acidovorax avenae

1. Acidovorax avenae subsp. Citrulli (Aac) ELISA Kit Acidovorax avenae subsp. Citrulli (Aac) ELISA Kit Cat.No: DEIAPB2 Lot. No. (See product label) Size 960 Tests Intended Use The test can be used to detect Aac in infected host plant tissue or seed as well as in pure bacterial cultures General Description Acidovorax avenae subsp. citrulli (Aac) is the causal agent of bacterial fruit blotch (BFB) in cucurbit crops, which is a serious threat to cucurbit crops (in particular melon and watermelon). Aac is naturally borne and transmitted by seed which usually serves as the primary inoculum source for BFB outbreaks. Bacterial fruit blotch can cause severe crop losses if allowed to progress in fields. Prevention is possible only by planting healthy seeds and eliminating infested seedlings. Principle of The Test During the first step of the assay the surface of a microtiter plate is coated with the antigen-specific coating-antibody (IgG). When an antigen-containing sample is added during the second step, the antigen binds to the immobilized IgG, forming an antibody-antigen complex. This complex reacts with the enzyme-labelled antibody-AP-conjugate during the third step by forming a double-antibody sandwich. During the fourth step the alkaline phosphatase (AP) reacts with the substrate 4- nitrophenylphosphate in an enzymatic reaction, resulting in yellow coloured 4-nitrophenol as product. This colour development can be evaluated visually or measured in a spectrophotometer at 405 nm after 1 and 2 hours. Reagents and Materials Provided 96 test format 96 test format 1. Antibody (IgG): 0.1 ml 2. Antibody-AP-conjugate: 0.1 ml 3. Positive Control: 10 tests 4. Negative Control: 10 tests 5. Coating Buffer: 1 liter 6. Wash Buffer: 1 x 5 liter 7. Conjugate/Sample Buffer: 1 x 1 liter 8. Substrate Buffer (5x): 1 x 25 ml 9. Substrate Tablets: 4 x 5 mg 10. Tween 20: 10 ml 11. High-binding ELISA plates: 12*8 wells 12. Sealing Cover: 1 Creative Diagnostics. All rights reserved 45-16 Ramsey Road Shirley, NY 11967, USA Tel: 631-624-4882 Fax: 1-631-938-8221 E-mail: info@creative-diagnostics.com www.creative-diagnostics.com 1

2. 480 test format 480 test format 1. Antibody (IgG): 0.5 ml 2. Antibody-AP-conjugate: 0.5 ml 3. Positive Control: 10 tests 4. Negative Control: 10 tests 5. Coating Buffer: 1 liter 6. Wash Buffer: 1 x 5 liter 7. Conjugate/Sample Buffer: 2 x 1 liter 8. Substrate Buffer (5x): 1 x 25 ml 9. Substrate Tablets: 5 x 20 mg 10. Tween 20: 10 ml 11. High-binding ELISA plates: 5 plates 12. Sealing Cover: 5 960 test format 960 test format 1. Antibody (IgG): 1.0 ml 2. Antibody-AP-conjugate: 1.0 ml 3. Positive Control: 20 tests 4. Negative Control: 20 tests 5. Coating Buffer: 1 liter 6. Wash Buffer: 2 x 5 liter 7. Conjugate/Sample Buffer: 4 x 1 liter 8. Substrate Buffer (5x): 2 x 25 ml 9. Substrate Tablets: 10 x 20 mg 10. Tween 20: 10 ml 11. High-binding ELISA plates: 10 plates 12. Sealing Cover: 10 Storage Our ELISA reagents are standardized for use at a dilution of 1:200 and a test volume of 200 μl/well. The products must be kept refrigerated (ca. 4°C) upon receipt. Once opened, we recommend using the reagents within 5 months. Reagent Preparation Coating Buffer Coating Buffer 1.59 g Na2CO3 2.93 g NaHCO3 Dissolve in distilled water and fill to 1L. Adjust pH 9.6. (Store refrigerated) Wash Buffer Wash Buffer 8.0 g NaCl 2.9 g Na2HPO4 x 12 H2O 0.2 g KH2PO4 0.2 g KCl 0.5 ml Tween 20 Dissolve in distilled water and fill to 1L. Adjust pH 7.2 - 7.4. (Store refrigerated) Conjugate/Sample Buffer for sample preparation and conjugate dilution Conjugate/Sample Buffer for sample preparation and conjugate dilution Creative Diagnostics. All rights reserved 45-16 Ramsey Road Shirley, NY 11967, USA Tel: 631-624-4882 Fax: 1-631-938-8221 E-mail: info@creative-diagnostics.com www.creative-diagnostics.com 2

3. Ingredients for Wash Buffer formulation (see above) and add: 20 g polyvinyl pyrrolidone (K10-K40) 2 g bovine serum albumin 0.1 g NaN3 (only if desired) Dissolve in distilled water and fill to 1L. Adjust pH 7.4. (Store refrigerated for no longer than 1 week. We recommend freezing aliquots and using the buffer solution as fresh as possible.) Substrate Buffer Substrate Buffer 97 ml diethanolamine 0.2 g MgCl2 x 6 H2O Dissolve in distilled water and fill to 1L. Adjust pH to 9.8 with 1 N HCl. (Store refrigerated) Substrate Solution Substrate Solution 1 mg/ml 4-nitrophenylphosphate-di-Na-salt in substrate buffer Prepare this solution immediately prior to use! Assay Procedure 1. Application of coating-antibody (IgG). Dilute IgG 1:200 from original vial in Coating Buffer, and add 0.2 ml per well to the plate, incubate at 37ºC for 4 h, then wash four times. 2. Sample application. Prepare samples at a 1:20 dilution in Sample Buffer, if not stated otherwise in the product certificate. (Dilute positive or negative controls in 2.1 ml Sample Buffer, if not specified otherwise.), and 0.2 ml per well to the plate, incubate at 37ºC for overnight, then wash four times. 3. Application of antibody-AP-conjugate. Dilute AP-conjugate 1:200 from original vial in Conjugate Buffer, and add 0.2 ml per well to the plate, incubate at 37ºC for 4 h, then wash four times. 4. Enzymatic assay. Add 0.2 ml Substrate Solution per well to the plate, incubate at room temperature for 1-2 h, then observe the results. Note: Washing is the process of removing the reagents with Wash Buffer after each incubation step. Quality Control Negative controls are made from a healthy host plant and are tested for the absence of the respective pathogen. They are lyophilized and sufficient for 10 test wells. Positive controls are made from infected plant material or bacterial cultures, if not stated otherwise. They are lyophilized and sufficient for 10 test wells. Inactivated positive controls are additionally tested for the absence of infectivity. Unspecific non-pathogenic 'method' controls are sold for some quarantine pathogens. Interpretation of Results We strongly advise to add positive and negative controls to the plate. To determine potential background of healthy plants, fresh non-infected extracts of the tested species, should be added to the plate. The positive/negative threshold needs to be determined by the user, as it depends on many factors, such as plant species and its physiological conditions (e.g. tissue type, age). Sensitivity Sensitivity of the ELISA is very high. Creative Diagnostics. All rights reserved 45-16 Ramsey Road Shirley, NY 11967, USA Tel: 631-624-4882 Fax: 1-631-938-8221 E-mail: info@creative-diagnostics.com www.creative-diagnostics.com 3

4. Precautions 1. Always wash hands thoroughly after using this product. 2. Prevent direct skin and eye contact with, or ingestion of, product components. Obtain medical attention in case of accidental ingestion of reagent components. Creative Diagnostics. All rights reserved 45-16 Ramsey Road Shirley, NY 11967, USA Tel: 631-624-4882 Fax: 1-631-938-8221 E-mail: info@creative-diagnostics.com www.creative-diagnostics.com 4