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TBA由肝脏分解代谢，其血清浓度升高反映肝实质性损伤。因此，TBA测定用于监测慢性肝病价值很大。胆汁酸被3α-羟甾醇脱氢酶(3α-HSD)以及氧化型β-硫代烟酰胺脲嘌呤二核苷酸(Thio-NAD)特异性氧化，生成3-酮类固醇以及还原型β-硫代烟酰胺脲嘌呤二核苷酸(Thio-NADH)。生成的3-酮类固醇在3α-羟甾醇脱氢酶及还原型β-烟酰胺脲嘌呤二核苷酸(Thio-NADH)存在下，再生成胆汁酸及氧化型β-烟酰胺脲嘌呤二核苷酸(NAD)。如上所述循环放大使检测灵敏度提高。测定在单位时间内生成的还原型β-硫代烟酰胺脲嘌呤二核苷酸(Thio-NADH)在405nm处的吸光度变化，以求得胆汁酸的含量。
注：本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
自备仪器和试剂
指标测定：
建议先选取1-3个差异大的样本（例如不同类型或分组）进行预实验，熟悉操作流程，根据预实验结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费！
1、样本提取：
① 液体样品：澄清的液体可直接检测；若浑浊则离心后取上清液检测。
② 组织样本：称取约0.1g组织样本加入研钵中，加入1mL无水乙醇，在冰上进行匀浆，若有损失则用无水乙醇补充到1mL，12000rpm，4°C或室温离心10min，取上清液待测。
③ 细胞样本：先收集细胞到离心管内，离心后弃上清；取约500万细菌或细胞加入1mL无水乙醇，超声波破碎细菌或细胞（冰浴，功率200W，超声3s，间隔10s，重复30次）；12000rpm 4°C离心10min，取上清，置冰上待测。
注：若增加样本量，可按照细菌/细胞数量（10⁴）：提取液（mL）为500~1000：1的比例进行提取。
2、检测步骤：
① 酶标仪预热30min（等待仪器过自检程序亦可），设置温度在37°C，设定波长到405nm。
② 所有试剂解冻至室温，在96孔板中依次加入：
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注：
1. 若A₂值大于0.8，须用生理盐水或蒸馏水对样本上清液进行稀释，稀释倍数D代入计算公式。
2. 若ΔA值小于0.005，可增加样本加样体积V₁（如由10μL增至20μL，空白管也由10μL增至20μL蒸馏水，标准管为10μL+10μL蒸馏水（总体积同测定管和空白管即20μL）；其他试剂均保持不变）；或者增加组织样本的取样质量W，则改变后的V₁和W代入公式重新计算。
结果计算：
1. 按照体积计算：
总胆汁酸 (TBA)(μmol/L)=(C标准×V₂)×(ΔA测定-ΔA空白)÷(ΔA标准-ΔA空)÷V₁×D =C标准×(ΔA测定-ΔA空白)÷(ΔA标准-ΔA空)×D
2. 按照蛋白浓度计算： 总胆汁酸 (TBA)(nmol/mg prot)=(C标准×V₂)×(ΔA测定-ΔA空白)÷(ΔA标准-ΔA空)÷(V₁÷V×Cpr)×D =C标准×(ΔA测定-ΔA空白)÷(ΔA标准-ΔA空)÷Cpr×D
3. 按照样本质量计算： 总胆汁酸 (TBA)(nmol/g 重量)=(C标准×V₂)×(ΔA测定-ΔA空白)÷(ΔA标准-ΔA空)÷(V₁÷V×W)×D =C标准×(ΔA测定-ΔA空白)÷(ΔA标准-ΔA空)÷W×D
4. 按细胞数量计算： 总胆汁酸 (TBA)(nmol/10⁴ cell)=(C标准×V₂)×(ΔA测定-ΔA空白)÷(ΔA标准-ΔA空)÷(V₁÷V×500)×D =C标准×(ΔA测定-ΔA空白)÷(ΔA标准-ΔA空)÷500×D
C标准---标品浓度；
V₁---加入样本体积，0.001mL；
V₂---加入标准品体积，0.001mL；
V---提取液体积，1mL；
500---细胞数量，万；
W---组织样本取样质量，g；
D---稀释倍数，未稀释即为1；
Cpr---样本蛋白质浓度，mg/mL。
TBA is catabolized in the liver, and elevated serum TBA concentrations indicate parenchymal liver injury. Therefore, TBA measurement is highly valuable for monitoring chronic liver diseases. Bile acids are specifically oxidized by 3α-hydroxysteroid dehydrogenase (3α-HSD) and oxidized β-thionicotinamide adenine dinucleotide (Thio-NAD) to produce 3-ketosteroids and reduced β-thionicotinamide adenine dinucleotide (Thio-NADH). The generated 3-ketosteroids are converted back to bile acids and oxidized β-nicotinamide adenine dinucleotide (NAD) in the presence of 3α-hydroxysteroid dehydrogenase and reduced β-thionicotinamide adenine dinucleotide (Thio-NADH). The cyclic amplification reaction described above improves assay sensitivity. The absorbance change of the produced reduced β-thionicotinamide adenine dinucleotide (Thio-NADH) at 405 nm within a unit time is measured to calculate the bile acid concentration.
Note: This kit is for research use only, and shall not be applied for clinical diagnosis or other purposes.
Required Instruments and Reagents (to be supplied by user)
⁕Full-wavelength microplate reader (capable of measuring absorbance at 405 nm)
⁕Mortar (homogenizer), ice box (ice maker), tabletop centrifuge, adjustable pipettes, water bath (drying oven, incubator, metal bath), 96-well plates, centrifuge tubes
⁕Distilled water (deionized water or ultrapure water is acceptable)
Assay Protocol:
It is recommended to select 1–3 samples with significant differences (e.g., different types or groups) for a pilot experiment to get familiar with the operating procedure. Adjust sample concentration according to pilot results to avoid unnecessary waste of samples and reagents!
1. Sample Extraction:
① Liquid samples: Clear liquid can be tested directly; turbid liquid shall be centrifuged and the supernatant collected for detection.
② Tissue samples: Weigh approximately 0.1 g tissue into a mortar, add 1 mL absolute ethanol, and homogenize on ice. Make up the final volume to 1 mL with absolute ethanol if liquid loss occurs. Centrifuge at 12,000 rpm for 10 min at 4 °C or room temperature, and collect the supernatant for testing.
③ Cell samples: Harvest cells into centrifuge tubes and discard supernatant after centrifugation. Add roughly 5 million bacteria or cells into 1 mL absolute ethanol, then lyse cells/bacteria by sonication on ice (power: 200 W, sonicate for 3 s, pause for 10 s, repeat 30 times). Centrifuge at 12,000 rpm, 4 °C for 10 min, transfer supernatant and keep on ice prior to measurement.
Note: If larger sample volume is needed, perform extraction at a ratio of 500~1000 : 1 (10⁴ bacteria/cells : mL extract solution).
2. Detection Procedure
① Preheat the microplate reader for 30 min (or wait for the instrument self-check process), set temperature to 37 °C and wavelength to 405 nm.
② Thaw all reagents to room temperature, then add reagents sequentially into the 96-well plate as follows:
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Notes:
1. If the value of A₂ is greater than 0.8, dilute the sample supernatant with normal saline or distilled water, and substitute the dilution factor D into the calculation formula.
2. If the value of ΔA is less than 0.005, you may increase the sample loading volume V₁ (e.g., increase from 10 μL to 20 μL; add 20 μL distilled water to the blank tube instead of 10 μL; for the standard tube, add 10 μL standard solution plus 10 μL distilled water to reach a total volume of 20 μL consistent with the assay tube and blank tube; all other reagent volumes remain unchanged). Alternatively, increase the weighed mass W of the tissue sample. Recalculate using the adjusted V₁ or W in the formula.
Calculation of Results:
1. Calculation by volume:
Total Bile Acid (TBA) (μmol/L) = (Cstd × V₂) × (ΔAtest − ΔAblank) ÷ (ΔAstd − ΔAblank) ÷ V₁ × D = Cstd × (ΔAtest − ΔAblank) ÷ (ΔAstd − ΔAblank) × D
2. Calculation by protein concentration:
Total Bile Acid (TBA) (nmol/mg prot) = (Cstd × V₂) × (ΔAtest − ΔAblank) ÷ (ΔAstd − ΔAblank) ÷ (V₁ ÷ V × Cpr) × D = Cstd × (ΔAtest − ΔAblank) ÷ (ΔAstd − ΔAblank) ÷ Cpr × D
3. Calculation by sample weight:
Total Bile Acid (TBA) (nmol/g tissue) = (Cstd × V₂) × (ΔAtest − ΔAblank) ÷ (ΔAstd − ΔAblank) ÷ (V₁ ÷ V × W) × D = Cstd × (ΔAtest − ΔAblank) ÷ (ΔAstd − ΔAblank) ÷ W × D
4. Calculation by cell count:
Total Bile Acid (TBA) (nmol/10⁴ cells) = (Cstd × V₂) × (ΔAtest − ΔAblank) ÷ (ΔAstd − ΔAblank) ÷ (V₁ ÷ V × 500) × D = Cstd × (ΔAtest − ΔAblank) ÷ (ΔAstd − ΔAblank) ÷ 500 × D
Cstd — Concentration of standard solution
V₁ — Volume of loaded sample, 0.001 mL
V₂ — Volume of loaded standard solution, 0.001 mL
V — Total volume of extraction solution, 1 mL
500 — Cell count unit: ten thousand cells
W — Weighed mass of tissue sample, g
D — Dilution factor; set as 1 if no dilution is performed
Cpr — Protein concentration of sample, mg/mL
| 项目号 | 物理外观 | 组件 | 96T | 储存 |
| T1522089A | 液体 | 试剂一 | 20 mL | 2-8℃. Store in the dark. |
| T1522089B | 液体 | 试剂二 | 5 mL | 2-8℃. Store in the dark. |
| T1522089C | 液体 | 标准管 | 0.2mL | 2-8℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737034 |
分析证书 | T1522089 |
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