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Mình hiện đang phân tích Cholinesterase trong não, cơ bằng máy so màu quang phổ Varian Cary 50 Conc, Bio-Rad (18cell) của Úc, trong 3 phút với bước sóng 412 anh chị nào có quy trình đó chính xác của máy này làm ơn giúp dùm, vì hiện tại hơi phiền phức ở chỗ chuẩn mẫu blank, theo cán bộ hướng dẫn thì đầu tiên sẽ cho máy chạy zero mẫu buffer pH7.4(sodium phosphate) trước sau đó chạy mẫu blank (gồm DTNB + buffer + cơ chất) nhưng có khi gặp rắc rối là mẫu blank ra kết quả âm nên phải chạy lại blank khác hoặc chạy zero lại, có khi vẫn không được thì cán bộ lại thay thế mẫu chạy zero là buffer bằng nước cất.Có khi phải lặp lại nhiều lần mới ra kết quả blank dương thì mới có thể bắt đầu đo mẫu não được làm tốn nhiều thời gian. Mình có cảm giác cán bộ hướng dẫn không chắc chắn lắm về quy trình (quy trình do phía Đan Mạch tài trợ hướng dẫn). Xin anh chị có kinh nghiệm chỉ giáo giúp.
Thứ 2 là đơn vị ABS có phải viết tắt của chữ Absorbance (độ hấp thu) không? Cám ơn!
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bạn lưu ý: mẫu trắng (blank) là DTNB và SET Zero. tốt nhất giữ nhiệt độ ổn định ở 24oC +-0.5oC
mình gửi bạn phương pháp xác định Cholinesterase. bên mình hiện có quy trình khá đầy đủ như sau:
A. Principle
Whole blood is diluted with pH 8.0 phosphate buffer and analyzed by an enzymatic method with acetylthiocholine as substrate. In presence of enzyme, acetylthiocholine yields thiocholine + acetate. Thiocholine is reacted with dithiobisnitrobenzoate to form yellow reaction product, thionitrobenzoic acid, which is monitored at 412 nm. Activity is calculated as µmoles of substrate hydrolyzed per mL of whole blood per min, µmole/mL/min.
B. Apparatus
(a) HITACHI UV-2900 Spectrophotometer, with Electronic thermostatted
cell holder.—Set at 412 nm for assay (410 nm for evaluation of spectrophotometer performance). double (preferred) beam, sensitive to 0.001 absorbance unit. With recording.
device to monitor absorbance change vs time.
(b) Cuvets.—1 cm, optical glass or disposable polystyrene.
(c) pH meter.—Accurate to 0.01 pH unit. Calibrate according to manufacturer’s instructions.
C. Reagents
(a) External control. —SeraChem Level 1 Clinical Chemistry Control Serum (Human) (Fisher Scientific), or equivalent normal serum control. Perform 10 or more replicate determinations to establish mean value. Acceptable range is ± 10% of that mean.
(b) Potassium hydroxide solution.—0.05N KOH. Dissolve 3.3 g reagent grade (85%) KOH in sufficient water to make 1 L.
(c) Chromate solution.—0.0400 g K2CrO4/L 0.05N KOH.
(d) Phosphate buffer solutions.—All solutions must be at room temperature prior to pH adjustment.
(1) Dibasic sodium phosphate solution.—0.1M. Dissolve 26.808g Na2HPO4•7H2O in water and dilute to 1 L.
(2) Monobasic potassium phosphate solution.—0.1M. Dissolve 13.609 g KH2PO4 in water and dilute to 1 L.
(3) Phosphate buffer, pH 7.0.—To 100 mL reagent 1, add sufficient reagent 2 (ca 150 mL) to adjust to pH 7.00. Keep solution refrigerated.
(4) Phosphate buffer, pH 8.0.—To ca 450 mL reagent 1, add sufficient reagent 2 (ca 50 mL) to adjust to pH 8.00. Keep refrigerated for long term storage. Warm to room temperature before use.
(e) Acetylthiocholine iodide (ATCI) solution. —0.075M. Dissolve 0.10835 g ATCI (Sigma Chemical Co. No. A 5751) in water and dilute to 5 mL. Keep frozen when not in use.
(f) Dithiobisnitrobenzoic acid (DTNB) solution.—0.01M. Dissolve 0.0396 g DTNB (Sigma Chemical Co. No. D 8130) in ca 9 mL pH 7.0 phosphate buffer, add ca 15 mg NaHCO3, and dilute to 10 mL with same buffer. Keep frozen when not in use.
D. Spectrophotometer Evaluation
Set wavelength to 410 nm. Adjust absorbance to zero with water in both sample and reference cuvets. Remove water from sample cuvet, rinse cuvet 3 times with ca 0.5 mL portions of K2CrO4 solution, and then fill cuvet with same solution. Absorbance reading at 410 nm should be 0.186–0.210. If reading is not within this range, recheck instrument performance before proceeding and, if necessary, recalibrate instrument according to manufacturer’s specifications
E. Preparation of Control and Sample Solutions
Prepare human serum control according to manufacturer’s instructions. Dilute well-mixed whole blood 1:1000 with pH 8.0 phosphate buffer (buffer should be 22.0–25.5°): Dilute 10 µL whole blood to 10 mL or 100 µL to 100 mL if 10 µL pipettor is not known to be accurate for blood. (Note: If air-displacement pipet is used, follow procedure for viscous samples.) Pipet 3 mL well-mixed, diluted blood into cuvet. Prepare second cuvet identical to first to serve as blank.
F. Determination
All reagents must be allowed to reach ambient laboratory temperature before performing assay; acceptable limits are 22.0–25.5°.
Add 50 µL DTNB to each cuvet and mix well. Place 1 cuvet in sample position and one in blank position. Set to zero absorbance at 412 nm.
Add 20 µL ATCI reagent to sample cuvet only and mix well. Follow absorbance increase over time. Allow 60-s delay prior to first reading to ensure linearity of reaction. Let reaction proceed minimum of 5 min and record A every 60 s. Calculate change in A for each time interval. A/min should be constant to ensure linear kinetics. Correlation coefficient (R2) for plots of A vs time should be 0.95–1.00.
Run external control in similar manner. Repeat assay if control value is not within acceptable range.