodynamically speaking, there must be a dissociation equilibrium between avidin and biotin, in spite of the avidity of the association. They determined that the formation and dissociation of the complex occurs simultaneously. The ramifications of this formation and dissociation with respect to the success of the multiplexed assays with NPs will be discussed.

Assays using the programmable array microscope

The solid phase for these assays was 15 well μ-slide Angiogenesis uncoated chamber for cell microscopy. The same assay protocol used for nanoparticles was carried out, but the secondary antibodies were goat anti-mouse IgG-QD 565 nm in a 1:10 dilution, and rat anti-mouse IgM-QD 650 nm (1:20 dilution). Each sample was analysed only once due to the size of the assay plate and time constraints.

Imaging spectroscopic measurements were carried out using a PAM installed on an inverted fluorescence microscope (IX 71 Olympus Germany Ltd., Hamburg, Germany). The 488 line of an argon ion laser (~ 200 mW, Coherent Innova 90, Spectra Physics 2000) was used for excitation and emitted light collected by D525/80 nm band pass filter and a 655/40 nm band pass filter (Chroma Technology GmbH, Fuerstenfeldbruck, Germany). The objective used was a water 40x NA 1.15. The camera (Ixon DV 897_BV) was cooled to -100 o_{C (Julabo} Hc F30, Ultratemp 200). EM gain was set at 200 and the exposure time was 250 ms. Data for the two different dyes were not collected simultaneously; the instrument was set to perform a z-slice through the plate in two different channels. This allowed the signal of the 565 nm QD to be read first, followed by the signal from the 650 nm QD. Data were collected as TIFF files and converted to numerical data using Image J.

Conventional ELISAs

ELISAs were performed on the mouse serum where mouse anti-dengue IgG and IgM were detected in separate wells of the assay plate. Standard ELISA protocol was followed. The enzyme used was horse radish peroxidise and the plate was read at 415 nm on a TECAN SpectraFluor plate reader (MTX Lab Systems Inc. Virginia, USA).