本产品为抗小鼠各个亚类及轻链特异性HRP标记抗体组合套装,用于鉴定细胞上清及腹水中单抗的亚型,使用者需要具备一定的ELISA操作技能和免疫学知识;否则,请购买本公司成品试剂盒。
产品特点:
本套装试剂特点:高灵敏度、高特异性、无交叉反应,检测样品范围广泛结果容易判定。当然您如果觉得很多试剂要自己准备很麻烦那您可以登陆网站选择我们为您准备的整套试剂盒产品。
保存条件:
-20℃避光保存效期不少于3年,过于在强光或不良环境中频繁开启管盖有可能因此使效期缩短。
组成成分:
Goat Anti-Mouse IgG1 (HRP conjugated) 100ul
Goat Anti-Mouse IgG2a (HRP conjugated) 100ul
Goat Anti-Mouse IgG2b (HRP conjugated) 100ul
Goat Anti-Mouse IgG3 (HRP conjugated) 100ul
Goat Anti-Mouse IgM (HRP conjugated) 100ul
Goat Anti-Mouse IgA (HRP conjugated) 100ul
本套装含有各1支共计6支,液体除含有酶标记物外还含有PBS、甘油、防腐剂和稳定剂,每支体积100μL。
使用范围:
用于鉴定细胞上清及腹水中小鼠单克隆抗体Ig类、亚类\亚型的鉴定以及相关内容的研究。
使用方法:
1.将抗原(待鉴定单克隆抗体亚型对应的抗原)用适当的缓冲液(CBS或PBS缓冲液)包被酶标微孔板,包被浓度在1ug/mL左右,置37℃温育2小时或4℃放置 12小时,随后用PBS-T洗一遍。含1% BSA的PBS-T封闭,每孔150ul,室温2小时,拍干;
2.将待测杂交瘤细胞株培养上清或用PBS-T稀释的单抗腹水(建议稀释1000倍)每株加6孔每孔100μl,置37℃温育30分;
3.温育结束后,用PBS-T洗5遍,将本产套装的6种酶标记物用含有0.5%BSA的PBS-T按1:1000稀释后每种加1孔,每孔100μl共6孔,置37℃温育30分;
4.用PBS-T洗5遍,随后加TMB显色液37℃避光显色20分钟即可判定结果。
本酶特异性很好一般本底极低,肉眼观察蓝色孔即为阳性;参看此孔对应的酶标二抗就知道此株单抗的Ig类别或亚型。如果需要记录数值,2M硫酸终止反应后用酶标仪450、630nm双波长读数判定;
如果同时多孔显色,取最高值作为亚型判定标准;
注:单抗属于杂交瘤融合的来,存在一定比例的变异,另外,如果待检样品浓度太高也容易造成背景偏高,原则上以高值孔对应的亚类为准!
注意事项:
1.使用前请查看瓶身确认试剂是否在有效期内;
2.本试剂为浓缩型,稀释1000倍后才可使用,本产品最大可以稀释为1000倍,仍可以正常工作,但建议您不要这样操作,由此带来的结果偏差及其他问题,售后不负责处理;
3.实验过程中自配试剂一定要确保准确,不能含有对酶活抑制成分(比如叠氮钠,酶抑制剂及各种强离子螯合剂);
4.稀释后的试剂,立即使用,不可存放下次再用;如果稀释液中含有合适的防腐剂和稳定剂可以存放在4℃保存,一年内仍可使用;专用稀释液可联系我们购买
5.本实验过程中所有的试剂溶液,如果不方便配制都可以联系我们购买。
特别提示:
不具备ELISA基础知识和操作的人员不要完成此实验或类似实验,因为这可能会给您带来错误的结果和不必要的损失。
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案例1:
Hepatitis B Virus Core Particles Containing a Conserved Region of the G Protein Combined with Interleukin-35 Protected Mice against Respiratory Syncytial Virus Infection without Vaccine-Enhanced Immunopathology
Jie Yang , Chen Ma , Yu Zhao , Anjing Fan , Xiufen Zou , Zishu Pan
J Virol. 2020 Jun 16;94(13):e00007-20. doi: 10.1128/JVI.00007-20. Print 2020 Jun 16.
…
RSV-specific antibodies (IgG, IgG1, and IgG2a) in the sera of mice were determined by
enzyme-linked immunosorbent assay (ELISA) using purified RSV as the coating antigen (38, 39). Briefly,96-well microtiter plates (Corning, Corning, NY, USA) were coated with 100 ? l of inactivated RSV (1 ? 10 5PFU/well) in coating buffer at 4°C overnight. Serial dilutions of mouse sera in PBS-Tween 20 containing1% bovine serum albumin were added to the wells, and then the plates were incubated for 1 h at 37°C.Horseradish peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Inc., Waltham, MA,USA) or IgG1 or IgG2a (Friendbio Science & Technology Co., Ltd., Wuhan, Hubei, China) was added to theplates, and the plates were incubated at 37°C for 1 h. The 3,3=,5,5=-tetramethylbenzidine (TMB; Sigma)substrate in a 100- ? l volume was added to each well, and the plates were incubated for 15 to 20 min atroom temperature. The reaction was stopped with 100 ? l of 2 M H 2 SO 4 , and the optical density at 450 nm was measured using an ELISA reader (Multidkan MK3; Thermo Fisher Scientific).