ey". ee Jere ELISA 7 oes cae Sa ELISA-TEK™ COOKED MEAT SPECIES KITS Dee’ ey: Ce tae Ea — ad gee ery foneKITS INCLUDED IN THESE INSTRUCTIONS FOR USE: ‘+ ELISA-TEK™ Cooked Pork Kit -510621 ‘« ELISATEK™ Cooked Poultry Kit - 510631 « ELISA-TEK™ Cooked Sheep Kit 510641 ISA-TEK™ Cooked Horse Kit - 510651 + ELISA-TEK™ Cooked Deer Kit- 510681 ISA-TEK™ Cooked 3 Species Kit - 510603) eu \-TEK™ Cooked 4 Species Kit - 910604 ISA-TEK™ Cooked Customs Kit- 510601 For other testing services visit elisa-tel.com_ SAFETY NOTE The techniques of “Good Laboratory Practice” should be employed, ‘wien wing this ki: if such practios are used, the reagents constitute a vey low potential isk to health, Any contact with skin/eyes should ‘be treated by washing/irrgation, ELISA-TEK™ COOKED MEAT SPECIATION KIT ELISA-TEK COOKED ‘MEAT SPECIATION KITS will detect, down to 1%, the species of animal ese sed as ingrediens in cooked meat Speciisty Kit Components Storage Instructions ‘TABLE OF CONTENTS Sample Prepavaian and Extraction Preparing Pate Plan Sremplsam Prepon OF Ri Mai. 6 oe eg eee 3 eee ee cee oa ea Ay Dicker u Sn Ge — BQISAJKELISA TECHNGLOGIES KIT COMPONENTS A. Microwell module in fil pouch (12 stsips of 8 wells each) B. Two or more vials of species control C. Three or more vials of anti-species biotinylated antibodies D. One vial of strepravidin- peroxidase conjugate E, One vial of ABTS. E One vial of peroxide citrate buffer G. One vial af stop solution. HE One boatle of wash solu- STORAGE INSTRUCTIONS ELISA-TEK™ Cooked Meat Speciation Kies shold be stored at 2-8 °C upon arvival. DO NOT FREEZE. Kit components hotel be removed from relrgeraton and brought to room temperature bere use. Siore the microwell module in the supplied lal pouch with Gesiceant. Performance eannot be guaranteed beyond the kit expiry ate oF when stored improperly. SPECIFICITY Bach at of species specific reagents has been tested against a panel of meat sampies fr evest-reacivity and has been four to produce negative responses 10 the heterologous species samples Te should be noted that samples f turkey wil produce a posdve sult in the cooked poultry assay Similarly, bison sample will produce a postive raul the cooked beef assay and a goat sarnple will produce a positive reponse inthe cooked sheep assy. Further etails of responses to other and eecely related species are available fom requestZGELISA TECHNOLOGIES SAMPLE PREPARATION & EXTRACTION Prepare a saline solution (0.9% w/ sodium chloride in deionized wae, eg, 9 g NaCl in | LH,O), 1 Homogenize/dice sample 2. Weigh 5 of sample into a plastic bag, tube, or beaker 3. Addl 10 mL of the prepared saline solution {4% w/v NaCl, 4.Mix 60s unl homogenized, 5. 6. Leave undisturbed for 1 bour at room temperature Heat dhe extract in a water bath at 95-100 °C for 15 mn (his step is optional for samples known to be cooked.) 7.Fltera portion of the liquid through a Whatinan 4 for similar) filter paper or centrifuge at 10,000 Xg for 10 main 8. Add NaOH or Hl as needed to adjust the pH of the extract 06.040. 9. The clear supernatant dssne extract may be used immediately ‘or stored overnight at 4° for use the nest day. PREPARING A PLATE PLAN Each assay should inchade replicates of normal saline, 100% positive tissue control,» 1% positive tissue control, ané ane ‘or mote negative tissue controls. For the posidive control, ise the appropiate kit contol (eg, park inthe pork test) ora lean tse ‘extract ofthe species of interes For the negative controls, use one or more appropriate kt co trols 4, bee in the pork test) and the matris-matched negative control prepared by the end user ‘Decide on the numberof replicates of each control and sample ‘extract w yan, For screening samples, single or duplicate microwells foreach control and sample extrac may be adequate. USDA-FSIS rotocols require use of quadruplicate microwells for each conteal and for sample extracts that are potevalvilatonsXGELISA PREPARATION OF KIT MATERIALS Alt components should be at rom temperature (~20-25 °C) prior to opening the foil pouch or performing the ass A. Antibody coated microwell module: Open the fil pouch, Place any exces strips back into the pouch for later use. B. Controls: Ready 10 use as 100% controls; dilute appropriate control to prepare 1% positive contrl eg, 10 pl of canta into 990 pL of prepared saline ©. Anti-species lates: Ready to use D. Streptavidin-peroxidase conjugate: Resdy to uss E, ABTS concentrate and peroxide citrate buffer: Mix contents of each separate vial by inversion. DO NOT SHAKE. ABTS is supplied asa 25% concentrate and must be diluted in peroxide citrate buffer prepare working ABTS solution, For cach stip of 8 st wells, add 20 pL ABTS concentrate 10 480 pl peroxide citrate buffer. If using the entre plate, add 4300 plot ABTS concentrate tothe entire 722 mL.of perox: ide citrate buffer. NOTE: Dilutions of ABTS concentrate should be made fest just prior to use (eg, during the aviin-peroxidase conjugate incubation E Stop solution: Ready to we. G. Wash solution concentrate: Dilute the LO wash solution, concentrate in dsiled/deionized water to prepare working ‘wash solution, ¢, 100 mL wash solution concentrate to 900 mL disilled/ deionized water. Working wash solution may be stored at 4 °C for up to one yearELISA ASSAY PROCEDURE Note: Gently svi plate after loading of samples and each reagent. L.A 100 pL of saline, samples, and controls into each well according to the prepared plate plan and incubate for 1h at room temperature, 2, Wash cach well 3x wih working wash solution, 3. Add 25 yl of anti-species biotinylate into all well nd incubate for 1 hat room temperature 4. Wash each well 3 with working wash solution 5. Add 25 pL of avidin peroxidase conjugate into alwels and incubate for 30 min at zoom temperature 6, Wash each well 6% with working wash solution. 7.Aild 50 pl-of working ABTS solution into ll wells and incubate for 30 min at room temperature ‘Alternately if using the USDA protocol, adjnst incubation dime unt the 414-192 OD. of posiive controls 0.450-0.500 (approximately 5.30 min), 8. Ad0 30 pil-of stop solution into all well ‘9. Measure the aworhance of each well at 4144nm with a reference of 492 nm using a miraplate reader XG INTERPRETATION OF RESULTS Measuce absorbance at 414 nm (405-420 nm acceptable) and subtract the reference wavelenge of 492 nm (485-500 nm acceptable). OD values are determined by subtracting the mean normal saline OD from the mean sample OD values forthe respective species: Mean OD of normal saline should be < 0.150, All subsequent OD values refer to mean blank-subtracted 414-192 nm var. {For the assay to be vale by the ELISA-TEK Protocol: the OD of the 1% positive control must be > 0.250, the OD of each neg- ative control must be < 0.150, and the standard deviation of the replicates mast be < 0,080 or 10% of the OD, whichever is greater 2.For the asay to be valid by the USDA Protocol: the OD of the 100% positive control must be > 0.600, the OD of the negative controls must be < 0.060, aid the standard eviation of the replicates must be $ 0.060 3.16 the assy is determined to be valid by the above parameters the ‘unknown samples may be clasified sfllows:JK ELISA TECHNOLOGIES INTERPRETATION OF RESULTS (cont) ELISA-TEK PROTOCOL: "There are owo eniteria to characterize unknown samples by this protocol Ie up tothe user to decide which criterion ie valid for their samples The Kis have been validated to criterion | in our laboratory CRITERION 1 — Any sample whose OD minus the standard deviations ix > 0.250 is positive; all others are negative CRITERION 2~ Any sample whose OD minus three standard vations is> 0.100 and higher than the OD of the highest negative ‘contol plus thier standard deviations x positive; all others are negative. The end user should extrac and test a matrx-matched negative contral o use this eiterion USDA PROTOCOL: Any suumple whose OD mings three standard deviations is > 0.250 s positive; all oder samples are negative ‘A positive result means that dhe indicated species was detected in the sample. A megative result means tha the indicated species was not detected at or above the 1% dhreshold for thie assay 0 DISCLAIMER ELISA Technologies, Ine. ensures that its produets are macle ftom high quality ave materials bt can make no warranty, expressed ‘or implied as to thei suitability other than to qualitatively decect, ‘cooked meat species content when used exactly in accordance with, these instructions. Reminders are included as tothe safe handling of materials and reagents, proper storage of material and reagents as well sto use “universal laboratory safety protocols and procedures Use of the kit for any other purpose is considered outside is intended use, Any damages, ndluding consequential or special damage or expense arising direily or incretly from using thi prodict, are Fmited to replacement Value ofthe kt at the discretion of ELISA. Technologies Ine‘ABOUT US: ELISA Technologies, Ine. ‘was founded in 198) to develop «enzyme immunoassay technology For use in the food indus (Our mission ito assure food {quality by creasing and supporing rapid, reliable, sensitive and costelletive testing solutions We are commited to using our expeatise in species allergens, ELISA TECHNOLOGIES amiibiotis, drugs, hormones, toxins and other aralytes to assist our cients with their analytic needs (Our commitment is to provide ‘our cients with accurate, con- fidential ficient, reliable and costetfective analytical options tomere their needs. Visit us today at clisa-tok.com