| CD14 is one of specific receptors for bacterial lipopolysaccharide (LPS) with high affinity. In addition to LPS, CD 14 has been reported to be receptors for a wide variety of bacterial products, such as peptidoglycan (PGN), lipoteichoic acid (LTA), lipoprotein (BLP), etc. Thus, CD 14 is an important component of the innate immune system and can be defined as pattern recognition receptor. CD 14 exists as both a cell surface receptor and soluble molecule found in normal blood serum, spinal fluid, etc. The level of sCD14 increase under some pathological conditions like infection, poly trauma, burns, and also SLE, etc. The increasing of sCD14 in serum is correlated to the development of some diseases. Now, sCD14 has been used as a marker for activation of monocyte/macrophage in monitoring the development of diseases. Less research on sCD14 has been done and no sCD14 ELISA Kit has been reported to be established up to now in China, while the Kit from US or Germany companies is too expensive to be widely used. For these reasons, we established a sandwich ELISA for the determination of soluble CD 14, which would be helpful to the research on sCD14 in China.A large amount of purified CD 14 protein was needed to prepare polyclonal and monoclonal antibodies which can recognize different epitopes of CD 14, and to study the relationship between CD 14 and Toll like receptors, and their roles in anti-infection immunity. Consequently, we purified native sCD14 from human plasma and expressed recombinant human CD 14 in Escherichia coli and Pichiapastoris. Our research can be divided into two parts: Part I Establishment of a sandwich ELISA for soluble CD14:Firstly, monoclonal and polyclonal antibodies to CD 14 were preparedand purified respectively. Ascitic fluid was purified with HiTrap protein G and 15mg purified MAb 60bca was obtained. Polyclonal antibody to CD 14 was got by immunizing rabbit with human monocyte transfected with human CD14 gene (THP1-CD14).Secondly, sandwich ELISA for the determination of soluble CD 14 was established using the antibodies described as above. The concentration of the capture mAb, polyclonal antibody and HRP-labeled goat-anti-rabbit IgG were determined. Serum from many healthy people was mixed together, which was used as the standard serum for ELISA system.Our kit has proven to be corresponding to that of foreign. The characteristic of the kit is high in specificity and sensitivity. The lowest detectable level is 5ng/ml, the range of detection is 5ng/ml~120ng/ml. The assay can be well repeated. The variation range is between 3.27%~9.72% and 6.7%~13.8% respectively in intra and inter assay. The level of sCD14 determined by our system is similar to that of IBL Kit.The serum level of sCD14 from 40 graduate students, 114 healthy old people, 100 patients of neuro-internal medicine and 300 patients with cardiovascular diseases were tested. The data shows that the concentration of serum sCD14 of normal people vary among different age groups. Compared with normal people, the level of serum sCD14 of patients with cerebral infarction or ACS increased significantly. Our research provides important evidence for further study in the mechanism of cerebral infarction and ACS. Part II Production of purified human CD14 proteinTo obtain native purified CD 14 protein, we separated sCD14 from human plasma using immune-affinity chromatography. The sCD14 affinity column was prepared by coupling purified mAb 60bca with CNBr-activated sepharose 4B, by which we got 400ug purified sCD14 from 200ml plasma.Although native sCD14 has more advantages on structure andbiological activities, the concentration is very low in human serum. So therecombinant CD 14 (rsCD14) had also been produced using expression systems of Escherichia coli and Pichiapastoris.Total RNA was isolated from THP1-CD14, and the gene of human CD 14 was obtained by RT-PCR. The sequence of PCR product is identical to that of CD 14 searched on GenBank. The products of the PCR were subcloned into pET28b and pPICZaA ve...
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