Preliminary Improvement Of CD1d Tetramer During The Preparation

TCR of NKT cells recognize the combination of thymus independent antigen and CD1d molecule.a-GalCer is the strongest glycolipid of NKT cell with high binding efficacy at present, which can be loaded into the CD1d antigen-binding groove。The CD1d tetramer technology which is established using this glycolipid and CD1d molecule to detect the NKT cell is the authoritative method.The liver is the biggest digestion and metabolism organ, however it is clear now its domain function in the immunology, even some people have taken the liver the independent immune organ, taking participation in immune response; NKT cells in the liver mononuclear cells ratio is 8%-15%. Our test use this special quality considering the ratio of the NKT cell in the liver, to evaluate the different experimental condition when establishment of the CD1d tetramer process,to carries on the optimization to its preparation condition.Tween-20 is commonly used laboratory organic flux, is the oil-in-water emulsion medicinal preparation, may serve as the solubilizing agent,stabilizer, lubricant and antistatic agent and so on. CD1d tetramer in preparation process including the Tween-20 PBS dispersion antigen whether to establish successful tetramer, but it can also have the adverse impact on the reacting system, thus while guaranteeing its dispersion effect should also control Tween-20 to find an ideal balance point in the entire reacting system quantity.a-GalCer and the fusion protein's bath time is rate-limiting step in the CD1d tetramer preparation process。nowadays bath time is all over 12 hours, this has limited the CD1d tetramer technology for fast application.This research establishes the CD1d tetramer technology to be use in the detection for iNKT cell in mouse thymus, spleen and liver. Analyzing the Tween-20 concentration in PBS and a-GalCer fusion protein bath time, hoping for it applying least Tween-20 and shortest bath time achieves the goal of good stable detection effect.MethodsTaking liver mononuclear cells as the examination object, using established CD1d tetramer involving Tween-20 different concentration in PBS to examine NKT ratio, through contrasting efficiency difference determines the most reasonable Tween-20 concentration.Taking the liver mononuclear cell as the examination object, using established CD1d tetramer involving different bath time of the glycolipid and the CD1d-IgG1 Fc fusion protein to carry on the detection for NKT, through contrasting different CD1d tetramer efficiency difference, determines the most reasonable bath time.ResultDetection of liver mononuclear cells of NKT cells With different concentration of Tween-20 PBS (0.00%,0.05%,0.10%,0.25%,0.50%,1.00%,2.00%) prepared process.Its detection efficiency in the 0.5 to 2%range increase, but the measured ratio of NKT cells decreased slightly when Tween-20 in 0.5,1%,2%.the detection ability of the three No statistically significant difference was found.0.5%Tween-20 can be used in prepare process and the detection ability of CD1d tetramer is good。The CD1d tetramer detection involving different a-GalCer and CD1d-IgG1 Fc fusion protein loading time (1h,2h,4h,6h,8h,12h and 24h) is carried on for the comparison to its detection efficiency. The data indicated that along with the loading time in 1h,2h,4h,6h and in the 8h scope continually increases, the NKT cell ratio continually to increase; In 8h,12h and in the 24h scope increases, NKT cell ratio to having the declining trend or the ratio slightly quite,。loading time 8h,12h and 24h is non-statistics difference, demonstrated loading time 8h can prepare the ability completely CD1d tetramer.ConclusionHas established the CD1d tetramer detection technology, and its examination efficiency is stable, reliable. CD1d tetramer detection efficiency and Tween-20 concentration have the close connection, based on this its examination efficiency with its concentration present related tendency, but after surpassing 0.5%, the efficiency do not have the difference. CD1d tetramer examination efficiency and the bath time of a-GalCer and CD1d-IgG1 Fc fusion protein to have the close connection。The examination efficiency and the bath time present related tendency, but after surpassing 8h, efficiency do not have the difference.