AFLATOXIN 總黃麴毒素-定量-ELISA-水溶性萃取-1~10 ppb

• 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
   
• 結果判讀
  偵測極限(limit of detection, LOD)： 0.8 ppb 
  定量極限(limit of quantification, LOQ)：1 ppb
  偵測範圍:1–10 ppb
   
• 樣品前置處理(SAMPLE PREPARATION)
  The sample to be tested should be collected according to accepted sampling techniques (see FGIS sampling protocol or contact your Neogen representative). Obtain a representative sample (minimum 100 g). Grind the entire sample so that at least 95% of the ground material passes through a 20 mesh sieve (about the particle size of a fine espresso). 
   
• 樣品萃取(SAMPLE EXTRACTION)
  1. Weigh out 10 g ± 0.1 g of sample into container cup. Pour contents of 1 MAX 2 Aqueous Extraction Packet into the container cup.
  2. Add 50 mL of distilled or deionized water over the sample and extraction powder.
  3. Vigorously shake, using hand or mechanical means, for 3 minutes. Let sample settle for 10 minutes. 
  4. Filter the extract with a filter syringe or through a Whatman #4 filter paper to collect a minimum of 3 mL sample filtrate into a sample collection tube. You may also pipette 1 mL of sample into a 2.0 mL micro-centrifuge tube, and centrifuge for 30 seconds using a micro-centrifuge.
  5. The sample is now ready for testing.
   
• 操作流程(TEST PROCEDURE)
  Allow all reagents to warm to room temperature 18–30°C (64–86°F) for at least two hours 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 which 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 right. 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

  1

  3

  5

  10

  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. Transfer 100 µL to the antibody-coated wells. Discard the red-marked mixing wells.
  7. Set timer for 15 minutes, mixing the wells for the first 30 seconds by sliding back and forth on a flat surface. Discard remaining substrate and rinse the reagent boat with water. Alternative method: Set plate on the plate shaker and continuously shake the plate for 15 minutes.
  8. 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 15 minutes, mixing the wells for the first 30 seconds by sliding back and forth on a flat surface. Discard remaining substrate and rinse the reagent boat with water.
  Alternative method: Set plate on the plate shaker and continuously shake the plate for 15 minutes.
  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 must 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 software.
  NOTE: Samples greater than 10 ppb need to be diluted and retested.
   
• 稀釋步驟(DILUTION PROCEDURE)
  Dilute the filtered sample 1:1 with supplied diluent (pink-labeled bottle) using a sample collection cup (i.e. 200 µL filtered sample with 200 µL dilution diluent). Mix well. Run diluted sample following the test procedure. Multiply your result by 2. 

• 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

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