Workshop and Training Program on Sampling and Detection Methods Applied to Transgenic Crops November 17 19, 2011, NIN, Hyderabad, India

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1 Workshop and raining Program on Sampling and Detection ethods Applied to ransgenic Crops odified (G) Plants and Food Detection ethods Dr. Clara. Alarcon Pioneer Hi-Bred Hyderabad, Plants 2.Seeds 3.Grain 4.Food 1 2 odified (G) Plants and Food Production of Antibodies argets for Detection DNA: Gene-specific PCR Event-specific PCR Southern blot Next Generation Sequencing antibodies (s): Plate format assay Lateral flow strip assay Western blot LC/S 3 Immunize the animals Collect the serum Process the serum and purify antibodies 4 Structure of Antibody ypes of Antibodies in Immunoassays Variable region Light chain Polyclonal antibodies [Ab] onoclonal antibodies [Ab] Heavy chain Dr. Jose Saldanha easurable signal Ag + Ab (Ag+Ab) 5 6

2 Workshop and raining Program on Sampling and Detection ethods Applied to ransgenic Crops Polyclonal and onoclonal Antibodies Antigen presentation and processing onoclonal vs Polyclonal aturity and clonal propagation Hybridoma technology onoclonal Antibodies (Single epitope) Polyclonal Antibodies (ultiple epitopes) onoclonal Lot-to-lot consistency Indefinite supply Highly specific Higher initial costs Polyclonal Lot-to-lot variability ore broadly reactive Often more sensitive Lower initial costs 7 8 Sensitivity of Immunoassay Sensitivity of Immunoassay Is a function of Affinity of antibody used in the assay (strength of Ag and Ab binding) Extraction efficiency Range from 1 ng/ml to 5 µg/ml How assay is developed & optimized. 9 Limit of Detection (LOD) he minimum concentration of protein that can be detected by the assay Limit of Quantification (LOQ) he smallest unknown concentration of protein that can be quantified reliably with specified accuracy and precision 10 Advantages of Immunoassay Easy sample preparation Economical and cost effective Robust, can be used in field or the lab Qualitative and quantitative applications Flexible formats available ulti-analyte capability 11 Disadvantages of Immunoassay Limited to samples for which we can produce a useful antibody Limited use in G detection in processed food denaturing protein Validation for each matrix required Cross reactivity Some Products may not express protein in seed/grain Low experssion argeted expression 12

3 Workshop and raining Program on Sampling and Detection ethods Applied to ransgenic Crops Commonly used Immunoassays Western blot Immunostaining odified (G) Plants and Food Western Blot and Immunohistochemistry are specialized techniques, usually used in the initial stages of gene discovery (Research). Not common for routine diagnostics Commonly used Immunoassays Immunoassay ethods Western blot Immunostaining he detection antibody is attached (conjugated) to an enzyme Enzyme substrate Enzyme-labeled antibody E Coated Antibody Y Colored product 15 Well of polystyrene 96-well plate 16 odified (G) Plants and Food Range of quantification 0.1 to 2.0% LOQ = 0.1% LOD = 0.05% - Quantitative estimation of G protein - Grain, animal feed, unprocessed food and in certain cases semi-processed food OD 650 nm Roundup Ready Soy (%) 17 18

4 Workshop and raining Program on Sampling and Detection ethods Applied to ransgenic Crops odified (G) Plants and Food odified (G) Plants and Food Advantages Quantitative-measure amount (%) of G protein Qualitative - Yes or No answer -Economical and robust -Easy to perform and transferred to laboratories -Qualitative and Quantitative - Seed Quality testing - Screening to preserve Identity - Sensitive, accurate, and reproducible - High throughput, can be automated - inimum lab equipment needed - ulti-analyte capability 19 - Widely accepted method 20 odified (G) Plants and Food odified (G) Plants and Food - Commercial kits Disadvantages - Requires several timed steps - Not very portable - Requires refrigerated storage Antibody coated plate Antibody conjugate Substrate Buffers Usually takes from 1 hours to 5 hours depending on the manufacturer Commonly used Immunoassays Absorbent Pad Western blot Immunostaining control positive Control Line est Line embrane Au Gold Pad Sample Au 23 Vial Filter Pad

5 Workshop and raining Program on Sampling and Detection ethods Applied to ransgenic Crops Immunoassay ethods Immunoassay ethods Sample Labeled specific antibodies Antigen complex to attached antibody Sandwich formed with capturing antibody Particle labeled antibody not bound to antigen arget [antigen] Antibody Conjugate Pad Wicking Pad Sample Pad Nitrocellulose embrane Backing aterial est Line Control Line Flexible designs Immunoassay ethods Advantages Immunoassay ethods Portable and can be used in the field Point of Care, fast, results in 5 minutes Robust and longer shelf life No refrigeration required inimal training and equipment Single strips Strip combs For 48 wells 27 Can be used for semi-quantification ulti-analyte capability 28 Disadvantages Immunoassay ethods Immunoassay ethods Leaf - Breeders and seed multiplication ore expensive than Less sensitive than Requires dry storage 29 Snap a leaf disc into the microfuge tube Add buffer and grind Add ImmunoStrip 30

6 Workshop and raining Program on Sampling and Detection ethods Applied to ransgenic Crops Immunoassay ethods Seed QC labs Seed quality testing Identity Preservation Adventitious Presence Identity Preservation Grain elevators where speed is critical Immunoassay ethods Immunoassay ethods - Grain testing G est in Food & Feed Supply Chain from truck to grader S t r i p e s t i n g P P P to tester Obtain representative sample devide weigh grind extract agitated Bt Cry 1Ab = Accurate est P EXPOR P P Bt Cry 9C Roundup Ready Bt Cry 3Bb Customer Customer Bt Cry 1F Liberty link Customer 33 Source: Grothaus et al. AOAC International. 2006, 89: odified (G) Plants and Food Readers easuring Response of Emerging echnologies Reflectance RUR ImmunoStrip Response Soy % Soy RUR 35 36

7 Workshop and raining Program on Sampling and Detection ethods Applied to ransgenic Crops Emerging echnologies ulti analyte capabilities Emerging echnologies ultiple trait strips Simultaneous determinations Anti-analyte 2 conjugate Anti-analyte 1 conjugate Antibody to analyte Antibody to analyte 1 Control Line Cry1Ab Cry2A PA/bar 8-strip combs 37 Source: EnviroLogix Inc., Portland, E ultiple trait strips Combined for Single Sample Extraction Common extraction of one large grain sample for maximum efficiency in Identity Preservation testing programs in the grain distribution system. Emerging echnologies Source: EnviroLogix Inc., Portland, E Barcode microarrays Glass slide format Fluorescence based detection Searchlight (Pierce) High protein binding plates Chemiluminescence SD (eso Scale Discovery) Special plates Electro-chemiluminescence (ECL) Luminex platform Colored beads Flow cytometer Emerging echnologies ultiplexed Platforms Signal dab Ag cab B B Antibody-based Array (SearchLight echnology) Emerging echnologies Luminex Platform Emerging echnologies Antibody coupled to individual bead 100 colored beads available Detect up to 16 proteins in one well Lasers FACS Analysis Detector Source:

8 Workshop and raining Program on Sampling and Detection ethods Applied to ransgenic Crops Emerging echnologies Homogenous Assays AlphaLISA bead-based technology relies on PerkinElmer's exclusive amplified luminescent proximity homogeneous assay. In the presence of the analyte, the two beads come into close proximity. he excitation of the donor beads at 680 nm generates singlet oxygen molecules that trigger a series of chemical reactions in the acceptor beads resulting in a sharp peak of light emission at 615 nm. Emerging echnologies Homogenous Assays Advantages over -like technologies Reduced hands-on and total assay times. No washing steps required Large dynamic range (approximately 3 5 logs) Easy to miniaturize (volumes as low as 5ul total assay) Easy to automate enabling High hroughput Screening Quantitative detection in 96-, 384- and well microplates possible Emerging echnologies Quant by ass Spec» arget proteins (and tens of thousands of other proteins) are digested with trypsin (or other proteases)» One/more signature peptides for target proteins are quantified by LC-S/S rypsin Digest Signature peptides are quantified as protein surrogates Emerging echnologies Quant by ass Spec» Immunoassays widely used for protein detection/quantification, but High quality antibodies are NO always available It takes months to generate good antibodies ypically it is challenging to differentiate mutants from wild type echnology that overcomes these challenges» Short turnaround time for method development» High sensitivity and high selectivity» ultiplexing capabilities Emerging echnologies Comparison of ultiplexed Platforms echnology icroarray Printer Sensitivity Dynamic Range ultiplexing No ~10 pg/ml 2-3 logs none odified (G) Plants and Food Questions? AlphaLISA No Low pg/ml 3-5 logs none Chip Yes Low pg/ml 3-4 logs Luminex No pg/ml 3 logs ~15-20 SD Yes Low pg/ml 5 log (claimed) up to 10 Searchlight Yes Low pg/ml 3-4 logs 16 S No Low pg/ml 3-4 logs > 50 48

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