BIOO ANIMAL HEALTH PRODUCTS NovareDxTM- EHDV Antibody Test Kit Manual Catalog #: 7006-01 (1 X 96 wells); 7006-02 (2 X 96 wells) ©BIOO Scientific Corp. • 2014 TABLE OF CONTENTS GENERAL INFORMATION .......................................................................................... 1 Product Description ........................................................................................................................ 1 Procedure Overview ........................................................................................................................ 1 Kit Contents, Storage and Shelf Life ............................................................................................... 2 Specificity (Cross-Reactivity) .......................................................................................................... 2 Required Materials Not Provided With the Kit ............................................................................... 2 Warnings and Precautions .............................................................................................................. 2 SAMPLE PREPARATION .............................................................................................. 3 Serum/Plasma ................................................................................................................................. 3 Cell culture Supernatant ................................................................................................................. 3 EHDV ANTIBODY ELISA TEST KIT PROTOCOL ................................................... 4 Reagent Preparation ....................................................................................................................... 4 ELISA Protocol ............................................................................................................................... 4 RESULTS: ....................................................................................................................................... 5 Interpretation of Controls ............................................................................................................... 5 Interpretation of Samples ................................................................................................................ 5 TROUBLESHOOTING ................................................................................................... 6 No Color Development or No Signals with Standards .................................................................... 6 Low Optical Density (OD) Readings in Positive Control ............................................................... 6 High Optical Density (OD) Readings in Negative Control ............................................................. 6 EXAMPLES OF THE KIT TEST FOR DEER SERUM .............................................. 7 The NovareDxTM EHDV Antibody ELISA Test Kit is intended for laboratory use only, unless otherwise indicated. This product is NOT for clinical diagnostic use. NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02 GENERAL INFORMATION GENERAL INFORMATION Product Description The NovareDxTM EHDV Antibody Test Kit is designed for the detection of antibodies specific to Epizootic Hemorrhagic Disease (EHD) virus in cervids. EHDV is an orbivirus that infects most wild and domestic ruminants. EHD is endemic in many countries, particularly affecting white-tailed deer in North America, with a morbidity and mortality rate as high as 90%. Deer, elk, and moose can seroconvert. Edema, hemorrhage, and ulcerations are seen in the mouth and lips in the acute form of the disease. In the chronic form of the disease, affected animals exhibit sloughing of the hoof wall, erosions, and scars in the rumen. Spontaneous abortions and stillbirths have also been reported in some epidemics. The detection of antibodies to EHDV is done using blood samples (serum or plasma) from infected hosts. The NovareDxTM EHDV Antibody Test Kit provides a simple, rapid, sensitive and cost-effective enzyme-based immunoassay (ELISA) screening method. This enables white-tailed deer breeders, ranchers, veterinarians, and government agencies to detect EHDV serogroup-specific antibodies. Bluetongue virus (BTV), a member of the genus Orbivirus, produces similar symptoms, and differential diagnosis is important to prevent widespread outbreak and economic loss. The ELISA method accurately detects antibodies specific to EHDV, without cross-reactivity between the serogroups of BTV and EHDV. Procedure Overview The method is based on a colorimetric ELISA. Purified EHDV antigens have been coated in the plate wells. During analysis, the serum or plasma sample is added. If antibodies to EHDV are present in the sample, it will bind to the coated antigen. The secondary antibody, tagged with a peroxidase enzyme, targets anti-EHDV antibodies, and the resulting color intensity, after the addition of substrate, is related to the amount of anti-EHDV antibodies in the sample. BIOO ANIMAL HEALTH • WWW.BIOOSCIENTIFIC.COM 1 NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02 Kit Contents, Storage and Shelf Life The NovareDxTM EHDV Antibody ELISA Test Kit has the capacity for testing of 46 samples (Cat # 7006-01) or 92 samples (Cat # 7006-02) in duplicate. Return any unused microwells to the foil bag and reseal them with the desiccant provided in the original package. The shelf life is 6 months when the kit is properly stored. Kit Contents EHDV antigen-coated Plate EHDV Positive Control EHDV Negative Control 50X HRP-Conjugated Antibody Solution Secondary Antibody Diluent Solution Serum/Plasma Diluent 20X Wash Solution TMB Substrate Stop Solution Cat # 7006-01 1 x 96-well plate 0.5 mL 0.5 mL Cat # 7006-02 2 x 96-well plate 1.0 mL 1.0 mL Storage 4°C -20°C -20°C 200 L 400 mL 10 mL 15 mL 20 mL 10 mL 10 mL 20 mL 30 mL 40 mL 20 mL 20 mL -20°C 4°C 4°C 4°C 4°C 4°C Specificity (Cross-Reactivity) This kit can broadly detect EHDV serogroup-specific antibodies. Required Materials Not Provided With the Kit Microplate reader (450 nm) Incubator (at 37°C) Vortex mixer 10, 20, 100 and 1000 L pipettes Multi-channel pipette: 50-300 L (Optional) Warnings and Precautions Bioo strongly recommends that you read the following warnings and precautions to ensure your full awareness of ELISA techniques and other details you should pay close attention to when running the assay. More information can be found in Troubleshooting section. Periodically, optimizations and revisions are made to the kit and manual. Therefore, it is important to follow the protocol that comes with the kit. If you need further assistance, you may contact your local distributor or Bioo Scientific at [email protected]. Handle the positive and negative controls with care. Do not use the kit past the expiration date. Do not mix reagents from different kits or lots. Antibodies and plates are lot and kit specific. Make sure that the HRP conjugated antibody and diluent are mixed in correct volumes. BIOO ANIMAL HEALTH • WWW.BIOOSCIENTIFIC.COM 2 NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02 Maintain a laboratory temperature of 20°– 25°C (68°– 77°F). Avoid running assays under or near air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays in direct sunlight, as this may cause excessive heat and evaporation. Water quality is very important; make sure you are using only distilled or deionized water. When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the lower corner of the well, making contact with the plastic. Incubations of assay plates should be timed as precisely as possible. Add your controls first, and then your samples. Always refrigerate plates in sealed bags with desiccant to maintain the plate(s) stability. Prevent condensation from forming on plates by allowing them equilibrate to room temperature (20 – 25C / 68 – 77F) while in the packaging. SAMPLE PREPARATION SAMPLE PREPARATION Serum/Plasma Dilute serum or plasma samples using the serum/plasma diluent provided in the kit (e.g. 50 L of serum/plasma is added to 500 L of diluent to obtain a 1:10 dilution). Perform dilution at room temperature and use immediately. Store any other serum samples at -20°C. Cell culture Supernatant Cell culture supernatants from hybridoma or engineered cell lines can be directly used in the assay. Frozen supernatants should be thawed at room temperature before use in the assay. BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. BIOO ANIMAL HEALTH • WWW.BIOOSCIENTIFIC.COM 3 NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02 EHDV ANTIBODY ELISA KIT PROTOCOL EHDV ANTIBODY ELISA TESTTEST KIT PROTOCOL Reagent Preparation IMPORTANT: All frozen reagents should be brought up to room temperature before use (1 – 2 hours at 20 – 25C / 68 – 77F); Make sure you read the “Warnings and Precautions” section on page 2-3. Solutions should be prepared just prior to performing the ELISA. All reagents should be mixed by inversion prior to use. Do not return used pipette tips or any reagents to the original stock tubes/bottles. Use disposable reservoirs when handling reagents to minimize the risk of contamination. 1. Preparation of 1X Wash Solution Mix 1 volume of 20X Wash Solution with 19 volumes of distilled water. 2. Preparation of 1X HRP-Conjugated Secondary Antibody Based on the needed amount, add 1 volume of 50X HRP-Conjugated Secondary Antibody solution to 49 volumes of the Secondary Antibody Diluent Solution and mix well by inverting. The Secondary Antibody solution is then ready to use. The diluted Secondary Antibody is stable for 2 weeks at -20C. ELISA Protocol Amount of reagent required for one reaction is shown in the table below. Component Volume per well Sample/Positive Control/Negative Control 100 L 1X HRP-Conjugated Antibody 100 L 1X Wash Solution 3 mL TMB Substrate 100 L Stop Solution 100 L Note: All samples and controls should be run in duplicate 1. 2. 3. Bring the EHDV antigen-coated plate and all reagent components to room temperature (20°– 25°C/ 68°– 77°F) for at least an hour. Aliquot EHDV Positive Control (100 L/well) to two wells of the antigen coated plate. Aliquot EHDV Negative Control (100 L/well) to two wells of the antigen coated plate. Aliquot the Positive and Negative Controls separate from each other, for e.g., the upper left-hand and bottom right-hand corner wells of the plate 4. Add 100 L of previously diluted (1:10) serum/plasma sample (or neat cell culture supernatant) per well in duplicate. 5. Cover plate with foil and incubate for 60 minutes at 37C. Avoid direct sunlight and air vents during incubation. BIOO ANIMAL HEALTH • WWW.BIOOSCIENTIFIC.COM 4 NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02 6. Discard the solution in the wells. 7. Wash the plate by adding 300 L of 1X Wash solution to each well of the plate. Discard the wash solution and tap dry on paper towels. Repeat four additional times for a total of five washes. Perform the next step immediately. Do not allow the plate to dry. 8. Add 100 L of 1X HRP-Conjugated Secondary Antibody to each well of the plate. Cover plate with foil and incubate for 60 minutes at 37C. 9. Wash plate with 1X Wash solution as described in Step 7. 10. Add 100 L of TMB Substrate to each well of the plate. Cover plate with foil and incubate for 5 minutes at room temperature (20 – 25C / 68 – 77F). 11. Add 100 L of Stop Solution to each well. Read the plate as soon as possible on a plate reader at 450 nm wavelength. RESULTS: Interpretation of Controls Positive Control: Absorbance readings at 450 nm for positive control wells should be greater than O.D of 1.0. Negative Control: Absorbance readings at 450 nm for negative control wells should be less than O.D of 0.4. Controls must be within the limits above for proper interpretation of samples. Interpretation of Samples O.D ≥ 1: Sample is interpreted as positive for antibodies to EHDV if the absorbance reading at 450 nm is close to greater than O.D of 1.0. O.D ≤ 0.4: Sample is interpreted as negative for antibodies to EHDV if the absorbance reading at 450 nm is close to or less than 0.4. O.D values between 0.4 and 1.0 can be interpreted as weak positive for antibodies to EHDV. BIOO ANIMAL HEALTH • WWW.BIOOSCIENTIFIC.COM 5 NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02 TROUBLESHOOTING TROUBLESHOOTING No Color Development or No Signals with Standards Possible Causes Recommended Action Reagents were used in the wrong order or a step was skipped. Wrong antibodies were used, or secondary antibody was prepared incorrectly or has deteriorated. TMB substrate has deteriorated. Follow the protocol carefully and repeat the assay. Make sure that the antibodies used are the ones that came with the kit. All antibodies are kit- and lot-specific. Make sure that the secondary antibody and diluent are mixed in correct volumes. Use a new set of BIOO TMB substrate. Low Optical Density (OD) Readings in Positive Control Possible Causes Recommended Action Reagents were expired or mixed with a different lot number. Wash solution was prepared incorrectly. Too many wash cycles were used. Incubation times were too short. Lab temperature was too low. Reagents and plates were too cold. Reader was at wrong wavelength, or reader was malfunctioning. Excessive kit stress has occurred. Assay plates were compromised Verify the expiration dates and lot numbers. Use the wash solution in the kit and make sure that it is prepared correctly. Make sure to use the number of washes per the protocol instruction. Time each plate separately to ensure accurate incubation times, follow protocol. Maintain the lab room temperature between 20°– 25°C (68°– 77°F). Do not run assays under air conditioning vents or near cold windows. Make sure plates and reagents are brought up to room temperature. Keep the kit components out of the kit box for at least 1 hour before starting the assay. Make sure the wavelength is 450 nm for the assay and read the plate again. Verify reader calibration and lamp alignment. Check records to see how many times the kit has cycled from the refrigerator. Check to see if the kit was left at extreme temperatures for too long. Always refrigerate plates in sealed bags with a desiccant to maintain stability. Prevent condensation from forming on plates by allowing them to equilibrate to room temperature (20 – 25C / 68 – 77F) while in the packaging. High Optical Density (OD) Readings in Negative Control Possible Causes Recommended Action Poor quality water was used in wash solution. Substrate solution has deteriorated. If water quality is questionable, try substituting an alternate distilled water source to prepare the wash solution. Make sure the substrate is colorless prior to addition to the plate. Use the number of washes per the protocol instruction. Make sure that at least 250 L of wash solution is dispensed per well per wash. Verify the performance of the washer system; have the system repaired if any ports drip, dispense or aspirate poorly. There was insufficient washing or poor washer performance. Reader was malfunctioning or not blanked properly. This is a high possibility if the OD readings were high and the color was light. Verify the reader’s performance using a calibration plate and check the lamp alignment. Verify the blanking procedure, if applicable, and reblank. Lab temperature was too high Maintain the room temperature between 20°– 25°C (68°– 77°F). Avoid running assays near heat sources or in direct sunlight. Reagents were intermixed, contaminated or prepared incorrectly. Ensure that the correct reagents were used, that working solutions were prepared correctly and that contamination has not occurred. BIOO ANIMAL HEALTH • WWW.BIOOSCIENTIFIC.COM 6 NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02 EXAMPLES OF THE FOR DEER SERUMSERUM EXAMPLES OFKIT THETEST KIT TEST FOR DEER Deer (n=292) exhibiting clinical signs for EHDV (edema, hemorrhage, ulcers) were tested for virus-specific antibody by NovareDxTM-EHDV Antibody Test Kit. Absorbance (at 450 nm) is plotted for each sample. Absorbance (450 nm) 3 2.5 2 1.5 1 0.5 0 1 41 81 121 161 201 241 Individual deer serum samples Independent field trial, January 2014 Bioo Scientific Corporation 7050 Burleson Rd Austin, TX 78744 USA Tel: 1.888.208.2246 Fax: (512) 707-8122 BIOO ANIMAL HEALTH • WWW.BIOOSCIENTIFIC.COM O.D Negative control Made in USA BIOO Animal Health Products [email protected] www.biooscientific.com 7 281
© Copyright 2024