- 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
組織胺(Histamine)是在腐敗水產魚肉中常見的一種化合物，常發生於已腐敗之鮪魚、鯖魚、鰹魚等鯖魚科魚類，故有時稱為鯖科魚類中毒症（scombrotoxicosis），鯖魚科魚類因魚肉中游離組胺酸含量較高，一旦鮮度保持不良，即會受到細菌作用，進而轉變成組織胺。此外，組織胺一旦產生，後續加熱也無法將其去除。因組織胺的中毒症狀與食物過敏的症狀十分相似(例如:皮膚或黏膜紅腫癢痛、噁心、嘔吐、腹痛、頭暈、頭痛...等)，故常會有誤判的情形發生。
根據衛福部-藥物食品檢驗局調查研究年報之調查，針對新鮮魚介類、魚類罐頭、水產罐頭之組織胺含量，多數抽樣之檢體的組織胺含量皆低於50ppm，且大多數的檢體(平均值)，皆是介於此套組之檢測範圍(2.5~40ppm)。
- 結果判讀
極限:
偵測極限(limit of detection, LOD)： 2 ppm
定量極限(limit of quantification, LOQ)：2.5 ppm
偵測範圍:2.5–40 ppm(樣品超過40ppm需重新稀釋及測量)
- 樣品準備(SAMPLE PREPARATION )
A. Canned and pouched tuna: AOAC 937.07b
➡️Place entire contents of can or pouch (meat and liquid) into a blender. Blend until homogenous. Store samples at 2–8° C (35–46° F) until analyzed.
B. Fresh or thawed frozen raw fish: AOAC 937.07a
➡️Clean and eviscerate three fish. Cut three cross-sectional pieces 2.5 cm (1 inch) thick, from back of the pectoral fin, halfway to vent and one posterior to the vent. De-bone slices and blend or grind combined samples until homogenous. Store samples at 2–8° C (35–46° F) until analyzed.
C. Dry Samples (example: Fishmeal)
➡️The sample to be tested should be collected according to accepted sampling techniques and be representative of the lot. After homogenization, grind part of the sample (minimum of 200 g) so at least 95% of the ground material passes through a 20 mesh sieve (about the particle size of fine espresso).
- 樣品萃取(SAMPLE EXTRACTION)
If using Neogen’s Veratox for Histamine Extraction Kit (Neogen item #9510), follow the instructions in that kit. Otherwise, continue with the instructions that follow.
NOTE: Glassware should not be used for extraction purposes as histamine may adhere to the glass.
Liquid/Wet Samples
1. Add 10 g of the homogenous mixture to a clean disposable extraction bottle containing 90 mL of distilled or deionized water.
2. Tightly cap and vigorously shake the bottle for 15–20 seconds in order to suspend the fish tissue in the water.
3. Wait approximately 5 minutes, then shake the bottle for 15–20 seconds in order to re-suspend the fish tissue.
4. Wait an additional 5 minutes, and again shake the bottle for 15–20 seconds in order to re-suspend the fish tissue. Allow the tissue to settle to the bottom of the bottle for about 30 seconds.
5. If necessary, filter the contents through folded filter paper or a histamine filter syringe (Neogen #9420H) into a clean container. The sample is now ready for extract dilution.
ALTERNATIVE: Centrifuge the sample and use the clear supernatant as the sample for extract dilution.
Dry Samples
1. Add 10 g of the homogenous mixture to a clean disposable extraction bottle containing 100 mL of distilled or deionized water.
2. Tightly cap and vigorously shake the bottle for 15–20 seconds in order to suspend the sample in the water.
3. Wait approximately 5 minutes, then shake the bottle for 15–20 seconds in order to re-suspend the sample.
4. Wait an additional 5 minutes, and again shake the bottle for 15–20 seconds in order to resuspend the sample. Allow the sample to settle to the bottom of the bottle for about 30 seconds.
5. If necessary, filter the contents through folded filter paper or a histamine filter syringe (Neogen #9420H) into a clean container. The sample is now ready for extract dilution.
ALTERNATIVE: Centrifuge the sample and use the clear supernatant as the sample for extract dilution.
- 樣品萃取之稀釋步驟(SAMPLE EXTRACT DILUTION)
Because of the sensitivity of the ELISA format, the sample extract must be diluted before performing the test (see Procedural Note 3 for preparing the sample extract diluent buffer).
1. Add 10 mL of sample extract diluent buffer to a clean test tube or bottle.
2. Using a clean pipette tip, add 100 µL of the extract to the sample extract diluent buffer. Gently swirl to mix.
3. The sample is now ready to test. Repeat for all samples.
NOTE: For optimal performance with expected results greater than 40 ppm, a further dilution of the sample extract using extract diluent buffer is recommended. Begin with a 1:1 dilution, repeating dilution and retesting if necessary until the new result (prior to application of the dilution factor) is less than 40 ppm. Corrected values require multiplying by 2x for total X repetitions of these additional 1:1 dilutions. Samples should be tested within 4 hours of extraction.
- 操作流程(TEST PROCEDURE)
Allow all reagents to warm to 18–30°C (64–86°F) 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 on the inside or bottom of the wells.
3. Mix each reagent by swirling the reagent bottle prior to use.
4. Add 100 µL of conjugate from the blue-labeled bottle to each red-marked mixing well.
5. The controls (see Procedural Note 2) are supplied ready to use—do not dilute. Using a new pipette tip for each, transfer 100 µL of controls and diluted samples to the red-marked mixing wells as shown:
If concerned about histamine levels between 2.5 and 40 ppm:0 2.5 10 20 50 S1 S2 S3 S4 S5 S6 S7 Strip 1 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 Strip 2
If concerned about histamine levels between 5 and 40 ppm:0 5 10 20 50 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 up and down 3 times. Transfer 100 µL to the antibody-coated wells.
7. Incubate for 10 minutes at room temperature 18–30°C (64–86°F) mixing for the first 10–20 seconds by sliding the microwell holder back and forth on a flat surface without splashing reagents from the wells. Discard red-marked mixing wells.
8. Shake out the contents of the antibody wells. Fill each antibody well with diluted wash buffer and dump them out. Repeat this step 3 times, then turn the wells upside down and tap out the remaining liquid on an absorbent towel.
9. Pour the needed volume of substrate from the green-labeled bottle into the green-labeled reagent boat, and with new tips on the 12-channel pipettor, pipette 100 µL of substrate into the wells.
10. Incubate for 10 minutes at room temperature 18–30°C (64–86°F) mixing 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.
11. Pour Red Stop solution from the red-labeled bottle (same volume as substrate) into the redlabeled reagent boat. Using the same pipette tips on the 12-channel pipettor as were used to dispense substrate, add 100 µL Red Stop to each well and 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 and Neogen Veratox software comparing to the standard curve to calculate results. Air bubbles should be eliminated, as they could affect analytical results. Results should be read within 20 minutes of completion of the test.
13. All materials are safe to be disposed of in the trash.
商品特色
商品規格
- 商品規格(38個檢測反應)
1️⃣48 antibody-coated microwells
2️⃣48 red-marked mixing wells
3️⃣6 yellow-labeled bottles of 0, 2.5, 5, 10, 20 and 50 ppm histamine controls
4️⃣1 blue-labeled bottle of histamine-HRP conjugate solution
5️⃣1 foil pouch of sample extract diluent buffer concentrate of 10 mM PBS dry powder
6️⃣1 bottle of 40 mL wash buffer concentrate of 10 mM PBS-Tween
7️⃣1 green-labeled bottle of K-Blue® Substrate solution
8️⃣1 red-labeled bottle of Red Stop solution
.png)
