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Brief Communications |
Correspondence: 1Corresponding Author: Mary T McBride, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA, 94551
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Key Words: Foot-and-mouth disease • immunoassay • multiplexed • serotyping
LuminexAuthor: This article has been lightly edited for grammar, style, and usage. Please compare it with your original document and make changes on these pages. Please limit your corrections to substantive changes that affect meaning. If no change is required in response to a question, please write "OK as set" in the margin. Copy editorEditor. Affiliations section, last sentence. The contributions footnote has been moved to the end of Affiliations. Should it be deleted entirely instead?Author. Abstract, sentence 2. Unable to verify spelling of "hyperimmunesera". Please check spelling. liquid array technology enables the simultaneous evaluation of a sample with multiple detection reagents (multiplexing) in a single assay. There are many examples of the creative use of this technology12 for the detection of biothreat agents13,14 and other infectious disease.10,11,15 Multiplexed analysis of a sample with numerous detection reagents gives increased confidence in assay results and eliminates well-to-well variation sometimes observed when comparing multiple single-well, singleplex assays. Multiplexing cuts down on reagent and consumable consumption, thereby reducing cost. In addition, significant reductions in labor costs and an expanded capability to deal with surge capacity are also possible when running multiplexed assays in single wells. Here, initial results demonstrating the feasibility of a liquid array, multiplexed immunoassay for foot-and-mouth disease (FMD) viral antigen detection, and serotype differentiation are presented.
Foot-and-mouth disease virus (FMDV) is a member of the picornaviridae family. It consists of a small, unenveloped capsidAuthor. Text, paragraph2, sentence 2. The spelling of "caspid" has been changed to "capsid." Please verify accuracy. and a single strand of positive sense RNA. There are 7 serotypes of the virus C, O, A, Asia, SAT1, SAT2, and SAT3 and many subtypes. FMD diagnosis and serotyping is most commonly carried out using an epithelial suspension but is also conducted with blood, saliva, and vesicular fluid using an enzyme-linked immunosorbent assay (ELISA),4,7,16 which enables a positive sample to be confirmed in a few hours. However, if the virus concentration is very low or inconclusive ELISA results are obtained, then cell culture is used to amplify the virus for analysis. If the multiplicity of infection is low, then it can take 2 to 4 days for a cytopathic effect to become apparent before subsequent cell culture supernatant samples are analyzed for the final diagnosis. All assays currently used for FMDV identification and serotyping require multiple assays, where each assay tests for a single serotype and the results are analyzed and interpreted as a composite to identify the serotype.Author. Text, paragraph 2, last sentence. Please ensure that edits have not changed meaning.
The multiplexed assay described here was based on a combination of guinea-pig (GP) and rabbit (Ra) FMD serotype-specific polyclonal antibodies, isolated from antisera using affinity columns. The multiplexed assay was assessed for its performance in the serotype differentiation of inactivated6 viral cell culture supernatant samples of 19 different FMD viral subtypes.
All reagent dilutions and assays were carried out using filtered (0.22 µm) PBS-TBN (phosphate-buffered saline, pH 7.4; Tween 20 0.02% vol/vol; bovine serum albumin [BSA] 0.1% wt/vol; sodium azide 0.02% wt/vol). Ra9 and GP5 antisera were produced as described previously. Antisera were prepared against specific subtypes: C1 Noville, O1 BFS 1860, A24 Cruzeiro, Asia 1 Shamir, SAT1 Ken 4/98, SAT2 Zim 5/81, and SAT3 Zim 4/81. Antisera samples (5 ml) were filtered using Steriflip-GP filters (0.22 µm).a Subsequently, antibodies were isolated from the antisera samples using Montage Antibody Purification Pro-sep A spin columnsa per kit instructions. Resulting antibody solutions were desalted and transferred into 
1.5-ml PBS/0.02% wt/vol NaN3 using Amicon Ultra 15 centrifugal filter units (MWCO 30 kDa)a Author. Text, paragraph 4, sentence beginning "Subsequently, antibodies." Please spell out "MWCO" and follow with acronym in brackets.per kit instructions and stored at 4°C. Recovered protein yields ranged from 6 µg/ml (GP SAT3) to 25 µg/ml (Ra SAT1) determined by UV absorbance at 280 nm (Nanodrop spectrophotometer).b One milliliter (1.25 x 107) of carboxylate beadsc was used as received and coated with the polyclonal antibodies as described previously13,15 using 0.5 ml antibody solution (125 µg/ml in 0.1 M 2[N-morpholino] ethane sulfonic acid buffer, pH 4.5). To determine successful antibody coupling, 2-µl beads were diluted into 98 µl PBS-TBN. Two microliters of anti-rabbit IgG (whole molecule)-R-phycoerythrin antibody produced in goatd and R-phycoerythrin AffiniPure F(ab')2 fragment donkey anti-guinea pig IgG (H+L)e were added to the Ra antibody–coated beads and GP antibody–coated beads, respectively, and incubated in the dark for 20 minutes. The samples were directly analyzed in a Bio-Plexf to 10,000 events per bead class with a 50-µl sample size to determine the median fluorescent intensity (MFI). Successful coating procedures yielded MFIs of 9,000 to 19,000 for Ra antibody–coated beads and 17,000 to 21,000 for GP antibody–coated beads. A set of 4 controls (instrument control [IC], fluorescent control [FC], antibody control [AC], and negative control [NC]) built into the bead mixture monitors and reports every step of the assay.14 The details and concentrations at which the control beads are coated have been described previously.13,15 The 10x bead mixtures were formulated in PBS-TBN to a final concentration of 7 x 105 of each bead class/ml. Following formulation, 10 µl of the 10x bead mixture diluted in 90 µl PBS-TBN was analyzed in duplicate in a Bio-Plexf to 10,000 events per bead class with a 50-µl sample size to determine if the bead counts in each class were approximately equal. If the bead count of a particular class was significantly (>30%) lower than the others, a compensatory amount of that bead was added to the bead mixture. The bead mixtures were stored at 4°C in the dark and diluted 10-fold directly before use. For the biotinylated detector antibody cocktail (b-Abc), typically, 2 ml of each antibody solution was prepared at 1 mg/ml in PBS/0.02% wt/vol NaN3. Two milligrams of EZ-Link sulfo-NHS-LC-bioting was freshly prepared in 364 µl distilled (DI) Author. Text, paragraph 4, sentence beginning "Two milligrams of EZ-Link." "DI" has been defined as "distilled." Please verify accuracy.water and 40.5 µl was added to each antibody solution and gently agitated for 30 minutes. Volumes and amounts were adjusted if a different volume/concentration of antibody solution was biotinylated. Excess, unreacted biotin was removed using 5-ml Zeba Desalt spin columnsg per kit instructions. The b-Abc was prepared at a working concentration as a mixture of the pertinent biotinylated Ra and/or GP antibodies, each at 3 µg/ml Author. Text, paragraph 3, sentence beginning "The b-Abc was prepared." Please spell out "SP" and follow with acronym in parentheses.and biotin-SP conjugated Affinipure rabbit anti-chicken IgY (IgG), Fc fragment specifice as a control at 0.02 µg/ml in PBS-TBN. Reporter reagent, streptavidin-R-phycoerythrin (SA-PE),h was prepared in PBS-TBN at 24 µg/ml and diluted 10-fold with PBS-TBN directly before use for a working concentration of 2.4 µg/ml. Viral cell culture supernatant samples were prepared8 and BEI-inactivated2 as described previously. Supernatant samples were stored at –80°C and were thawed and diluted in PBS-TBN directly before use where appropriate.
Assays were carried out as described previously.15 In brief, 100 µl of sample was incubated with 50 µ- bead mixture for 20 minutes. Samples were washed twice and resuspended in 100 µl PBS-TBN and incubated with 50 µl b-Abc for 15 minutes. Samples were washed and resuspended in 100 µl PBS-TBN and incubated with 50 µl SA-PE for 5 minutes. The samples were washed and resuspended in 100 µl PBS-TBN. Finally, the suspended beads were transferred to a round-bottomed 96-well platei for analysis with a Bio-Plexf configured to count a minimum of 100 beads per class and a 50-µl sample size.
Figure 1 depicts the basis of liquid array technology for multiplexed immunoassay-based serotype differentiation. Serotype-specific capture antibodies are each covalently attached to a unique bead class. These antibodies capture the specific antigen, and detection is via a cocktail of biotinylated serotype-specific antibodies and the subsequent addition of a reporter molecule, SA-PE. The complex is analyzed in a flow cytometer, and the MFI of the reporter is used to quantify the amount of antigen captured on each serotype-specific antibody-coated bead. The ELISA routinely used by the WRL Author. Text, paragraph 6, sentence 5. Please spell out "WRL" and follow with acronym in parentheses.for FMD for antigen detection and serotype differentiation is based on serotype-specific Ra antisera as a capture agent and GP serotype-specific antisera as a detector reagent.3,4 For this initial development of a multiplexed serotyping immunoassay, 7 serotype-specific polyclonal Ra and 7 serotype-specific polyclonal GP antibodies were isolated from antisera and were evaluated against cell culture supernatant samples from the 7 major serotypes and various subtypes of FMDV. Each antibody was employed as both a capture and as a detector antibody, and all possible combinations were evaluated. With consideration given to homotypic and heterotypic reactions with each antibody as a capture and detector reagent, a set of capture-detector antibodies exhibiting the highest homotypic and lowest heterotypic reactions was then selected for further analysis. The intensity of the response and the serotype differentiation for SAT1 was unfortunately extremely low with the antibody pairs available, and therefore SAT1 was eliminated from this further evaluation. Therefore, the bead mixture comprised capture antibodies GP C; Ra O, GP A, GP Asia, GP SAT2, and GP SAT3 plus controls IC, FC, AC, and NC. The b-Abc combination was GP C, Ra O, GP A, GP Asia, Ra SAT2, and GP SAT3 plus control. This combination was used to analyze undiluted viral cell culture supernatant samples obtained from the same viral subtype as the capture and detector antibodies, and the results are shown in Figs. 2a–2f. From comparison of the absolute MFI values on beads C, O, A, and Asia samples (Figs. 2a–2d, respectively), homotypic reactions were significantly stronger (more than double) than heterotypic reactions. Results for SAT2 and SAT3 samples (Figs. 2e and 2f, respectively) are not as distinct, with lower intensity responses and greater heterotypic reactions. However, while the SAT2 and SAT3 assays were not as clearly differential as Author. Figure 1 legend. The numbers 1 to 5 have been changed to bold-faced lowercased letters per journal style. Please ensure that edits have not affected meaning.the others, the assay still provided a conclusive result. The responses from the control beads were monitored throughout all experiments (data not shown) and remained consistent across all experiments, demonstrating consistency in assay procedure; a consistently low negative control indicated a lack of nonspecific binding in the complex sample matrix.
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a. Millipore Corp., Billerica, MA.
b. Nanodrop Technologies, Inc., Wilmington, DE.
e. Jackson ImmunoResearch Laboratories, West Grove, PA.
f. Bio-Rad Laboratories, Hercules, CA.
g. Pierce Biotechnology Inc., Rockford, IL.
h. Caltag Laboratories, Invitrogen, Carlsbad, CA.
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