BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光。低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
血浆中低密度脂蛋白（LDL）是运输内源性胆固醇的主要载体，通过结合其细胞膜上的低密度脂 蛋白受体（LDL-R）被降解和转化，是运输胆固醇到肝外组织的主要运载工具，当低密度脂蛋白，尤其是氧化修饰的低密度脂蛋白（OX-LDL）过量时，其携带的胆固醇便积存在动脉壁上，会增加动脉粥样硬化的风险，而动脉粥样硬化是大多数心脑血管疾病发生的前期病理基础和危险因素。
检测原理：在胆固醇酯酶（CHER）和胆固醇氧化酶（CHOD）存在的胆固醇测定系统中，加入特异的表面活性剂，选择性地使LDL-C溶解，以测定LDL-胆固醇。其他脂蛋白（HDL、 VLDL、乳糜微粒）由于受到表面活性剂和糖化合物的阻碍而不反应，在反应液中以脂蛋白形式残存。基于这个原理，可以直接测定LDL-胆固醇。接着利用酯酶催化胆固醇酯水解生成游离胆固醇（FC），FC在胆固醇氧化酶作用下被氧化生成 4-胆甾烯酮和过氧化氢；接着与4-氨基氨替吡啉等反应生成红色醌类化合物，其在546nm处有特征吸收峰，通过检测546nm处吸光值即可得出LDL-C含量。
| Component | 96T | Storage |
| 试剂一 | 18 mL | 2-8℃. Store in the dark. |
| 试剂二 | 6 mL | 2-8℃. Store in the dark. |
| 标准品 | 1EA | 2-8℃. Store in the dark. |
标准品（粉剂1支）：
1、临用前8000g 4℃离心2min使试剂落入管底；
2、加0.1ml蒸馏水，一周内用完， 配成的浓度见标签。
自备仪器和试剂
研钵（匀浆机）、天平、冰盒（制冰机）、台式离心机、可调式移液枪、水浴锅（烘箱、培 养箱、金属浴）、96孔板、离心管、酶标仪、蒸馏水（去离子水、超纯水均可）、乙醇。
实验流程
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
1.1 组织样本
称取约0.1g组织样本加入研钵中，加入1mL乙醇，进行冰浴匀浆，12000rpm，4℃或室温离心10min，取上清液待测。
注：若增加样本量，可按照组织质量 (g)：提取液 (mL) 为1:5~10的比例进行提取。
1.2 液体样本
澄清的液体样本直接测定，若浑浊则离心后取上清检测。
1.3 细菌/细胞样本
先收集细菌或细胞到离心管内，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℃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
2.1 酶标仪预热30min（等待仪器过自检程序亦可），设定波长到546m。
2.2 所有试剂解冻至室温（25℃），在96孔板中按下表依次加入：
| 试剂组分（μL） | 测定管 | 标准管 （仅做一次） | 空白管 （仅做一次） |
| 样本 | 2.5 | ||
| 标准品 | 2.5 | ||
| 蒸馏水 | 2.5 | ||
| 试剂一 | 180 | 180 | 180 |
混匀，37℃孵育5min，于波长546nm处读取各管吸光值A 1 。
| 试剂二 | 60 | 60 | 60 |
混匀，37℃孵育10min，于546nm处读取各管吸光值A 2 。ΔA=A 2 -A 1 。
注：
(1) 若测定管的A 2 值大于1，则需将样本用乙醇进行稀释，稀释倍数D需代入公式重新计算。
(2) 若ΔA 测定 低于ΔA 空白 ，可增加加样体积V 1 （如测定管的样本量和空白管的蒸馏水增至5μL或更多，则试剂一和二保持不变；标准品仍为2.5μL，额外加2.5μL蒸馏水补齐）；或增加样本取样质量W（如增至0.2g或更多），则改变的V1 和W则代入公式重新计算。
3、结果计算
3.1 按样本质量计算
LDL-C (μmol/g 重量)=(C标准 ×V 2 )×(ΔA 测定 -ΔA 空白 )÷(ΔA 标准 - ΔA 空白 )÷(W×V 1 ÷V)×D=C标准 ×(ΔA 测定 -ΔA 空白 )÷(ΔA标准 -ΔA 空白 )÷W×D
3.2 按蛋白含量计算
LDL-C (μmol/mg prot)=(C标准 ×V 2 )×(ΔA测定 -ΔA空白 )÷(ΔA 标准 -ΔA 空白 )÷(Cpr×V 1 ÷V)×D =C标准 ×(ΔA测定 -ΔA空白 )÷(ΔA标准 -ΔA空白 )÷Cpr×D
3.3 液体中LDL-C含量计算
LDL-C (mmol/L)=(C 标准 ×V 2 )×(ΔA 测定 -ΔA 空白 )÷(ΔA 标准 -ΔA 空白 )÷V 1 ×D =C 标准 ×(ΔA 测定 -ΔA 空白 )÷(ΔA 标准 -ΔA 空白 )×D
3.4 按细胞数量计算
LDL-C (nmol/10⁴ cell)=(C 标准 ×V 2 )×10³×(ΔA 测定 -ΔA 空白 )÷(ΔA 标准 -ΔA 空白 )÷(500×V 1 ÷V)×D =2×C 标准 ×(ΔA 测定 -ΔA 空白 )÷(ΔA 标准 -ΔA 空白 )×D
参数说明
C 标准 ：见标签；
V 1 ：样本加入体积，0.0025mL；
V：提取液体积，1mL；
V 2 ：标准品加入体积，0.0025mL；
D：稀释倍数，未稀释即为1；
2：血清前处理中的稀释倍数；
500：细胞数量，万；
W：样本取样质量，g；
Cpr：上清液蛋白浓度，mg/mL，推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
注意事项
1、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。
2、本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
Low-Density Lipoprotein (LDL) in plasma is the main carrier of endogenous cholesterol. It is degraded and metabolized by binding to the LDL receptor (LDL-R) on cell membranes and serves as the primary vehicle for transporting cholesterol to peripheral tissues. However, when LDL, especially oxidized LDL (OX-LDL), is present in excess, the cholesterol it carries accumulates in the arterial walls, increasing the risk of atherosclerosis. Atherosclerosis is the pathological basis and risk factor for the majority of cardiovascular and cerebrovascular diseases.
Detection Principle: In a cholesterol assay system containing cholesterol esterase (CHER) and cholesterol oxidase (CHOD), specific surfactants are added to selectively solubilize LDL-C for the determination of LDL-cholesterol. Other lipoproteins (HDL, VLDL, chylomicrons) do not react due to inhibition by the surfactants and sugar compounds, remaining in the form of lipoproteins in the reaction mixture. Based on this principle, LDL-cholesterol can be measured directly. Subsequently, esterase catalyzes the hydrolysis of cholesterol esters to generate Free Cholesterol (FC). FC is oxidized by cholesterol oxidase to produce 4-cholestenone and hydrogen peroxide. Hydrogen peroxide then reacts with 4-aminoantipyrine and other components to produce a red quinoneimine compound, which has a characteristic absorption peak at 546 nm. The LDL-C content is determined by measuring the absorbance at 546 nm.
| Component | 96T | Storage |
| Reagent 1 | 18 mL | 2-8℃. Store in the dark. |
| Reagent 2 | 6 mL | 2-8℃. Store in the dark. |
| Standard | 1EA | 2-8℃. Store in the dark. |
Standard (Powder, 1 vial) Preparation:
1. Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom of the tube.
2. Add 0.1 mL of distilled water to dissolve. Use within one week. The prepared concentration is as indicated on the label.
User-Prepared Instruments and Reagents:
Mortar (Homogenizer), balance, ice box (ice maker), benchtop centrifuge, adjustable micropipettes, water bath (oven, incubator, metal bath), 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water are acceptable), ethanol.
Experimental Procedure
It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.
1. Sample Extraction
1.1 Tissue Samples
Weigh approximately 0.1 g of tissue sample and place it in a mortar. Add 1 mL of ethanol and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C or room temperature for 10 minutes. Collect the supernatant for assay.
Note: If increasing the sample amount, maintain a tissue mass (g) to ethanol volume (mL) ratio between 1:5 and 1:10.
1.2 Liquid Samples
Assay clear liquid samples directly. If turbid, centrifuge and use the supernatant for assay.
1.3 Bacterial/Cell Samples
Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of ethanol per approximately 5 million bacteria/cells. Disrupt the bacteria or cells by sonication in an ice bath (power 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
*Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to ethanol volume (mL) ratio between 500:1 and 1000:1.*
2. Assay Steps
2.1 Preheat the microplate reader for 30 minutes (or wait for the instrument to complete its self-check). Set the wavelength to 546 nm.
2.2 Thaw all reagents to room temperature (25°C). Add reagents sequentially to a 96-well plate as follows:
| Reagent (μL) | Test Tube | Standard Tube (once) | Blank Tube (once) |
| Sample | 2.5 | ||
| Standard | 2.5 | ||
| Distilled Water | 2.5 | ||
| Reagent 1 | 180 | 180 | 180 |
Mix well and incubate at 37°C for 5 minutes. Read the absorbance at 546 nm for each tube (A1 ).
| Reagent 2 | 60 | 60 | 60 |
Mix well and incubate at 37°C for 10 minutes. Read the absorbance at 546 nm for each tube (A2 ). Calculate ΔA = A2 - A1 for each tube.
Note:
(1) If the A2 value for the Test Tube is greater than 1, dilute the sample with ethanol. The dilution factor (D) must be substituted into the calculation formula.
(2) If ΔA for the Test Tube is lower than ΔA for the Blank Tube, consider increasing the sample volume V1 (e.g., increase the sample volume in the Test Tube and the water volume in the Blank Tube to 5 μL or more, keeping Reagents 1 and 2 volumes unchanged; for the Standard Tube, keep at 2.5 μL and add 2.5 μL distilled water to make up volume) or increasing the sample weight W (e.g., to 0.2 g or more). The changed V1 or W must then be substituted into the calculation formula.
3. Calculation of Results
3.1 Based on Sample Mass
Derived Formula:
LDL-C (μmol/g weight) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (W × V1 ÷ V) × D
Simplified Formula:
LDL-C (μmol/g weight) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ W × D
3.2 Based on Protein Content
Derived Formula:
LDL-C (μmol/mg prot) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (Cpr × V1 ÷ V) × D
Simplified Formula:
LDL-C (μmol/mg prot) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ Cpr × D
3.3 LDL-C Content in Liquids
Derived Formula:
LDL-C (mmol/L) = (CStandard × V2 ) × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ V1 × D
Simplified Formula:
LDL-C (mmol/L) = CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) × D
3.4 Based on Cell Count
Derived Formula:
LDL-C (nmol/10⁴ cells) = (CStandard × V2 ) × 10³ × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) ÷ (500 × V1 ÷ V) × D
Simplified Formula:
LDL-C (nmol/10⁴ cells) = 2 × CStandard × (ΔATest - ΔABlank ) ÷ (ΔAStandard - ΔABlank ) × D
Parameter Definitions:
CStandard : Concentration as indicated on the label (mmol/L or μmol/mL)
V1 : Volume of sample added (0.0025 mL)
V: Volume of extraction buffer (ethanol) added (1 mL)
V2 : Volume of standard added (0.0025 mL)
D: Dilution factor (1 if not diluted)
500: Number of cells (in units of 10⁴)
W: Sample weight (g)
Cpr: Protein concentration of the supernatant (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.
Precautions
1. It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents.
2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.
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