Fumonisins伏馬毒素-定量-ELISA-甲醇萃取-0.5~6ppm

• 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
  
  伏馬鐮孢毒素（Fumonisins)又稱伏馬毒素，此毒素主要由真菌-鐮孢菌（Fusarium spp.）產生，伏馬毒素B₁+B₂是最常被檢出的種類，其檢出含量也常最高，約佔總伏馬毒素量之80到90%，故B₁ 和 B₂ 為伏馬毒素群中最具代表性之毒素。此毒素常出現於飼料或食物原料(例如:小麥、米類、玉米)，且會造成馬腦白質軟化症（Equine leukoence phalomalacia, LEM），亦被證實會引起小鼠及其他動物之肝癌，因而倍受重視。
   
• 結果判讀
  極限:
  偵測極限(limit of detection, LOD)： 0.2 ppm 
  定量極限(limit of quantification, LOQ)：0.5 ppm
  偵測範圍:0.5–6 ppm(樣品超過6ppm需重新稀釋及測量)
   
• 樣品準備與萃取(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. 
  1. If not using Neogen’s prepared solution, prepare a 70% methanol solution by mixing 7 parts ACS Grade methanol with 3 parts 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. Extract at a ratio of 1 part sample to 5 parts 70% methanol. Example: Blend 25 grams of ground sample with 125 mL of 70% methanol for 2 minutes in a high-speed blender. 
  Alternative method: Add 5 grams of ground sample to 25 mL of 70% methanol and shake vigorously for 3 minutes.
  4. Filter the extract by pouring at least 5 mL through a Whatman #1 filter (or Neogen filter syringe) and collecting the filtrate as a sample.
  5. Dilute sample by adding 100 µL of extract to a prefilled sample dilution bottle and mix well by swirling bottle. The sample is now ready for testing without further preparation. Repeat for each sample, making sure to label each bottle.
   
• 操作流程(TEST PROCEDURE)
  Allow all reagents to warm to room temperature 18–30oC (64–86oF) before 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 which will not be used immediately to the foil pack with desiccant. Reseal the 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 100 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

  0.5

  1

  3

  6

  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 3 times. Transfer 100 µL to the antibody-coated wells. Discard red-marked mixing wells.
  7. Set timer for 5 minutes, mixing the wells for the first minute of the room temperature incubation by sliding the microwell holder back and forth on a flat surface without splashing reagents from the wells.
  8. The initial reaction is now complete. 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.
  9. Pour the needed volume of substrate from the green-labeled bottle into the green-labeled reagent boat.
  10. With new tips on the 12-channel pipettor, prime and pipette 100 µL of substrate into the wells.
  11. Set timer for 10 minutes, mixing the wells for the first minute by sliding the microwell holder back and forth on a flat surface. Discard remaining substrate and rinse the reagent boat with water.
  12. Pour Red Stop solution from the red-labeled bottle into the red-labeled reagent boat.
  13. 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.
  14. 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.
  15. Read and calculate results using Neogen’s Stat Fax microwell reader, or equivalent. If using a strip/plate reader, calculate results using Neogen’s Veratox for Windows 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