Multiplex Innovations & Introduction to SPF Animal

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1 Multiplex Innovations & Introduction to SPF Animal 30th June 2015 Conference Room Level 5, Block B, KL Campus (South Wing), UCSI University Organised By :

2 Instant ELISA Sharing Nur Afifah Samsudin

3 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

4 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 affinitybind to the antigens. Signal Measurement: detection of the signal generated via the direct or secondary tag on the specific antibody.

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6 Antigen Detection

7 Sandwish ELISA Workflow

8 Immunoassay platforms

9 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

10 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.

11 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.

12 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

13 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 highsensitivity.

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15 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)

16 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

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19 Advantages Time saving: Shorter setup of Instant time of only 15 minutes ELISA 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

20 Available products Cayman Ebioscience Abcam Biovision BioAssay Abnova

21 Multiplex Innovation Surani Sukor

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23 Multiplex Immunoassay Surani Sukor

24 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 µl of samples Reduced reagent, labor and expenses 24

25 Multiplexing Increases Throughput ELISA Multiplex 25

26 Maximizes limited samples Minimizes experimental variability Optimizes productivity 26

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28 How Does It Work? 28

29 How Does It Work? 29

30 How Does It Work? 30

31 How Does It Work? 31

32 How Does It Work?

33 How Does It Work? 33

34 How Does It Work? 34

35 How Does It Work? 2ak 35

36 Open platform format 36

37 QuantiGene Plex 2.0 (QGP) Surani Sukor

38 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 38

39 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 39

40 QGP vs. RT-PCR 40

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43 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. 43

44 Video reference ky 44

45 Housekeeping Genes 45

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47 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.

48 Hardware and analyzer Multiplexing capabilities FLEXMAP 3D Luminex 200 MAGPIX Price and throughput 48

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51 Thank You Surani Sukor Prima Nexus Sdn Bhd Mobile:

52 Introduction to SPF rodents, SPF Housing and AAALAC Accreditation Cindy Shiu

53 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

54 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

55 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

56 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 Facility design (standardize facility, constant environment) Care breed microorganism) Quality control (genetic, health, environment) Reduce stress Avoid contamination research 3Rs concept Physical plant/operation (operational technique)

57 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

58 Different Type of Rodent Housing Open Caging. Systems Individual microisolator caging. Positive Pressure (commonly used) Negative Pressure (to control rare or BSL3 type of pathogens) Isolator

59 IVC 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.

60 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.

61 Factors in Selecting Rodent Housing System 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.

62 Integration of Rodent Housing Systems to the Facility Building 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.

63 How to set up AAALAC certified animal facility What is a program?

64 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

65 Breeding Center of BioLASCO Outside the center Lunch Room Barrier Room Corridor Side Corridor RO Room Isolator Entrance The Memorial Stele Rest Area Boiler Center vacuum System

66 Breeding Center of The Jackson Laboratory

67 Breeding Center of Charles River Laboratory

68 Price comparison from different sources

69 Thanks for Your Attention!!