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

surani@primanexus.com.my

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!!