Navigation Links
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

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.

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 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).

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).

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.

back to top


Page: All 1 2 3 4

Related biology technology :

1. The let-7 microRNA Family Regulates RAS: Implications for Development and Oncogenesis
2. Rapid Method Development With the BioLogic DuoFlow Chromatography System for the Purification of His-Tagged Proteins
3. A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis
4. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
5. Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3, 5 monophosphate and its validation for pharmacological screening at G protein-coupled receptors
6. Development of an NK2 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System
7. Development of an adrenergic α2a receptor binding assay using the LEADseeker Multimodality Imaging System
8. Development of a CRF1 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System
9. Development of Radioligand Binding Assays for the Motilin Receptor Using ScreenReady Targets.
10. Mx4000 Multiplex Quantitative PCR System*,PPP
11. QIAGEN Multiplex PCR Handbook
Post Your Comments:

(Date:10/8/2015)... ... 2015 , ... Cystic fibrosis (CF) is a genetic disease ... results in dysfunction of the lungs and the gastrointestinal (GI) tract. In CF ... very difficult to clear, blocks the airways and the glands, and creates an ...
(Date:10/7/2015)... ... , ... Neil Riordan, PhD to Discuss Medicinal Effects of Mesenchymal Stem Cells ... 3:50 pm to 4:30 pm. , Dr. Riordan is one of the early pioneers ... the Co-Founder and Chief Science Officer of the Riordan-McKenna Institute of Regenerative ...
(Date:10/7/2015)... Oct. 7, 2015  ContraVir Pharmaceuticals, Inc. (Nasdaq: ... focused on the development and commercialization of targeted antiviral ... offering of 5,000,000 shares of its common stock and ... Company,s common stock at a fixed combined price to ... and warrants will be issued separately.  The warrants will ...
(Date:10/7/2015)... Oct. 7, 2015   VWR (NASDAQ: ... laboratory products, services and solutions, announced today that ... specialty solvent company that performs high volume purification ... enhance VWR,s growing laboratory and production chemical manufacturing ... manufacturing customers. Manuel Brocke-Benz , President ...
Breaking Biology Technology:
... SANFORD, Maine, Jan. 31, 2012  Maine Manufacturing, LLC announced ... GE Healthcare granting Maine Manufacturing the exclusive right to ... line.  Maine Manufacturing, which has been manufacturing the FAST ... FAST Slides and accessories including FAST Quant, direct to ...
... Jan. 26, 2012 Life Technologies Corporation (NASDAQ: ... company,s presentations at the following upcoming investment conferences in ... Life Technologies will present at the Leerink Swann 2012 ... a.m. ET. Life Technologies will also present ...
... 2012  ZyGEM Corp. Ltd., a developer and marketer of ... nucleic acids, today announced that it has signed an ... DNA and RNA extraction kits.  SDL was established by ... as part of the Saudi Arabian government,s drive to ...
Cached Biology Technology:
(Date:10/8/2015)... 8, 2015 NXT-ID, Inc. ... a biometric authentication company focused on the growing ... smart wallet announces that revenues for the three ... compared with $113,00 for the three months ended ... ended September 30, 2015 were approximately $520,000. ...
(Date:10/6/2015)... LAKE CITY , Oct. 6, 2015 ... company, announced today that it has signed a contract ... monitoring services across the full range of sentences under ... Group,s President of the Americas. "This contract with the ... region of the US and advances our position as ...
(Date:10/2/2015)... DUBLIN , Oct. 02 2015 ... addition of the "Enforcing the Law Using ... ) has announced the addition of ... report to their offering. --> Research ... addition of the "Enforcing the Law Using ...
Breaking Biology News(10 mins):
... that ever walked the earthIndricotherium transouralicum, a hornless ... stood about eighteen feet high at the shoulderlived ... largest mammal that ever walked the earth Indricotherium ... approximately seventeen tons and stood about eighteen feet ...
... and other pathogens result in global crop losses of over ... Institution,s Department of Plant Biology developed a novel trick for ... organism. They discovered a novel family of pores that transport ... pores to access the plant sugar for food. The first ...
... , In the scorching summer heat of the Chihuahuan ... 97F (36C) while at the surface of the soil ... extreme temperatures, plants must utilize creative methods to not ... lethal conditions. This new work by Dr. Gretchen ...
Cached Biology News:
Mouse monoclonal [3G8] to MDMA ( Abpromise for all tested applications)....
Ly-49C and Ly-49I Biotin Anti-Mouse clone 5E6, Isotype Mouse IgG 2a , kappa, 0.1 mg Consult technical datasheet for details....
Fluorescein isothiocyanate (FITC) mouse anti-human IFN-alpha2 25 tests...
Dog polyclonal to Porcine Pancreatic Islets of Langerhans Cells ( Abpromise for all tested applications). Antigen: Tissue / cell preparation (Pig): Sonicated crude membrane fraction of porcine p...
Biology Products: