Ochratoxin 赭麴毒素-定量-ELISA-2~25ppb-穀物專用(cereal grains)

• 原理
  酵素連結免疫分析法Enzyme-linked immunosorbent assay (ELISA)，利用抗原和抗體之間專一性鍵結的特性，並加入顯色酵素和酵素受質，利用顯色的深淺，對檢體進行檢測的分析方法。ELISA可使用標準曲線法 (standard curve) 進行定量，透過檢體的吸光值與標準品進行比對，即可得到檢體的對應濃度。競爭法(competitive ELISA) 一般用於分子量較小的抗原檢測，在抗體數量固定的塑膠孔盤上，加入可鍵結的酵素抗原及檢體抗原，兩種抗原皆會競爭塑膠孔盤上抗體鍵結，當檢體中抗原含量越多，顯色也就越淺
  
  Ochratoxin 赭麴毒素，依其在薄層層析法上螢光顏色的不同，通常分成A、B、C三種類型，其中以Ochratoxin A 毒性最強且產量最高，故一般赭麴毒素多以ochratoxin A為代表。赭麴毒素主要是真菌Aspergillus ochraceus、Penicillium verrucosum產生的二次(次級)代謝物。穀類(例如:米、玉米、麥類)、咖啡、香辛植物...等相關加工品或原料可能會受到赭麴毒素的汙染，目前在動物實驗上，已證實赭麴毒素 A 的暴露，會造成腎臟毒性，並引發腎臟癌
   
• 結果判讀
  極限:
  偵測極限(limit of detection, LOD)： 1 ppb 
  定量極限(limit of quantification, LOQ)：2 ppb
  偵測範圍:2-25 ppb(樣品超過25ppb需重新稀釋及測量)
• 樣品準備和萃取(SAMPLE PREPARATION AND EXTRACTION)
  The sample to be tested should be collected according to accepted sampling techniques. The sample should be ground and thoroughly mixed prior to proceeding with the extraction. Store samples at 2–8°C (35–46°F) until analyzed. If using Neogen’s Mycotoxin Extraction Kit, follow the instructions in that kit for the extraction procedure. If preparing the extraction solution in house, continue with the following instructions.
  
  1. Prepare a 50% methanol solution by mixing 1 part ACS grade methanol with 1 part distilled or deionized water for each sample to be tested.
  2. Obtain a representative sample. Grind the entire sample so that at least 75% of the ground material passes through a 20 mesh sieve, the particle size of a fine instant coffee.
  3. Blend 25 g of ground sample with 100 mL of 50% methanol/water solution for 3 minutes in a high-speed blender. 
  ALTERNATIVE: Add 10 g of ground sample to 40 mL of 50% methanol/water and shake vigorously for 3 minutes.
  4. Filter the extract by pouring at least 5 mL through a Whatman no. 1 filter (or Neogen filter syringe) and collecting the filtrate as a sample.
  5. After filtration, dilute the sample extract 1:1 with 50% methanol (1 mL extract with 1 mL 50% methanol).
   
• 操作流程(TEST PROCEDURE)
  Allow reagents to warm to room temperature 18-30oC (64-86oF) prior to use.
  1. Remove 1 red-marked mixing well for each sample to be tested plus 5 red-marked wells for controls and place in the well holder.
  2. Remove an equal number of antibody-coated wells. Return antibody wells that will not be used immediately to the foil pack with desiccant. Reseal the foil pack to protect the antibody. Mark one end of strip with a “1” and place strip in the well holder with the marked end on the left. Do not mark the inside or bottom of the wells.
  3. Mix each reagent by swirling the reagent bottle prior to use.
  4. Place 200 µL of conjugate from the blue-labeled bottle in each red-marked mixing well.
  5. Using a new pipette tip for each, transfer 100 µL of controls and samples to the red-marked mixing wells as described below. 

  0

  2

  5

  10

  25

  S1

  S2

  S3

  S4

  S5

  S6

  S7

  strip 1

  S8

  S9

  S10

  S11

  S12

  S13

  S14

  S15

  S16

  S17

  S18

  S19

  strip 2

  6. Using a 12-channel pipettor, mix the liquid in the wells by pipetting it up and down 5 times. Prime and pipette 100 µL of the mixture into the wells. Set the timer for a 10 minute room temperature (18–30°C, 64–86°F) incubation, mixing the wells for the first 30 seconds of the incubation by sliding the microwell holder back and forth on a flat surface without splashing reagents from the wells. Discard the red-marked mixing wells.
  7. Shake out the contents of the antibody wells. Fill the wells with distilled or deionized water and dump them out. Repeat this step 5 times, then turn the wells upside-down and tap out on a paper towel until the remaining water has been removed.
  8. Pour the needed volume of substrate from the green-labeled bottle into the green-labeled reagent boat.
  9. Add new tips to a 12-channel pipettor. Prime and pipette 100 µL of the substrate into the wells. Set the timer for a 10 minute room temperature (18–30°C, 64–86°F) incubation, mixing the wells for the first 30 seconds of the incubation by sliding the microwell holder back and forth on a flat surface without splashing reagents from the wells. Discard the red-marked mixing wells.
  10. Pour Red Stop solution from the red-labeled bottle into the red-labeled reagent boat.
  11. Eject the excess substrate from the 12-channel pipettor, prime the tips, and pipette 100 µL of Red Stop to each well. Mix by sliding back and forth on a flat surface. Discard the tips.
  12. Wipe the bottom of the microwells with a dry cloth or towel and read in a microwell reader using a 650 nm filter. Air bubbles should be eliminated, as they could affect analytical results. Results should be read within 20 minutes after the addition of Red Stop.
  13. Read and calculate results using Neogen's Awareness StatFax microwell reader, or equivalent. If using an EL301 reader or other strip/plate reader, calculate results using Neogen's Veratox software.

• 產品說明書：連結
• 操作手冊：連結
• 物質安全資料表(Safety Data Sheet; SDS)：中文連結、英文連結

參考規範：食品中污染物質及毒素衛生標準