Antibody Microarray Technology And Application In Liver-proteomics

With the genome sequences of several organisms now in public databases, the scientific community has realized that it is time to prepare for the next step: the understanding of biological systems or systems biology. Gene expression microarray technology has exploded in the last five years with completion of the Human Genome Project. Its use has been shown across a wide range of fields including but not limited to biomarker discovery, predicting disease outcomes and response to treatment, assessing coregulation via time course and/or dose-response experiments, and detecting molecular mechanisms and /or pathways associated with a particular disease state. Whereas genes contain the information for life, the encoded proteins and RNAs fulfill nearly all the functions, from replication to regulation. At present, there is a perceived demand for high-throughput and parallel analytical devices as research tools in systems biology, and, in addition, for new concepts to extract knowledge and value from these data. For this reason, a new technology called antibody microarrays has been developed to assess differential expression directly at the protein level. Antibody microarrays can be used for expression profiling of hundreds of thousands of proteins simultaneously, with the goal of identifying disease/protein or protein/protein relationships.This topic is the sub-item of National Key Technologies R&D Program — "The instauration and application of liver-protein antibody database". Its main task is: using the acquired mono-antibody to manufacture the antibody chip that keeping with liver-protein expression chart and function research, investigating the method of raise the labeling efficiency and the chip sensitivity, researching to manufacture the antibody chip and kits, and apply in the liver-protein's researches such as observational measurement, quantitative determination and interaction etc.. In this thesis, spotting concentration, protein immobilization substrates, target labeling and affinity reaction condition were investigated, and the antibody chip of liver-protein researches were applied. The major contributions include the following aspects:1. After pro-10 spot to order, the fluorescence measured value of each protein sample is tend in stability. Then We can spot formally.2. After spotting the fixed time take 24-48h as proper. Both long and short time will cause the fluorescence measured value low.3. It's the best choice using EA solution to remove unbound protein. The best antigen-antibody particularity combines temperature is 37 °C, time is 2h,choosing the 1 mg/ml dose the optimal spotting concentration.4. When spotting concentration was definite, more the labeling target concentration, higher the fluorescence signal. And the target concentration has a liner relationship with the fluorescence signal in 1ng/ml-10ug/ml.5. Apply the antibody chip technique to analyze the person's four kinds of totalprotein (Adult human liver homogenate proteins* Adult human liver plasma proteins, Fetal human liver nuclei proteins, Fetal human liver mitochondrial proteins), make the antibody chip with 32 kinds of mono-antibody and total 432 spots for researching analysis of the liver protein. Get the following initial conclusion: CBR expresses the plentiful degree higher in fetal human liver mitochondrial proteins, lowest in adult human liver plasma proteins. DCXR expresses the plentiful degree higher in adult human liver plasma proteins. ALB is in the different sample(adult human liver or fetal human liver) that same growth expect the expression is tend in stability, but higher in adult human liver than in fetal human liver. TF expresses the plentiful degree higher in adult human liver homogenate proteins. A2M expresses the plentiful degree highest in adult human liver homogenate proteins, lowest in adult human liver plasma proteins.6. Investigating the new method of QD fluorescence marking protein. Using the NHS to dress QD, which can covalence coupling with protein. Turn the functional QD labeling protein to used for the antibody chip, which prove that can be apply in the bio-microarray to detect.7. Proceed from research need to manufacture the eyes-see examination chip, we investigated the gold-paramagnetic microsphere to label protein. It can use for antibody chip detection by the labeling protein. Also the result can be observed by the naked eye. This method is simple, operate in brief.