- 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
極限:
偵測極限(limit of detection, LOD)： 1.4 ppb
定量極限(limit of quantification, LOQ)：5 ppb
偵測範圍:5–50 ppb(樣品超過50ppb需重新稀釋及測量) - 樣品前置處理和萃取(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. NOTE: If you are using NEOGEN’s Mycotoxin Extraction Kit, follow the instructions in that kit for the extraction procedure. If you are preparing your own extraction solution, continue with the instructions that follow.
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 95% of the ground material passes through a 20 mesh sieve, the particle size of fine espresso.
3. Vigorously shake, using hand or mechanical means, 5 g of ground sample in 25 mL of 70% methanol for 3 minutes.
AOAC Method: Vigorously shake, using hand or mechanical means, 50 g ground sample in 250 mL of 70% methanol for 3 minutes.
USDA/FGIS Method: Blend 50 g of ground sample with 250 mL of 70% methanol for 1 minute in a high-speed blender.
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. 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) before use.
1. Remove 1 red-marked mixing well for each sample to be tested plus 4 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 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 5 15 50 S1 S2 S3 S4 S5 S6 S7 S8 Strip 1 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 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 the red-marked mixing wells.
7. Set timer for 2 minutes, mixing the wells for the first 10–20 seconds of the room temperature incubations by sliding the microwell holder back and forth on a flat surface without splashing reagents from wells.
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 3 minutes, mixing the wells for the first 10–20 seconds by sliding 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 software.
商品特色
商品規格
- 商品規格(48個檢測反應)
1️⃣48 antibody-coated wells
2️⃣48 red-marked mixing wells
3️⃣04 yellow-labeled bottles of 0, 5, 15, and 50 ppb aflatoxin controls
4️⃣01 blue-labeled bottle of aflatoxin HRP conjugate solution
5️⃣01 green-labeled bottle of K-Blue® Substrate solution
6️⃣01 red-labeled bottle of Red Stop Solution
