德国IBL ASP ELISA试剂盒使用说明（二）
Assay principle The ASP ELISA assay is in a direct competition format, where free DA in the sample competes with DA-conjugated protein coated on plastic wells for binding to anti-DA antibodies free in the solution (Fig. 2). The polyclonal ovine anti-DA antibodies are conjugated to horseradish peroxidase (HRP). Sample diluted in buffer is incubated in the wells with the anti-DA-antibody-HRP conjugate. After washing, the amount of conjugate remaining bound to the well is measured by incubation with a substrate that gives a blue product upon reaction with the HRP enzyme. Addition of acid stops the reaction and changes the product colour from blue to yellow. The colour intensity is measured spectrophotometrically on a platereader at 450 nm, and is inversely proportional to the concentration of DA in the sample solution. The assay is calibrated using dilutions of a DA calibration solution supplied with the kit. The calibrated range of the assay (I(20) – I(80)) is approximately 10 to 300 pg/mL of DA. The ASP ELISA kit can either be used in 2 rounds of analysis for the quantification of 12 individual sample dilutions in duplicate (plus calibration solutions, A(max) and blanks), or for the quantification of 36 sample dilutions in duplicate using all 8 strips. The working range for ASP toxins in shellfish is 0.01mg/ kg up to at least 200 mg/kg. 
F I G U R E 2 : A S S A Y F O R M A T F O R T H E C O M P E T I T I V E A S P E L I S A
B . S A F E T Y I N S T R U C T I O N S As all chemicals should be considered potentially hazardous, always wear suitable protective clothing during handling of the kit. CAUTION: Domoic acid is a neurotoxin that is harmful by inhalation and ingestion. Avoid contact with skin, eyes and clothing. Wash hands thoroughly after handling. Beware of the hazardous nature of methanol and sulfuric acid. Please refer to the manufacturers Material Safety Data Sheet for these reagents. C . S T O R A G E A N D S T A B I L I T Y Store the kit at 2-8°C upon arrival. Do not freeze. See expiry date on the kit box for stability of the kit. D . W A R R A N T Y A N D L I M I T A T I O N O F R E M E D Y Biosense Laboratories AS (hereafter: Biosense) makes no warranty of any kind, expressed or implied, including, but not limited to, the warranties of fitness for a particular purpose and merchantability, which extends beyond the description of the chemicals on the face hereof, except that the material will meet our specifications at the time of delivery. Buyer’s exclusive remedy and Biosense´s sole liability hereunder shall be limited to refund of the purchase price of, or at Biosense´s option, the replacement of, all material that does not meet our specifications. Biosense shall not be liable otherwise or for incidental or consequential damages, including, but not limited to, the costs of handling. Said refund or replacement is conditioned on Buyer giving written notice to Biosense within thirty (30) days after arrival of the material at its destination, and Buyer treating the material as outlined in the product data sheet and/or kit insert after arrival. Failure of Buyer to give said notice within said thirty (30) days, or failure of Buyer in treating the material as outlined in the product data sheet and/or kit insert shall constitute a waiver by Buyer of all claims hereunder with respect to said material. The responsibility of all patent considerations in the use of our products rests solely with the user.
E . K I T C O N T E N T S Number: A) 12-well microplate strip modules 2 sealed pouches - 4 strips each (Precoated with DA-protein conjugate) B) Plate sealers 2 C) PBS/Tween tablets 2 D) Domoic Acid standard, 100 ng/mL 2 vials (derived from NRC CRM-DA-e) E) Anti-DA-HRP conjugate, 2 vials (6x concentrated) F) Ovalbumin 2 vials G) TMB peroxidase substrate 2 vials F . ADDITIONAL REAGENTS AND EQUIPMENT REQUIRED In addition to the reagents supplied with the kit, the following reagents and equipment are required and/or recommended to perform the assay: • Kitchen blender. • Centrifuge. • Vortex mixer. • Micropipettes. • Microplate spectrophotometer equipped with a 450 nm filter. • Water; distilled and deionised (e.g. Milli-Q water, Millipore). • Methanol (analytical grade). • 0.3 M H2SO4.
G . IMPORTANT NOTES 1. Read the complete procedure before starting the assay. 2. Protect vials and microwell strips containing DA standard dilutions and samples from direct light during incubations. 3. The plate sealers are used to seal the strips during incubation and care must be taken when removing them from the strips. 4. Positive displacement pipettes (50 μL) are recommended for dispensing methanolic extracts. 5. As in every quantitative ELISA, consistent and precise pipetting at each step in the procedure is essential in order to obtain reliable results. 6. Reproducibility in any ELISA is also dependent upon consistent washing of the microwells. 7. After each wash, the wells are emptied by inverting the strips over a sink and then tap dry the wells against a pile of paper towels to remove all of the remaining liquid. 8. Avoid prolonged intervals between the working steps of the procedure, and do not allow the microwells to dry out totally during the assay procedure. Definitions Blank wells: Background absorbance of the TMB peroxidase substrate; approximately 0.05 A.U. (Absorbance Units). Amax wells: Maximum absorbance; no standard or sample is added to these wells allowing maximum binding of the anti-DA-HRP conjugate to the plate-coated DAconjugate; approximately 1.0 A.U. (Absorbance Units).
