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


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-Plex 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.


back to top
'"/>

Source:


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:7/1/2015)... ... July 01, 2015 , ... It is now possible to ... single photon emission computed tomography (SPECT) according to researchers from Amen Clinics, ... over 20,000 brain scans and is believed to be the largest brain imaging study ...
(Date:7/1/2015)... BANGALORE, India , July 1, 2015  Strand ... next generation sequencing technology to empower cancer care, today ... as chief executive officer. In his expanded role as ... all strategy, business expansion and worldwide operations effective immediately.  ... Strand,s chairman and CEO since its founding in 2000, ...
(Date:7/1/2015)... ... ... A newly launched business plan contest will ask startup companies from around the ... R&D. The Pistoia Alliance President’s Startup Challenge 2015 will see small and medium enterprises ... mentorship from a senior industry figure drawn from the Pistoia Alliance membership. , The ...
(Date:7/1/2015)... , July 1, 2015  UCB, a multinational ... a global leader in designing, transforming, and running ... Award in recognition of their close collaboration in ... UCB,s global business services (GBS) finance organization. ... and clients that work together effectively to embrace ...
Breaking Biology Technology:Brain Scans Can Tell Post-Traumatic Stress Disorder and Traumatic Brain Injury Apart According to New Research by Amen Clinics 2Brain Scans Can Tell Post-Traumatic Stress Disorder and Traumatic Brain Injury Apart According to New Research by Amen Clinics 3Brain Scans Can Tell Post-Traumatic Stress Disorder and Traumatic Brain Injury Apart According to New Research by Amen Clinics 4Strand Life Sciences Names Scott A. Storrer CEO as Company Continues Global Expansion with Focus on Precision Medicine Product 2Strand Life Sciences Names Scott A. Storrer CEO as Company Continues Global Expansion with Focus on Precision Medicine Product 3Pistoia Alliance Launches Startup Challenge 2Pistoia Alliance Launches Startup Challenge 3UCB and Genpact Receive Aecus Innovation Award for Success in Global Business Services 2UCB and Genpact Receive Aecus Innovation Award for Success in Global Business Services 3UCB and Genpact Receive Aecus Innovation Award for Success in Global Business Services 4UCB and Genpact Receive Aecus Innovation Award for Success in Global Business Services 5
... ... Analysis in Influence Mapping, Lnx Pharma Research Finds Pharmaceutical Companies May Be Ignoring Up ... ... research results shatter current best practice for companies in pharmaceutical and biotechnology research who ...
... Intarcia Therapeutics, Inc. today presented final results from ... continuous delivery of exenatide) for the treatment of type ... Association for the Study of Diabetes in Vienna, Austria. ... plasma glucose (FPG) within 24 hours of the start ...
... with One-of-a-kind Video from Big Bend National Park , ... to: "What Do America,s National Parks Mean To You?" , ... announced today that DoctorLoopy , Doug ... of the " Your Parks" Video Challenge after online ...
Cached Biology Technology:In Key Opinion Leader Management, Quantity Does Not Equal Quality in Evaluating a Scientist's Real Importance 2In Key Opinion Leader Management, Quantity Does Not Equal Quality in Evaluating a Scientist's Real Importance 3Intarcia Therapeutics, Inc. Presents Positive Results of ITCA 650 Phase 1b Study in Type 2 Diabetes at the European Association for the Study of Diabetes Conference 2Intarcia Therapeutics, Inc. Presents Positive Results of ITCA 650 Phase 1b Study in Type 2 Diabetes at the European Association for the Study of Diabetes Conference 3Intarcia Therapeutics, Inc. Presents Positive Results of ITCA 650 Phase 1b Study in Type 2 Diabetes at the European Association for the Study of Diabetes Conference 4Intarcia Therapeutics, Inc. Presents Positive Results of ITCA 650 Phase 1b Study in Type 2 Diabetes at the European Association for the Study of Diabetes Conference 5The National Park Foundation and Olympus Announce Winner of the First-Ever 'Your Parks' Video Challenge 2The National Park Foundation and Olympus Announce Winner of the First-Ever 'Your Parks' Video Challenge 3The National Park Foundation and Olympus Announce Winner of the First-Ever 'Your Parks' Video Challenge 4
(Date:6/23/2015)... , June 23, 2015   Valencell, ... the results of a recent study that illustrates ... the wrist during activity. In a study conducted ... compared along with the Apple Watch against a ... heart rate during activity. The study demonstrated that ...
(Date:6/17/2015)... , Deutschland und GERMANTOWN, Maryland , ... ®   Produktlinie mit DNA-Tests verbessert ... N.V. (NASDAQ: QGEN ; Frankfurt Prime Standard: QIA) ... neue STR-Testkits der Produktlinie Investigator ® ... den Markt eingeführt. Die neuen Kits zur Erstellung genetischer ...
(Date:6/16/2015)... /CNW Telbec/ - handyem Inc. and FlowMetric Diagnostics ... of handyem,s HPC-150 portable flow cytometer within FlowMetric ... unprecedented model of mobile laboratory platform is presented ... Pennsylvania Convention Center, ... to 18 th 2015.  By virtue of ...
Breaking Biology News(10 mins):Valencell Delivers Breakthrough Heart Rate Accuracy on the Wrist 2Valencell Delivers Breakthrough Heart Rate Accuracy on the Wrist 3QIAGEN bringt in den USA neue Kits für die forensische DNA-Analyse auf den Markt 2QIAGEN bringt in den USA neue Kits für die forensische DNA-Analyse auf den Markt 3QIAGEN bringt in den USA neue Kits für die forensische DNA-Analyse auf den Markt 4QIAGEN bringt in den USA neue Kits für die forensische DNA-Analyse auf den Markt 5QIAGEN bringt in den USA neue Kits für die forensische DNA-Analyse auf den Markt 6handyem and FlowMetric Diagnostics announce their partnership in the development of Flow Cytometry- Based Solutions in diagnostic capabilities 2handyem and FlowMetric Diagnostics announce their partnership in the development of Flow Cytometry- Based Solutions in diagnostic capabilities 3
... WASHINGTONSeptember 8, 2010Research!America today called on Congress to ... human embryonic stem cell research to proceed, in light ... lift his recent injunction on federally funded research using ... Stem Cell Research Advancement Act (H.R. 4808) introduced in ...
... AUSTIN, Texas, Sept. 8 /PRNewswire/ -- ... agreement with BMC Software that allows ... Compliance Manager, which has been tightly integrated with ... BMC,s enterprise customers access to the industry,s leading ...
... just published in the scientific journal , Osteoporosis International ... used to describe the current and future burden of ... model, published by researchers from the UK on behalf ... was developed and validated using Swedish data. It can ...
Cached Biology News:Research!America asks Congress to support embryonic stem cell research now 2SailPoint Signs International Reseller Agreement with BMC Software 2SailPoint Signs International Reseller Agreement with BMC Software 3
Mouse Anti-Human Killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 4 (KIR2DL4 Monoclonal Antibody Family: New...
BD BioCoat Laminin 150 mm Culture Dishes...
...
Chicken Anti-Human DNA-directed RNA Polymerase II 7.6 Kd Polypeptide (POLR2L Polyclonal AntibodyOther Sub-Family: not assigned-Other Applications: ELISA, Western Blot...
Biology Products: