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Development of a Multiplex Bead-Based Assay for Antibody Screening of a Nonhuman Primate Colony on the Bio-Plex System

LaRene Kuller, David Anderson, Dominic Andrada, and Richard Grant, National Primate Research Center, University of Washington, Seattle, WA 98195 USA


Introduction
We have been using four separate enzyme-linked immunosorbent assays (ELISAs) to screen our primate colony for the viruses of greatest concern: simian retrovirus (SRV), simian T-cell leukemia virus (STLV), herpes B, and simian immunodeficiency virus (SIV). To minimize the labor involved, testing of antibody responses to these viruses requires that these individual assays be performed on a limited sample volume. As we add more viruses to our screening protocol, serological testing by ELISA will become more time-consuming.

The Bio-Plex suspension array system, which is based on Luminex xMAP technology, is a multiplex flow cytometricbased system that utilizes up to 100 color-coded 5.6 m polystyrene bead sets. Each bead set is internally dyed with different ratios of two spectrally distinct fluorophores. Each of the bead sets can be conjugated with a unique protein, peptide, antibody, or, in our case, disrupted whole virus. The conjugated beads are pooled together in the wells of a microplate with the sample to be tested (plasma), followed by the addition of a detection antibody, forming a capture immunoassay that is then read by the Bio-Plex suspension array reader. Each separate reaction is identified and quantitated based on the bead color. This allows a single sample to be tested simultaneously for reaction to a number of different viruses.

Our goal was to determine whether the Bio-Plex system yields results comparable to those of our current ELISA assays. The capability to multiplex would allow us to screen animals for multiple viruses simultaneously, greatly reducing the time and labor required to perform our assays while also conserving limited samples.


Methods
Viral Disruption for Conjugation
To develop a method for viral conjugation, we began with our whole-virus ELISA protocol, in which the virus is disrupted using SDS. For the first bead conjugation, SRV was disrupted with SDS. We also tried undisrupted whole virus and whole virus disrupted with Triton X-100 detergent. To compare the results obtained with the various disruption methods, the beads were run on the Bio-Plex system.

Optimizing Conjugation by Virus/Plasma Titration
After determining that we could successfully conjugate whole disrupted virus to the beads, the next step was to optimize the amount of virus for bead conjugation, as well as to determine the optimum plasma concentration. The protocol provided with the Bio-Plex amine coupling kit suggested using 512 g of protein for conjugation. We conjugated beads with 2, 5, 8, 10, and 12 g of virus (disrupted with Triton X-100), and then reacted the conjugated beads with both positive and negative plasma at dilutions of 1:50, 1:100, and 1:200.

ELISA Assay
ELISA assays followed conventional ELISA protocols.

Bio-Plex Assay
Multiplexing was performed according to the following protocol:
1. Prepare plasma samples.
2. Load beads into wells.
3. Wash beads.
4. Incubate plasma with antigen-conjugated beads.
5. Wash beads.
6. Incubate beads with phycoerythrin detection antibody (Figure 1).
7. Wash beads.
8. Read on Bio-P lex suspension array reader.


Results and Discussion
Viral Disruption
We were unable to find any reports of whole virus being conjugated to beads for use in this type of multiplex assay. While our approach using SDS-disrupted whole virus did work, it resulted in multiple fragmented beads. Comparing all three approaches, undisrupted, SDS-disrupted, and Triton X-100-disrupted virus, the Triton X-100 treatment gave lower background (as determined by the negative plasma) and a higher signal to the positive plasma than either the SDSdisrupted or undisrupted virus, and with no bead fragmentation (Figure 2).

Virus/Plasma Titration
The results of the virus/plasma titration studies are shown in Figure 3.

Correlation With ELISA
The results we obtained showed a strong correlation between the Bio-Plex and ELISA assays of positive and negative control plasma (Table 1).

Cross-Reactivity
When control plasma was incubated with the bead mixture and then analyzed for each separate bead type, there was no cross-reactivity between the different bead types (Table 2).

Single-Bead vs. Multiplex Assays
Because we were concerned that sensitivity might be lost when multiplex assays were compared to a single-bead assay, both single- and multiple-bead assays were performed; however, as shown in Table 3, we found no loss of sensitivity.

Sensitivity and Specificity
We determined the sensitivity and specificity of the assays by evaluating the number of true and false readings (Table 4).


Conclusions
There was an excellent correlation between the multiplex suspension assay and the ELISA (r2 = 0.960.99)
The CV was lower in the multiplex assay than in the ELISA
There was no loss of sensitivity when multiplexing
Fewer false positives were detected with the multiplex assay than with the ELISA
The Bio-Plex assay had equivalent specificity to that of the ELISA, and slightly higher sensitivity

Triton is a trademark of Union Carbide. xMAP is a trademark of Luminex Corporation. The Bio-Plex suspension array system includes fluorescently labeled microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation.

Information in this tech note was current as of the date of writing (2004) and not necessarily the date this version (Rev B, 2005) was published.


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