BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
白蛋白（Ovalbumin, OVA）是鸡蛋清中的主要磷酸化糖蛋白，广泛应用于ELISA、免疫印迹、免疫组化等生化实验。作为经典球蛋白，该蛋白性质稳定、溶解性优异，批次一致性高。此外，这种分子量约45 kDa的蛋白大小适中、结构清晰，具备适中且背景清晰的免疫原性，每个OVA分子可偶联多个半抗原分子，以此提升抗原免疫原性，提高免疫动物针对目标表位产生高滴度抗血清的概率，因此，OVA可用作载体蛋白与半抗原及其他抗原偶联，使后者更具免疫原性，用于免疫接种。
马来酰亚胺活化OVA偶联试剂盒采用简便快捷的流程，实现含巯基半抗原与载体蛋白OVA的偶联。该载体蛋白经过活化，暴露出可用于反应的马来酰亚胺基团，与含巯基的半抗原反应，偶联物可用于动物免疫制备特异性抗体。试剂盒提供马来酰亚胺活化的OVA(SMCC-OVA)、偶联所需的所有试剂、纯化填料和离心柱管，以及详细的分步操作指南。本试剂盒包含两种规格，可支持1次或5次独立偶联反应。
图1 马来酰亚胺活化OVA偶联试剂盒 (M1524129)偶联原理图
产品组份
| M1524129 | 组分 | 物理外观 | 1 reaction | 5 reactions | 储存温度 | 每次反应用量 |
| M1524129A | SMCC Activated OVA | 冻干粉 | 2 mg | 5×2mg | 2-8℃，避光，禁止冷冻 | 2mg SMCC-OVA可用于偶联2mg的半抗原 |
| M1524129B | 偶联缓冲液 | 无色透明液体 | 6 mL | 30 mL | 2-8℃ | 请按说明配制 |
| M1524129C | 纯化缓冲液 | 无色透明液体 | 10 mL | 50 mL | 2-8℃ | 请按说明配制 |
| M1524129D | 纯化填料 | 白色微珠混悬液 | 3 mL | 15 mL | 2-8℃，禁止冷冻 | 每次反应需使用3mL |
| M1524129E | 离心柱管 | 透明管体 | 1 EA | 5 EA | 2-8℃ | 每次反应需使用1个 |
其他所需试剂
1. 离心机。
2. 用于标记的半抗原。
3. PBS缓冲液（pH 7.2-7.4）。
重要须知
1. 本方案为通用流程，适用于绝大多数半抗原；需根据半抗原分子量、空间结构优化条件以提升偶联效率。
2. 体系中半抗原摩尔量过量可保证偶联充分；通常半抗原与OVA等质量投料即可实现摩尔过量。
3. 反应体系添加过DMSO时，平衡柱子的纯化缓冲液需加入同等比例DMSO，避免脱盐过程蛋白析出。
4. 若偶联反应出现沉淀：离心收集上清单独纯化，沉淀留存；纯化完成后将沉淀与纯化液合并使用。
5. 待纯化反应液体积应控制在0.4-0.7 mL，保证纯化柱的最佳去除效果。
6. 纯化填料建议一次性使用。
7. 在正式实验前，需将试剂恢复至室温，在使用前将试剂进行离心，较低的温度和离心管壁试剂残留均会影响偶联效果；
8. 操作过程中，务必穿戴实验服、一次性手套及防护装备；
9. 所有产品仅限于研究用途。
使用说明
1. 配制SMCC-OVA溶液（10 mg/mL）：向SMCC Activated OVA中加入200 μL纯水，轻轻摇晃直至全部溶解。
注意：复溶的SMCC Activated OVA溶液须现用现配。
2. 制备半抗原溶液（4-10mg/mL）：取2 mg半抗原，加入200-500 μL偶联缓冲液。
注意：部分半抗原水溶性差，可用DMSO助溶，DMSO在终反应体系中占比＜30%；过高浓度DMSO会造成OVA不可逆变性。
3. 立即将半抗原溶液全部加入到200 μL SMCC-OVA溶液中，混匀得到半抗原-OVA混合液，室温避光孵育2 h。
注意：反应进行期间，可提前进行步骤4的操作，完成脱盐柱的平衡。
4. 脱盐柱预平衡
通过脱盐纯化去除EDTA和叠氮化钠，这些物质不应注射到实验动物体内。脱盐无法将未偶联的载体蛋白与偶联蛋白分离开；但由于本方案中使用了大大过量的半抗原，因此不太可能存在大量未偶联的载体蛋白。
4.1 将一个空的离心柱放入一个 15 mL 收集管中。
4.2 通过反复倒置试剂瓶重悬纯化树脂填料。然后取3.0 mL悬液加入柱中，让树脂沉降。让柱缓冲液依靠重力从柱中排出。初始时，可能需要施加一些压力使最初的几滴缓冲液流出。以1000 × g离心柱子2分钟，弃去储存缓冲液，将柱子放回同一个收集管中。
注意：纯化树脂以20%乙醇的悬液形式提供，树脂与乙醇的体积比为3:1(v/v)。
4.3 向脱盐柱树脂顶部加入2 mL PBS，1000×g离心2分钟，弃去流出液。
注意：若偶联物短期1周内使用：可使用PBS完成脱盐；如需冻存，必须使用试剂盒配套的纯化缓冲液，可避免冻融过程蛋白失活析出
4.4 重复步骤4.3两次，第三次洗涤时加入PBS，直到进入下一步骤时再进行离心。
注意：使用定角转子时，在柱子背对转子中心的一侧做标记。在后续所有离心步骤中，将柱子放入离心机时，标记面应背对转子中心。
4.5 第三次洗涤离心后，脱盐柱中应无溶液残留，如有残留，可短暂离心直至溶液完全排出；
5. 偶联物脱盐纯化
5.1将准备好的脱盐柱放入新的15 mL离心管中，并将步骤3中的全部样品加入脱盐柱中。
注意：若反应出现沉淀，离心收集上清单独纯化，沉淀留存；纯化完成后将沉淀与纯化液合并使用。
5.2 脱盐柱1000 × g离心2分钟，收集管中的即为纯化偶联物。
6. 保存
6.1 纯化后可直接用于动物免疫；若存放超过数日，无菌过滤后置于无菌容器，4℃短期保存或分装-20℃冻存。
Ovalbumin (OVA) is the major phosphorylated glycoprotein in egg white, that has numerous biochemical applications including ELISAs (Enzyme-Linked Immunosorbent Assay), immunoblots, and immunohistochemistry. As a classic globular protein, it exhibits high stability, excellent solubility, and high batch-to-batch consistency. In addition, this protein with a molecular weight of approximately 45 kDa has a moderate molecular size and well-defined structure, coupled with moderate immunogenicity that yields low assay background. Several hapten molecules can be conjugated to each OVA molecule to increase their immunogenicity and likelihood of eliciting immunized animals to produce high-titer antisera to the epitopes of interest. Consequently, OVA can be used as a carrier protein for conjugation with haptens and other antigens to enhance the immunogenicity of the latter for immunization.
Maleimide Activated OVA Conjugation Kit uses a simple and quick process for OVA conjugation of haptens. The carrier proteins are activated to present an available maleimide group that reacts with sulfhydryl-containing haptens. The kit provides SMCC activated OVA, all the reagents, purification resin and empty spin columns, as well as a detailed step-by-step protocol. This kit is available in two sizes, supporting either 1 or 5 independent conjugation reactions.
Fig. 1 Maleimide Activated OVA Conjugation Kit (M1524129) Labeling Principle Schematic
Contents and storage
| M1524129 | Component | Appearance | 1 reaction | 5 reactions | Storage | Quantity Per reaction |
| M1524129A | SMCC Activated OVA | Lyophilized powder | 2 mg | 5×2mg | 2-8℃. Store in the dark. Do not freeze. | 2 mg SMCC-OVA for conjugation with 2 mg hapten |
| M1524129B | Conjugation Buffer | Colorless clear liquid | 6 mL | 30 mL | 2-8℃. | Prepare according to instructions |
| M1524129C | Purification Buffer | Colorless clear liquid | 10 mL | 50 mL | 2-8℃. | Prepare according to instructions |
| M1524129D | Purification Resin | White bead slurry | 3 mL | 15 mL | 2-8℃. Do not freeze. | 3 mL for 1 reaction |
| M1524129E | Empty Spin Column | Transparent tube | 1 EA | 5 EA | 2-8℃. | 1 EA for 1 reaction |
Required materials not supplied
1. Microcentrifuge capable of 1000 × g.
2. Desired hapten for labeling.
3. PBS buffer (pH 7.2-7.4).
Important
1. The following protocol is a general protocol for a wide variety of haptens. Optimize the protocol accordingly for the conjugation efficiencies based on the size and structure of your hapten.
2. Using a molar excess of hapten over carrier protein ensures efficient conjugation. In general, a reaction with equal mass amounts of hapten and carrier protein will achieve sufficient molar excess.
3. If DMSO was used in the conjugation, prepare the Purification Buffer Salts with the same percentage of DMSO used for the conjugation. This minimizes precipitation in the column during desalting.
4. If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the precipitate. Purify only the supernatant. Combine the purified conjugate to the precipitate.
5. The volume of the reaction solution to be purified should be controlled between 0.4 mL and 0.7 mL to ensure optimal removal efficiency using the purification column.
6. Do not reuse the purification resin.
7. Before formal experimentation, the reagents must be returned to room temperature and centrifuged before use. Both lower temperatures and reagent residue on the centrifuge tube walls may impair conjugation efficiency.
8. During operation, always wear a lab coat, disposable gloves, and protective equipment.
9. All products are for research use only.
Instruction for use
1. Prepare SMCC-OVA solution (10 mg/mL): Add 200 µL of ultrapure water to the SMCC Activated OVA. Gently swirl the vial to achieve full dissolution.
Note: The SMCC-OVA solution must be prepared fresh before each use.
2. Prepare hapten solution (4-10 mg/mL): Dissolve 2 mg hapten in 200-500 µL Conjugation Buffer.
Note: For haptens with limited solubility, use ≤ 30% DMSO in the final conjugation solution. Using a higher concentration of DMSO might irreversibly denature the carrier protein.
3. Immediately add the hapten solution to the 200 μL SMCC-OVA solution to have Hapten-OVA mixture solution. Incubate at room temperature for 2 hours, protected from light.
Note: During the incubation period, proceed to steps 4 below, to prepare a spin column for the purification of the labeled protein.
4. Prepare the spin column
Purify the conjugate by Purification Resin to remove EDTA and sodium azide, which should not be injected into laboratory animals. Desalting will not separate non-conjugated carrier protein from conjugated protein; however, a large excess of hapten is used in this protocol, making it unlikely that non-conjugated carrier exists in significant quantity.
4.1 Place an empty spin column in a 15 mL collection tube.
4.2 Suspend the Purification Resin by repeated inversion of the reagent bottle. Then add 3.0 mL of the suspension into the column and allow the resin to settle. Allow the column storage buffer to drain from the column by gravity. Initially, some pressure may be required to cause the first few drops of buffer to elute. Centrifuge the column at 1000 × g for 2 minutes, discard the storage buffer and return column to the same collection tube.
Note: The purification resin is supplied as a slurry in 20% ethanol, with a resin-to-ethanol volume ratio of 3:1 (v/v).
4.3 Add 2 mL of PBS Buffer, then centrifuge the column-tube assembly at 1000 × g for 2 minutes to equilibrate the column.
Note: If the conjugate is to be used for injection within one week, PBS may be used for purification. If the conjugate will be frozen, use the Imject Purification Buffer Salts for purification, which will preserve the product during freeze-thaw cycles.
4.4 Repeat step 4.3 for two additional times. Add PBS for the third wash but wait to centrifuge until immediately prior to proceeding to step.
Note: When using a fixed-angle rotor, place a mark on the side of the column that faces away from the rotor center. For all subsequent centrifugation steps, place the column in the microcentrifuge with the mark facing away from the rotor center.
4.5 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
5. Purification with Desalting Column
5.1 Place column into a new 15 mL collection tube and apply the entire sample from step 3 to the column.
Note: If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the precipitate. Purify only the supernatant. Combine the purified conjugate to the precipitate.
5.2 Centrifuge the column-tube assembly at 1000 × g for 2 minutes. The purified conjugate is in the collection tube.
6. Strorage
6.1 The hapten-carrier conjugate can now be used for immunization. If the hapten-carrier conjugate is to be stored for more than a few days, sterile filter the conjugate and store in a sterile container at 4℃ or -20℃
| M1524129 | Component | Appearance | 1 reaction | 5 reactions | Storage | Quantity Per reaction |
| M1524129A | SMCC Activated OVA | Lyophilized powder | 2 mg | 5×2mg | 2-8℃. Store in the dark. Do not freeze. | 2 mg SMCC-OVA for conjugation with 2 mg hapten |
| M1524129B | Conjugation Buffer | Colorless clear liquid | 6 mL | 30 mL | 2-8℃. | Prepare according to instructions |
| M1524129C | Purification Buffer | Colorless clear liquid | 10 mL | 50 mL | 2-8℃. | Prepare according to instructions |
| M1524129D | Purification Resin | White bead slurry | 3 mL | 15 mL | 2-8℃. Do not freeze. | 3 mL for 1 reaction |
| M1524129E | Empty Spin Column | Transparent tube | 1 EA | 5 EA | 2-8℃. | 1 EA for 1 reaction |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939429 |
分析证书 | M1524129 |
| ZJ26F0939428 |
分析证书 | M1524129 |
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