Capture antibody coupling to microspheres.
Carboxylated magnetic microspheres (Luminex, Austin, TX) were conjugated with feline IFN-γ, IL-10, and IL-12/IL-23 p40 capture antibodies from DuoSet ELISA Development kits (R&D Systems, Minneapolis, MN) according to the manufacturer's recommendations for two-step carbodiimide coupling of protein to MagPlex-C magnetic carboxylated microspheres (Luminex) (Table ). Briefly, lyophilized capture antibody was resuspended in 1 ml of sterile phosphate-buffered saline (PBS). Magnetic microspheres were washed with 100 μl of distilled water and resuspended in 80 μl of activation buffer (0.1 M sodium phosphate monobasic, anhydrous, pH 6.2). Carboxyl groups on the surface of the microspheres were chemically activated using 10 μl N-hydroxysulfosuccinimide (50 mg/ml) and 10 μl 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (50 mg/ml) (Fisher Scientific, Pittsburgh, PA). Activated microspheres were washed twice with 250 μl of coupling buffer [0.05 M 2(N-morpholino)ethanesulfonic acid, pH 5.0] and then resuspended in 100 μl of the buffer. Capture antibody was added, and the final volume was brought to 500 μl with coupling buffer. The mixture was incubated for 2 h at room temperature in the dark with gentle rotation. After incubation, coupled microspheres were washed three times (500 μl and two 1-ml washes) with blocking/storage buffer (PBS, 0.1% bovine serum albumin [BSA], 0.02% Tween 20, 0.05% azide, pH 7.4). The microspheres were then resuspended in blocking/storage buffer (150 μl per 106 microspheres) and stored at 4°C in the dark for 2 to 4 months. Microsphere concentrations were determined using an automated counter (Invitrogen, Carlsbad, CA).
Components used for the development of the domestic cat microsphere assaya
To confirm antibody coupling, microspheres (~5,000) were incubated with a phycoerythrin (PE)-labeled secondary antibody (R&D Systems) according to the manufacturer's recommendations (Bio-Plex amine coupling kit instruction manual: protein coupling validation [Bio-Rad, Hercules, CA]). A median fluorescence intensity (MFI) of >2,000 indicated successful coupling.
Microsphere assay protocol.
The immunoassay protocol used in these experiments was modified from a protocol for a washed capture sandwich immunoassay using magnetic microspheres (Luminex). Aliquots of coupled microspheres were combined in assay buffer (PBS, 1% BSA, pH 7.4) for a final concentration of either 50 or 100 microspheres/μl per analyte. A 50-μl aliquot of the multiplex microsphere solution (i.e., ~5,000 or 2,500 microspheres/analyte per well) was then added to each well in a 96-well, round-bottom polystyrene plate (Fisher Scientific). Wells were washed twice with 100 μl assay buffer using a 96-well magnetic separator (Ambion, Austin, TX) and again after each subsequent incubation. Plates were incubated for 30 min at ~800 rpm in the dark at room temperature. Microspheres were incubated in 50 μl of solution in the following order: (i) sample, standard, spike, or cell culture medium control (5 to 20% heat-inactivated fetal bovine serum, 2% glutamine, 0.5% 2-mercapto ethanol, 1% penicillin-streptomycin, quantity sufficient [q.s.] RPMI 1640); (ii) detection antibody; and (iii) streptavidin-PE (Invitrogen). After the final incubation and washes, the microspheres were resuspended in 100 μl of assay buffer and analyzed.
A Bio-Plex 200 instrument (Bio-Rad) was calibrated before each analysis and validated monthly to ensure optimal performance of optics, reporters, fluidics, and classification per the manufacturer's recommendations. Each plate included an eight-point standard curve (2-fold dilution series) and four control wells, in addition to samples with known or unknown analyte concentrations. All standards and samples were run in duplicate. Median fluorescence intensity was calculated from ≥100 microspheres per analyte per well. Bio-Plex Manager 5.0 (Bio-Rad) was used for data collection and analysis. A five-parameter logistic standard curve was generated for each analyte from the standards by plotting concentration versus fluorescence. The computer program calculated the concentration of each sample or spike using the respective analyte standard curve. Acceptable standard recovery was 70 to 130% of the nominal value (Bio-Plex cytokine assay instruction manual; Bio-Rad).
Microsphere assay development.
Assay development and optimization were conducted in the following order: (i) optimization of capture antibody concentrations, (ii) optimization of detection antibody concentrations, (iii) determination of standard curve ranges, (iv) evaluation of cross-reactivity among analytes, (v) comparison of MFI between single and multiplex standard curves, (vi) optimization of streptavidin-PE concentration, and (vii) optimization of the number of microspheres per well. Capture antibody concentrations tested were 1, 2.5, 5, 7.5, 10, and 15 μg per 106 microspheres. Detection antibody concentrations tested were 1×, 2×, and 3× the recommended concentration for ELISA protocols. Standard curve ranges evaluated were based upon the recommendations of the ELISA protocols (7-point curves), with three additional lower concentrations (i.e., a 10-point curve was tested). To test for cross-reactivity among analytes, each cytokine standard curve was individually tested with multiplex microspheres and detection antibody. Additionally, to determine if the MFI was affected by multiplexing analytes, single and multiplex standard curves were compared. Streptavidin-PE concentrations tested were 2, 3, and 4 μg/ml. The numbers of microspheres tested per well were approximately 1,000, 2,000, 2,500, and 5,000 microspheres per analyte to determine the minimum number required per well.
Multiplex assay validation.
Intra- and interassay experiments were conducted to determine both the accuracy and precision of the multiplex assay. In these experiments, the spike concentrations tested were based upon the dilution series of the standard curve. The stock solution of each standard provided by the ELISA kit manufacturer was diluted to three concentrations and spiked into cell culture medium to test the accuracy of detecting sample concentrations based upon the standard curves generated. Spike concentrations were as follows: (i) 31, 250, and 1,000 pg/ml for IFN-γ; (ii) 63, 500, and 2,000 pg/ml for IL-10; and (iii) 39, 156, and 625 pg/ml for IL-12/IL-23 p40. For the intra-assay experiment, 10 wells with each spike concentration were run in a single experiment. For the interassay experiments, spiked samples (duplicate or quadruplicate wells) were run in nine separate experiments.
Acceptable spike recovery was 70 to 130% of the nominal value (Assay qualification guidelines for Luminex assays; Panomics, Santa Clara, CA). The coefficient of variation (CV) for the mean spike recoveries at each concentration was required to be <20% for the intra-assay experiment and <30% for the interassay experiments (Bio-Rad, personal communication). The lower limit of quantitation (LLOQ) was the lowest concentration tested that was repeatedly recovered. The upper limit of quantitation (ULOQ) was the highest concentration tested that was repeatedly recovered.
Generation of supernatant from domestic cat PBMC.
We compared cytokine values determined by commercially available, unvalidated ELISAs (DuoSet ELISA Development kits; R&D Systems) to those determined by the multiplex assay using cell culture supernatant samples generated from domestic cat PBMC. The ELISA kits are marketed as a reagent resource for analysis of cytokines in cell culture supernatant. Cell culture supernatant samples generated in our laboratory were tested to verify that the multiplex assay was capable of detecting natural cytokines produced by domestic cat cells (i.e., other than the recombinant cytokine standards).
Specific-pathogen-free domestic cats were housed in Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC, Intl.) approved facilities and handled under protocols approved by the Colorado State University Institutional Animal Care and Use Committee (IACUC). Heparinized blood was collected from both naïve and FIV-positive cats sedated with ketamine-acepromazine. PBMC were isolated using a Histopaque gradient (Sigma-Aldrich, St. Louis, MO) and resuspended in medium. Cells were grown in the presence of 0, 0.5, or 5 μg/ml of concanavalin A (ConA) (Sigma-Aldrich). Additionally, PBMCs from naïve cats were infected in vitro with one of two FIV strains, as a component of other ongoing experiments. Supernatants were harvested from 1 to 10 days postculture and stored in aliquots of 0.5 to 1 ml at −80°C until tested with both assays.
ELISA and multiplex cytokine analysis of supernatant samples.
The manufacturer's protocol was followed for each ELISA, although the standard curve range was altered to reflect the range used in the microsphere assay. IFN-γ, IL-10, and IL-12/IL-23 p40 concentrations in all samples were tested simultaneously by both assays to minimize freeze-thaw and sample storage variability. Samples were included in the assay comparison analysis if the concentrations obtained from both assays were within the standard range tested (i.e., a sample was excluded if one or both assays resulted in a concentration above or below the standard range). Values were compared using a Spearman's rank correlation for nonparametric data and 95% prediction intervals (GraphPad Prism 5.0; GraphPad, La Jolla, CA).