D-氨基酸氧化酶 (D-Amino acid oxidase, D-AAO)是一种黄素酶，能够催化D-氨基酸氧化脱氨，生成α-酮酸和过氧化氢 (H₂O₂)，在哺乳动物的肠道微生物代谢、内源性D-氨基酸的分解与利用、神经系统的调节和衰老中发挥重要作用。D-AAO可以调节不同D-氨基酸的水平，而不同的D-氨基酸因其结构差异和代谢途径的多样性在神经系统、免疫调节和能量代谢中发挥不同的作用。研究表明，D-AAO的活性在脑组织、血清和脊髓液中会随各种病理状态发生改变。例如，D-丝氨酸是大脑中N-甲基D-天冬氨酸 (N-methyl-D-aspartic acid, NMDA)受体的内源性共激动剂，参与学习、记忆和行为调节。当D-AAO活性异常升高时，会导致D-丝氨酸过度降解，进而影响NMDA受体的功能。这一机制被认为与精神分裂症的阳性症状 (如幻觉和妄想)、阴性症状 (如情感淡漠)和认知功能障碍有关。因此，D-AAO活性检测可能对临床相关疾病的早期诊断和病程监测具有重要意义。
在D-氨基酸氧化酶 (D-AAO) 的催化下，D-氨基酸 (D-Amino acid, D-AA)脱氨形成α-酮酸 (Alphaketo acid)、NH₃和 H₂O₂ ，再通过检测 H₂O₂与Amplex Red的反应产物试卤灵 (Resorufin)的荧光强度或吸光度来最终计算样品中D-氨基酸氧化酶的活力。通过检测试卤灵荧光强度 (560 nm 激发、590 nm 发射）或吸光度 (570 nm），即可实现D-氨基酸氧化酶活性的高灵敏定量检测，信号强度与D-氨基酸氧化酶活性成正比。
本试剂盒可采用分光光度法和荧光法两种方法进行检测，具有检测灵敏度高、线性范围宽、样品用量少等特点。在样品体积为20 µL 时，采用分光光度法检测在 5-500 μM 浓度范围内有良好的线性关系；采用荧光法检测在1-100 μM 浓度范围内有良好的线性关系。
组分货号 | 组分名称 | 物理外观 | 50T | 100 T | 储存 | 单次测试使用量 |
A1510319A | 酶混合液 | 液体 | 0.2 mL | 0.3 mL | -20℃.避光 | 3.0 μL |
A1510319B | 阳性对照 | 液体 | 10.0 μL | 10.0 μL | -20℃.避光 | 按需 |
A1510319C | Amplex Red | 液体 | 0.1 mL | 0.2 mL | -20℃.避光 | 2.0 μL |
A1510319D | 底物 | 液体 | 0.5 mL | 1.0 mL | -20℃.避光 | 10.0 μL |
A1510319E | 过氧化氢 (3%) | 液体 | 0.5 mL | 1.0 mL | -20℃.避光 | 按需 |
A1510319F | 检测缓冲液 | 液体 | 10.0 mL | 20.0 mL | -20℃. | 65.0 μL |
使用方法
1. 试剂盒的准备
1) 融解Amplex Red和检测缓冲液 ，平衡至室温后混匀备用。其它试剂存放于冰浴备用，使用完毕后宜立即按照试剂盒要求的条件保存。
2) H₂O₂ 标准品的校准
由于 H₂O₂ 不是非常稳定，使用前需自行测定 H₂O₂ 的实际浓度以进行校准。把试剂盒中过氧化氢 (3%) 用水稀释100倍，使 H₂O₂ 的浓度约为10 mM，测定 A₂₄₀。A₂₄₀的测定可采用如下的任一方法。
(a) 普通紫外分光光度计法。使用含比色皿架的紫外分光光度计、NanoDrop 2000C、QuickDrop等仪器，配套石英比色皿。确定比色皿光程一般为1 cm。用比色皿检测的 H₂O₂ 浓度最接近实际浓度。
(b) 微量紫外分光光度计法。如NanoDrop 2000、NanoDrop One、QuickDrop、含超微量检测板µDrop Plate的 Varioskan等仪器。确定光程：对于NanoDrop 2000、NanoDrop One等，需要取消‘‘自动化光程’’，此时光程一般为0.1 cm；Varioskan的超微量检测板µDrop Plate的光程一般为0.05 cm。具体的微量紫外分光光度计的光程请参考仪器参数。
(c) 96孔紫外酶标仪法 (须能检测240 nm波长)。根据96孔板的参数确定光程，一般200微升样品的光程为0.552 cm (样品体积除以96孔单孔孔内横截面面积)。一般建议使用专用的96孔紫外检测板，设置含等量双蒸水的孔作为空白对照 (一般200 μL水在该类96孔板的 A₂₄₀在3.8左右)，计算时须减去该空白对照。 在使用非紫外检测专用板的情况下，由于96孔酶标仪在240 nm的检测上限有限，建议将 H₂O₂ 稀释至约10 mM左右后再进 行浓度测定。
注：以上所有方法都需要设置等量双蒸水或超纯水作为空白对照，并在计算时减去该空白对照。
d) 浓度计算公式：c=A/(ε×b)。
c为样品浓度(单位为mol/L或M)；
A为吸光值；
ε为波长依赖的摩尔消光系数(单位为 L·mol⁻¹·cm⁻¹ 或 M⁻¹·cm⁻¹ )，过氧化氢的摩尔消光系数为 43.6 M⁻¹·cm⁻¹ ；b=光程 (单位为cm)。
因此： H₂O₂ 浓度(M)= A₂₄₀/(43.6×b)；即： H₂O₂ 浓度(mM)=22.94× A₂₄₀ /b 从而计算出本试剂盒提供过氧化氢(3%) 的实际浓度，并根据实际测定出来的浓度进行后续的标准曲线的设置。
示例：将本试剂盒提供的过氧化氢(3%) 用双蒸水稀释100倍后，用96孔酶标仪及一般的96孔板进行检测，每孔200微升，每组3个平行。双蒸水对照组的平均A₂₄₀为3.750，过氧化氢样品组的平均 A₂₄₀为3.974，差值为0.224，200微升样品的光程为 0.552 cm。代入公式， H₂O₂ 浓度 (mM)=22.94×0.224/0.552=9.31，则实际本试剂盒提供的过氧化氢 (3%)浓度为0.931 M。
2. 标准曲线的测定
1) H₂O₂ 标准曲线设置吸光度或荧光检测，可选取其中的一种，对于样品量较少或浓度较低的情况，优先推荐采用荧光检测。
a) 吸光度检测。根据校准确定的 H₂O₂ 标准品浓度，取适量的 H₂O₂标准品 ，用检测缓冲液混匀，配制成浓度为500 μM的 H₂O₂ 标准溶液。分别取500 μM的 H₂O₂ 标准溶液0、0.2、0.4、0.8、2、4、8、20 μL加入96孔板的标准品孔中，并相应地用检测缓冲液补足至20 μL，此时，标准曲线的浓度分别为0、5、10、20、50、100、200、500 μM。
b) 荧光检测。根据校准确定的 H₂O₂ 标准品浓度，取适量的 H₂O₂标准品 ，用检测缓冲液混匀，配制成浓度为100 μM的 H₂O₂ 标准溶液。分别取100 μM的 H₂O₂ 标准溶液0、0.2、0.4、1、2、4、10、20 μL加入96孔板 的标准品孔中，并相应地用检测缓冲液补足至20 μL，此时，标准曲线的浓度分别为0、1、2、5、10、20、50、100 μM。
2) 阳性对照的设置
根据选择检测方法不同，通过稀释不同梯度的阳性对照，使其落在检测范围之内。
a) 吸光度检测
例如取阳性对照5 μL，加入检测缓冲液进行梯度稀释。不同的缓冲体系稀释可能检测的酶活有所不同，推荐用该试剂盒缓冲液稀释，为了减少误差范围推荐梯度稀释。例如1 μL+ 499 μL稀释500倍，然后再取50 μL+950 μL 稀释20倍混匀, 再取该混合液20 μL作为1× 阳性对照可进行吸光度发检测。
b) 荧光检测
可以将吸光度法配制的1× 阳性对照母液进行2倍稀释进行荧光检测，如取20 μL1× 的阳性对照加入20 μL检测缓冲液，作为荧光检测阳性对照，可根据试验进行调整，保证合理落在曲线范围之内即可。
注：阳性对照高浓度条件下稳定性良好，稀释后的低浓度可放在四度储存，如要长期保存，推荐用带有高浓度的甘油稀释后进行保存，不同浓度下的甘油会影响酶活检测数据。作为阳性对照组使用检测1-2次即可。
注:吸光度检测时建议使用透明96孔板/ 荧光法检测时建议使用96孔黑板。
3. 样本制备
推荐使用新鲜样本，若不立即实验，-80℃可保存 1 个月，使用时冰上解冻。
1) 动物组织用冷 PBS 清洗组织，尽可能去除血液。吸干水分，称取 0.1 g，加入 1 mL 预冷的裂解液，冰浴匀浆。10000 g，4℃离心 5 min，取上清置冰上待测。
2) 植物组织称取约 0.1 g 样本，加入 1 mL 预冷的裂解液捣碎，冰浴超声破碎 5 min (功率 20% 或 200 W，超声 3 s，间隔 7 s，重复 30 次），10000 g，4℃离心 5 min，取上清置冰上待测。
3) 细胞（菌）收集每 1×10⁶ 个细胞或细菌，用冷 PBS 清洗后，加入 0.1-0.2 mL 预冷的裂解液冰浴匀浆或超声破碎 5 min。12000 g，4℃离心 5 min，取上清置冰上待测。
4) 血浆 、血清、尿液等生物体液去除蛋白后取上清：可使用 10 kDa 超滤管过滤取滤液；或将样本：30% ZnSO₄溶液 = 20:1 混合涡旋，10000 g 室温离心 5 min，取上清。
4. Amplex Red反应工作液的配制
按照每个反应80 µL的体积配制适量的Amplex Red反应工作液。均匀混合65 µL检测缓冲液 、2 µL Amplex Red、3 µL酶混合液 、10 µL底物，即可配制成80 µL Amplex Red反应工作液。根据待检测样品(包括标准品)的数量，配制适量的Amplex Red反应工作液。配制好的 Amplex Red反应工作液置于4ºC或冰浴避光保存，可以在当天使用，建议尽量现配现用。
5. 样本中酶活性测定
1) 按以下体系加样：
试剂 (µL) | 对照孔 | 测定孔 |
Amplex Red | 2 | 2 |
酶混合液 | 3 | 3 |
底物 | / | 10 |
样品 | 20 | 20 |
检测缓冲液 | 75 | 65 |
注：
a) H₂O₂ 的存在会对D-氨基酸氧化酶的检测产生干扰。如果样品含有 H₂O₂ ，须同时设置背景对照孔，加入不含底物的Amplex Red反应工作液，即配制Amplex Red反应工作液时10 µL底物用检测缓冲液替代。
b)为确保样品数值在标准曲线范围内，建议设置多个稀释倍数，以确定样品中大致酶活，如果数值不在标准曲线范围内，请使用检测缓冲液稀释。样品总稀释倍数记n(例如本步骤中对样品进行10倍稀释，加入的 ‘‘稀释后的样品’’为20 µL，则n=10)。
2) 反应体系充分混匀后25℃避光孵育 20-30 min。吸光度法检测，读取570 nm，记为A；如果使用荧光检测，设置激发波长为560 nm，发射波长为590 nm进行荧光强度检测，记为RFU 。
注：随着反应时间的延长，高浓度标准品孔的信号值可能会达到平台期，从而导致其不在标准曲线的线性范围内，此时可以舍去异常点的数据，取在线性范围内的数据来拟合标准曲线。
6. 结果计算
1) 建立标准曲线，将A或RFU带入标准曲线，即可算出在反应时间内样品中D-氨基酸氧化酶催化产生的 H₂O₂ 的浓度 (B)，如果样品的背景对照信号比较高，样品的信号值应减去样品背景对照的信号值。H₂O₂ 标准曲线，吸光度检测在5-500 μM浓度范围内有良好的线性关系，荧光检测在1-100 μM浓度范围内有良好的线性关系。D-氨基酸氧化酶活力的计算公式如下：
D-AAO (U/L)=B×n/T
注：B为根据标准曲线确定的 H₂O₂ 浓度 (μM)； n为样品总稀释倍数； T为的反应时间 (min)。
2) D-氨基酸氧化酶活性单位的定义为：1个酶活力单位 (unit, U)为在25ºC条件下，在1分钟内可以催化D-氨基酸反应生成1.0 μmol的 H₂O₂ 。
注： 如有必要，可以测定样品溶液中的蛋白浓度，这样可以计算出单位蛋白量中的D-氨基酸氧化酶酶活性。
注意事项
1) Amplex Red 试剂光敏、易氧化，全程避光，开瓶配制后尽快使用。
2) 过氧化物酶浓度过高会导致信号下降，样品需提前稀释。
3) H₂O₂ 工作液不稳定，必须现配现用。
4) 反应体系避免含 DTT、2 - 巯基乙醇等还原剂，否则干扰检测。
5) 每次实验均需重新配制标准品并做标准曲线。
6) 阳性对照稀释后可放四度保存，避免反复冻融。
7)为了您的安全和健康，请穿实验服并戴一次性手套操作。
8)本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品。
D-Amino acid oxidase (D-AAO) is a flavoenzyme that catalyzes the oxidative deamination of D-amino acids to produce α-keto acids and hydrogen peroxide (H₂O₂). It plays vital roles in intestinal microbial metabolism, endogenous D-amino acid catabolism and utilization, neural regulation, and senescence in mammals. D-AAO modulates the concentrations of various D-amino acids; due to structural differences and divergent metabolic pathways, distinct D-amino acids exert disparate effects on the nervous system, immune regulation and energy metabolism. Studies have demonstrated that D-AAO activity in brain tissue, serum and cerebrospinal fluid alters under diverse pathological conditions. For instance, D-serine acts as an endogenous co-agonist of N-methyl-D-aspartic acid (NMDA) receptors in the brain and participates in the regulation of learning, memory and behaviors. Excessively elevated D-AAO activity accelerates the degradation of D-serine, thereby impairing NMDA receptor function. This mechanism is implicated in the positive symptoms (hallucinations and delusions), negative symptoms (apathy) and cognitive dysfunction of schizophrenia. Accordingly, the detection of D-AAO activity is of great significance for the early diagnosis and disease progression monitoring of relevant clinical disorders.
Under the catalysis of D-amino acid oxidase (D-AAO), D-amino acids (D-AAs) undergo deamination to generate α-keto acids, ammonia (NH₃) and hydrogen peroxide (H₂O₂). The activity of D-amino acid oxidase in samples is quantified by measuring the fluorescence intensity or absorbance of resorufin, the reaction product formed between H₂O₂ and Amplex Red. Highly sensitive quantitative detection of D-AAO activity can be achieved by measuring the fluorescence intensity of resorufin (excitation at 560 nm, emission at 590 nm) or its absorbance (570 nm), and the signal intensity is directly proportional to D-AAO activity.
The kit supports both absorbance and fluorescence detection, featuring high sensitivity, wide linear range, and low sample consumption. With a sample volume of 20 µL:
- Absorbance assay: good linearity from 5-500 µM
- Fluorescence assay: good linearity from 1-100 µM
Component No. | Component Name | Appearance | 50 T | 100 T | Storage | Quantity Per Test |
A1510319A | Enzyme Solution | Liquid | 0.2 mL | 0.3 mL | -20℃.Store in the dark. | 3.0 μL |
A1510319B | Positive Control | Liquid | 10.0 μL | 10.0 μL | -20℃.Store in the dark. | As needed |
A1510319C | Amplex Red | Liquid | 0.1 mL | 0.2 mL | -20℃.Store in the dark. | 2.0 μL |
A1510319D | Substrate | Liquid | 0.5 mL | 1.0 mL | -20℃.Store in the dark. | 10.0 μL |
A1510319E | H₂O₂ (3%) | Liquid | 0.5 mL | 1.0 mL | -20℃.Store in the dark. | As needed |
A1510319F | Assay Buffer | Liquid | 10.0 mL | 20.0 mL | -20℃. | 65.0 μL |
Usage Method
1. Kit Preparation
1) Thaw Amplex Red and Assay Buffer, equilibrate them to room temperature and mix well for later use. Keep other reagents on ice until use. Immediately store all reagents under the conditions specified in the kit manual after the experiment is completed.
2) Calibration of Hydrogen Peroxide (H₂O₂) Standard
H₂O₂ is relatively unstable; its actual concentration must be determined for calibration prior to use. Dilute the H₂O₂ (3%) 100-fold with water to obtain an approximate 10 mM H₂O₂ working solution, then measure its absorbance at 240 nm (A₂₄₀). Any one of the following methods can be adopted for A₂₄₀ measurement:
a) Conventional UV Spectrophotometer Method
Use a UV spectrophotometer equipped with a cuvette holder, NanoDrop 2000C, QuickDrop or equivalent instruments paired with quartz cuvettes. The standard path length of cuvettes is 1 cm. Concentrations measured via cuvettes are closest to the true concentration of H₂O₂ (3%) .
b) Microvolume UV Spectrophotometer Method
Applicable instruments include NanoDrop 2000, NanoDrop One, QuickDrop, and Varioskan fitted with µDrop Plate microvolume detection plates. Path length setup: for NanoDrop 2000, NanoDrop One and similar models, disable the "automatic path length" function, which sets a fixed path length of 0.1 cm; the µDrop Plate for Varioskan has a standard path length of 0.05 cm. Refer to your instrument’s specifications for the exact path length of your microvolume UV spectrophotometer.
c) 96-Well UV Microplate Reader Method (capable of reading absorbance at 240 nm)
Determine the path length according to your 96-well plate specifications. For a sample volume of 200 µL per well, the path length is typically 0.552 cm (calculated as sample volume divided by the cross-sectional area of a single well in the 96-well plate). UV-transparent 96-well plates are strongly recommended. Wells loaded with an equal volume of double-distilled water serve as blank controls (the A₂₄₀ value of 200 µL pure water in such plates is approximately 3.8), and blank absorbance must be subtracted during calculation.
If non-UV-transparent microplates are used, the detection limit of the microplate reader at 240 nm is constrained. In this case, dilute H₂O₂ to roughly 10 mM before concentration measurement.
Note: For all above methods, prepare a blank control with an equal volume of double-distilled water or ultrapure water, and subtract the blank absorbance value in all calculations.
Concentration Calculation Formula: c=A/(ε×b)
· c = sample concentration (mol/L, or M)
· A = absorbance value
· ε = wavelength-dependent molar extinction coefficient (L·mol⁻¹·cm⁻¹ or M⁻¹·cm⁻¹); the molar extinction coefficient of hydrogen peroxide is 43.6 M⁻¹·cm⁻¹
· b = optical path length (cm)
Derived formulas: concentration (M)
H₂O₂ concentration (M) = A₂₄₀ /(43.6×b)
H₂O₂ concentration (mM) = 22.94× A₂₄₀ /b
Calculate the true concentration of the H₂O₂ (3%) supplied with this kit using the above formula, then prepare standard curves based on the measured actual concentration.
Calculation Example
The H₂O₂ (3%) provided in the kit is diluted 100-fold with double-distilled water, and measured in a standard 96-well microplate reader with 200 µL sample per well, three technical replicates per group.
The average blank A₂₄₀ of double-distilled water = 3.750
The average sample A₂₄₀ of diluted H₂O₂ = 3.974
Absorbance difference = 0.224
Path length for 200 µL sample = 0.552 cm
Substitute into the formula:
H₂O₂ concentration (mM) =22.94×0.224/0.552=9.31
Therefore, the actual concentration of the undiluted H₂O₂ (3%) from the kit is 0.931 M.
2. Standard Preparation
1) Either absorbance detection or fluorescence detection can be selected for establishing the H₂O₂ standard curve. Fluorescence detection is highly recommended when sample volume is limited or target concentration is low.
a) Absorbance Detection
Based on the calibrated actual concentration of H₂O₂ standard stock, pipette an appropriate volume of H₂O₂ Standard and dilute it with Assay Buffer to prepare a 500 μM H₂O₂ working standard solution.Add 0, 0.2, 0.4, 0.8, 2, 4, 8, 20 μL of the 500 μM H₂O₂ working standard into respective standard wells of a 96-well plate, then top up each well to a total volume of 20 μL with Assay Buffer. The final gradient concentrations of the standard curve are 0, 5, 10, 20, 50, 100, 200, 500 μM.
b) Fluorescence Detection
Based on the calibrated actual concentration of H₂O₂ standard stock, pipette an appropriate volume of H₂O₂ Standard and dilute it with Assay Buffer to prepare a 50 μM H₂O₂ working standard solution.Add 0, 0.2, 0.4, 1, 2, 4, 10, 20 μL of the 100 μM H₂O₂ working standard into respective standard wells of a 96-well plate, then top up each well to a total volume of 50 μL with Assay Buffer. The final gradient concentrations of the standard curve are 0, 1, 2, 5, 10, 20, 50, 100 μM.
2) Establishment of positive control
Depending on the selected detection method, dilute the positive control at different gradients to bring its values within the detection range.
a) Absorbance Detection
Example: Take 5 μL positive control and dilute it with assay buffer. Different buffer systems may lead to variations in measured enzyme activity. Dilution with the kit‑supplied assay buffer is recommended. Serial dilution is suggested to minimize assay error. Serial dilution procedure: Mix 1 μL positive control with 499 μL kit assay buffer for a 500‑fold dilution. Then take 50 μL of the above diluted solution and add 950 μL kit assay buffer to achieve an additional 20‑fold dilution, mix thoroughly. Take 20 μL of this mixture as 1× positive control for absorbance‑based measurement.
b) Fluorescence detection
Prepare a 2‑fold dilution from the 1× positive control stock prepared for absorbance assay. For instance, mix 20 μL of 1× positive control with 20 μL assay buffer to obtain the positive control for fluorescence detection. Adjust the dilution factor according to experimental requirements to ensure signals fall within the valid range of the standard curve.
Notes：The undiluted high-concentration positive control exhibits good stability. Diluted low-concentration positive control can be stored at 4 °C. For long-term preservation, dilute the stock with high-concentration glycerol; glycerol at varying concentrations may interfere with enzyme activity test results. The diluted positive control is only for 1-2 replicate assays as a reference control.
3. Sample Preparation
Fresh samples are recommended. If not assayed immediately, store samples at -80℃ for up to 1 month; thaw on ice before use.
1) Animal Tissue
Rinse tissue with cold PBS to remove blood; blot dry. Weigh 0.1 g, add 1 mL of cold lysis buffer , and homogenize on ice. Centrifuge at 10,000 g, 4℃ for 5 min. Collect supernatant on ice for assay.
2) Plant Tissu
Weigh ~0.1 g sample, add 1 mL of cold lysis buffer r, and homogenize. Sonicate on ice for 5 min (20% or 200 W; 3 sec on, 7 sec off; 30 cycles). Centrifuge at 10,000 g, 4℃ for 5 min. Collect supernatant on ice for assay.
3) Cells / Bacteria
Collect 1×10⁶ cells or bacteria; wash with cold PBS. Add 0.1-0.2 mL of cold lysis buffer , then homogenize or sonicate on ice for 5 min. Centrifuge at 12,000 g, 4℃ for 5 min. Collect supernatant on ice for assay.
4) Plasma, Serum, Urine, and Other Biological Fluids
Deproteinize before use:Filter through a 10 kDa ultrafiltration tube and collect filtrate; or
Mix sample : 30% ZnSO₄ solution = 20:1, vortex, centrifuge at 10,000 g, room temperature for 5 min, collect supernatant.
4. Detection Working Solution Preparation
Prepare an appropriate volume of Amplex Red working solution based on a reaction volume of 80 µL per test. Mix 65 µL Assay Buffer, 2 µL Amplex Red, 3 µL Enzyme Solution and 10 µL Substrate thoroughly to prepare 80 µL Amplex Red working solution. Prepare sufficient working solution according to the total number of samples to be tested (including standards).
The prepared Amplex Red working solution should be stored at 4 °C or on ice under light protection and used within the same day. Fresh preparation prior to use is highly recommended.
5. Measurement of Sample Enzyme Activity
1) Set up reactions as follows:
Reagent(μL) | Control Well | Sample Well |
Amplex Red | 2 | 2 |
Enzyme Solution | 3 | 3 |
Substrate | / | 10 |
Sample | 20 | 20 |
Assay Buffer | 75 | 65 |
Note :
a）H₂O₂ interferes with the detection of D-amino acid oxidase. If the sample contains endogenous H₂O₂, background control wells must be set up in parallel. For background controls, prepare Amplex Red working solution without Substrate, replace the 10 µL Substrate with an equal volume of Assay Buffer during working solution preparation
b) To ensure sample readings fall within the linear range of the standard curve, serial dilution gradients are recommended for preliminary estimation of approximate enzyme activity in samples. If the measured value exceeds the standard curve range, dilute the sample with Assay Buffer. Record the total dilution factor of the sample as n (if the sample is diluted 10-fold in advance and 20 µL of this diluted sample is added to the reaction system, then n = 10).
2) Thoroughly mix the reaction system and incubate at 25 °C protected from light for 20-30 min. Absorbance detection is performed by measuring the absorbance at 570 nm, denoted as A. For fluorescence detection, the excitation wavelength is set to 560 nm and the emission wavelength to 590 nm to record the relative fluorescence unit (RFU).
Note: As the reaction time increases, the signal values of wells containing high‑concentration standards may reach a plateau, falling outside the linear range of the standard curve. Under such circumstances, data from abnormal points can be discarded, and only data within the linear range shall be used for standard‑curve fitting.
6. Data Analysis
1) Construct a standard curve, then substitute the absorbance (A) or relative fluorescence unit (RFU) value into the curve to calculate the concentration of H₂O₂ (denoted as B) generated by D-amino acid oxidase catalysis in the sample within the reaction duration. If the background control signal of the sample is high, subtract the background signal value from the raw sample signal. For the H₂O₂ standard curve, good linearity is obtained from 5-500 μM via absorbance measurement, and from 1-100 μM via fluorescence measurement.
The formula for calculating D-amino acid oxidase activity is as follows:
D-Amino acid oxidase activity (U/L) = =B×n/T
Note:
- B = H₂O₂ concentration (μM) calculated from the standard curve.
- n = total dilution factor of the sample.
- T = reaction incubation time (min).
- Plot the standard curve with standard concentration as the X-axis and absorbance (A) as the Y-axis to calculate D-AAO activity in samples.
2) Definition of D-Amino Acid Oxidase Activity Unit
- One unit (U) of D-Amino acid oxidase activity is defined as the amount of enzyme that catalyzes the oxidation of D-amino acid to produce 1.0 μmol H₂O₂ per minute at 25 °C.
Note: If necessary, the protein concentration of the sample solution can be determined to calculate the D-amino acid oxidase activity per unit mass of protein.
Precautions
1.Amplex Red reagent is light‑sensitive and prone to oxidation. Protect from light throughout the procedure and use promptly after reconstitution upon vial opening.
2.Excessively high peroxidase concentration will cause signal decay; samples shall be pre‑diluted before assay.
3.Working solution of H₂O₂ is unstable and must be freshly prepared right before use.
4.Avoid reducing agents such as DTT and 2‑mercaptoethanol in the reaction system, as they will interfere with detection.
5.Prepare fresh standards and generate a standard curve for each independent experiment.
6.The diluted positive control can be stored at 4 °C; avoid repeated freeze‑thaw cycles.
7.For your safety and health, wear a lab coat and disposable gloves during operation.
8.This product is intended for scientific research use by professionals only. It shall not be used for clinical diagnosis or therapy, nor for food or pharmaceutical applications.
ZJ26F0939002
危险品化学品经营许可证（带存储）