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Multiplex Innovations & Introduction to SPF Animal 30th June 2015 Conference Room Level 5, Block B, KL Campus (South Wing), UCSI University Organised By :
Multiplex Innovations
& Introduction to
SPF Animal 30th June 2015
Conference Room Level 5, Block B, KL Campus
(South Wing), UCSI University
Organised By :
Instant ELISA
Sharing
Nur Afifah Samsudin
What is an ELISA?
•The enzyme linked immunosorbent assay (ELISA) is a powerful method for
detecting and quantifying a specific protein in a complex mixture.
Analysis of protein samples immobilized in microplate wells using specific
antibodies
The basic elements of ELISA
•Coating/Capture: direct or indirect immobilization of antigens to the surface of
polystyrene microplate wells.
•Plate Blocking: addition of irrelevant protein or other molecule to cover all
unsaturated surface-binding sites of the microplate wells.
•Probing/Detection: incubation with antigen-specific antibodies that affinity-
bind to the antigens.
•Signal Measurement: detection of the signal generated via the direct or
secondary tag on the specific antibody.
Antigen Detection
Sandwish ELISA Workflow
Immunoassay platforms
Coated-It-Yourself ELISA
Ready-SET-Go!® ELISA Kit
- Keep costs low. Each affordable, coat-it-yourself ELISA set includes the
reagents required to prepare and run the ELISA
Advantages of Ready-SET-Go! ELISA
● Flexible – available in variety of package sizes, from 2, 10 or 20 plates to
meet the demands of your research
● Complete and easy-to-use–sets include optimized antibody pairs, and
unlike other ELISA Sets, eBioscience Ready-SET-Go!® Sets also include
recombinant protein standards, TMB substrate, and other essential
reagents to perform your assay.
● Affordable – priced to accommodate the most demanding budgets,
maximizing your research dollars.
Pre-coated ELISA
Platinum® ELISA Kit
Ideal for labs who need a comprehensively tested
kit that requires no additional validation.
>Highly validated pre-coated ELISA kits, with
simple protocols.
> Most comprehensive range of ELISA kits for
Th17 research.
> Available for broad range of analyte targets for
human, mouse, rat, monkey and pig.
Advantages of Platinum ELISA
➢ Easy-to-use: Includes all required (color-coded) assays buffer and
reagents and a straightforward protocol
➢ Highly Validated: Optimized for serum, plasma and cell culture
supernatants
High Sensitivity® ELISA Kits
•Detect low-abundant cytokines. Amplified sandwich ELISAs allow for detection limits
below 1 pg/mL without loss of resolution or increase in background
• Biotinyl-tyramide signal amplification technology detects < 1.0 pg/ml.
• Requires only 50 μl of sample for reproducible, accurate results from precious
samples.
• Shorter incubation time than comparable ELISA kits.
• Includes pre-coated plates, ancillary reagents and a simple protocol.
Designed for labs who need optimal ELISA performance for targets that demand high-
sensitivity.
Advantages of High Sensitivity
ELISA
➢ Sensitive: Detect low cytokine concentration <1.0pg/mL for reliable
quantification
➢ Requires minimal sample: 50microL sample volume ideal for limited
sample resources
➢ Easy-to-use: Includes all required reagents and a straightforwards
protocol
➢ Compatible instrumentation: Uses standard colorimetric plate reader
(absorption measurement at 450nm)
1-wash Elisa
Instant® ELISA Kit
•The name „Instant‟ says it all: the addition of sample is all that is needed to start the assay. There is
no laborious preparation of reagents, serial dilutions of standards, or their sequential addition to the
plate. In contrast to conventional ELISA protocols, the Instant ELISA® plate contains coating antibody
and lyophilized detection antibody, streptavidin-HRP, and sample diluent. Additional wells containing
the ready-to-use standard curve are provided separately.
•Save time and reduce steps. Our 1-hour, 1-wash cell signaling assay detects total and
phosphorylated targets with sensitivity exceeding industry standards
Advantages of Instant ELISA
➢ Time saving: Shorter setup time of only 15 minutes
➢ Maximum accuracy: No need to add antibody or do serial dilution of
standards; reduced handling means less error and more consistent results
➢ Better value: Standard curve data is generated in parallel with additional
well strips provided to enable use of all 96 wells for your samples
Available products
● Cayman
● Ebioscience
● Abcam
● Biovision
● BioAssay
● Abnova
Multiplex
Innovation
Surani Sukor
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Multiplex
Immunoassay
Surani Sukor
Reasons
Benefits:
❏ Increased amount of information in much shorter
duration ● Multiplex > 50plex
● Human, Mouse, Rat, Non-human Primate, Canine, Porcine
❏ Decreased sample volume ● Require only 25 - 50 µL of samples
❏ Reduced reagent, labor and expenses
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Multiplexing Increases Throughput
ELISA Multiplex
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● Maximizes limited samples
● Minimizes experimental variability
● Optimizes productivity
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How Does It Work?
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How Does It Work?
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How Does It Work?
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How Does It Work?
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How Does It Work?
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1 2 3
How Does It Work?
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How Does It Work?
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How Does It Work?
https://www.youtube.com/watch?v=S4qFXeeM
2ak
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Open platform format
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QuantiGene Plex
2.0 (QGP)
Surani Sukor
What is QGP?
1. The QuantiGene Plex Assay provides DIRECT from
lysate measurement of 3 to 80 target RNAs per well
with unparalleled accuracy and precision.
2. Quantitatively measure multiple RNA targets
simultaneously.
3. Quantitation of original RNA population directly from
lysates avoids biases, sample loss, false
positive/negative results associated with: ● RNA isolation
● cDNA synthesis
● PCR amplification
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Uniques Benefits
● High accuracy and precision using branch DNA (bDNA) signal
amplification versus target amplification (PCR)
● High precision - coefficient of variation (CV) of less than 15% for
entire process (sample isolation to results)
● High accuracy - distinguish percentage differences in RNA copy
number - linear assay with low CVs
● No optimization required for multiplexing 3 to 80 target RNAs
● Excellent results in difficult clinical research samples, including,
H&E-stained FFPE, blood, and skin
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QGP vs. RT-PCR
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Steps
Step 1-Sample Preparation: Samples are lysed to release and stabilize RNA‟s. The RNA assay works with a variety of
samples such as: cultured cells, human, plant and animal tissues, FFPE tissues, whole blood and PAXGene blood, or
purified RNA.
Step 2-Target Hybridization: Overnight hybridization in the 96-well plates with the target specific probe sets panel
(Capture extenders – CE‟s, Label Extenders – LE‟s and blocking probes).
Step 3-Signal Amplification: Signal amplification is achieved using branch DNA (bDNA) technology. A Pre-Amplifier
(PreAmp) molecule hybridizes to each pair of Label Extenders, but not to individual probes. Then, multiple Amplifier
(Amp) molecules hybridize to each PreAmp. Finally, multiple Label Probe oligonucleotides hybridize to each Amp.
Step 4-Detection: Addition of streptavidin phycoerythrin (SAPE) generates a signal that is proportional with the amount
of target RNA present in the sample. The signal is read using a Luminex instrument.
Video reference
https://www.youtube.com/watch?v=Of78a6PJo
kY
https://www.youtube.com/watch?v=-bIJt7zsx2k
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Housekeeping Genes
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QGP vs. RT-PCR
Samples from LPS-treated mice were tested
using QuantiGene Plex and qPCR. Both
QuantiGene Plex and qPCR assays showed
similar patterns of up- and down-regulation
upon treatment. However, the QuantiGene Plex
assay provided better accuracy and precision
than did qPCR. In this study, 14 qPCR plates
had to to be processed to provide as much
information as one QuantiGene Plex plate. Ebsworth K. Gene expression analysis by Quantigene Plex: validation and applications. Paper
presented at: Planet xMAP USA; 2007 March 12-14; Laguna Hills, CA.
Hardware and analyzer
MAGPIX
Luminex 200
FLEXMAP 3D
Price and
throughput
Multiplexing capabilities
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Thank You
Surani Sukor
Prima Nexus Sdn Bhd
Mobile: 017 - 305 1191
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Introduction to SPF rodents, SPF Housing and AAALAC Accreditation
Cindy Shiu
What is SPF?
■ Animals without a “specific pathogen” distributed or
infected within them or onto their surfaces.
■ Majority of animals used in research
■ Test negative for:
Most or all known exogenous viruses
Pathogenic parasites
Limited list bacteria that may cause disease or otherwise
interfere with research
Immunocompetent: Primary Pathogens
Immunocompromised: Opportunists
Why use SPF animals?
■ infections:
Interfere with research
Cause Disease
Contaminate biological materials
Cause subtle changes that alter
Experimental responses &
Phenotye in Genetically Modified animals
Are zoonotic agents pose a risk to public health
Are subclinical in natural host
LCMV, hantavirus, S. moniliformis
■ Commercially the most accepted quality by users for drug safety testing purpose
Advantages of using SPF animals
■ Do not have clinical disease
■ No exposure to disease causing pathogens
■ Certified to be free of specific disease causing pathogen, such as
● MHV(mouse hepatitis virus)
● MRM(murine respiratory mycoplamosis)
● SDA(Sialodacryoadentitis virus)
● PVM(Pneumonia virus of mice)
● Sendi virus
● Pinworms
Effect of Animal Quality in Research Changes in animal quality (due to genetic changes, infection, or
environmental factors), resulting in the change of physiological functions,
or cause animal morbidity or mortality, affect the success or failure of the
experiment, results and interpretation.
Key Issues in Research Quality as follow:
Production Shipping Use in
research Facility design (standardize facility,
constant environment)
Reduce
stress
3Rs concept
Care breed、
microorganism)
Avoid
contamination
Physical
plant/operation
(operational
technique)
Quality control (genetic, health,
environment)
What is Biosecurity?
All measures taken to increase :
■ Bioexclusion (i.e., prevention) or
■ Biocontainment (i.e., limit spread) or
■ By Eradication or
■ Minimize Adventitious infections
• Open Caging.
• Individual microisolator caging.
– Positive Pressure (commonly used)
– Negative Pressure (to control rare or BSL3 type of pathogens)
• Isolator
Different Type of Rodent Housing
Systems
• Each cage is individually ventilated and works independently from each
other.
• Air Supply and Exhaust are HEPA-filtered by blowers on top of rack.
• Require electricity to operate.
IVC Rodent Housing Systems
Isolator Rodent Housing System
Typical use in animal quarantine, housing and production of immunodeficient animals, and maintenance of foundation colonies of genetically-modified animals. Isolators also provide a cost-effective method of biocontainment or bioexclusion for critical research studies.
• Institutional exclusion list of organisms potentially affect the studies.
• Institutional response to positive findings / contaminations.
• Research requirements.
• Available physical facilities.
• Operational limitations – capital vs running cost.
• Level of risk that is acceptable.
Final decision is complex depending on capital investment and
running costs.
Factors in Selecting Rodent Housing
System
• Barrier facility versus non-barrier facility.
• Barrier facility
– Open caging system, IVC or isolator
• Non-barrier facility
– IVC or isolator
• Ways to integrate IVC to the building.
Integration of Rodent Housing Systems
to the Facility Building
How to set up AAALAC certified animal
facility
What is a
program?
Program needs
■ Animal procedures - vivarium or laboratories
■ Surgical or diagnostic radiography suites
■ In-house diagnostic needs
■ Need for floor drains
■ Containment/contamination control
■ Imaging requirements
■ Sizing major installed equipment
■ Impact of design on labor costs
Lunch Room Outside the center
Barrier
Room
Corridor
Side Corridor
RO Room
Entrance The Memorial Stele Rest Area Boiler Center vacuum System
Isolator
Breeding Center of BioLASCO
Breeding Center of The Jackson Laboratory
Breeding Center of Charles River Laboratory
Price comparison from different
sources
Thanks for
Your Attention!!
