Development Of The Amplified Luminescent Proximity Homogeneous Immunoassay For The Interaction Of Protein And Pathways

JAK (Janus kinase) is a class of non-receptor tyrosine kinase (PTK), JAK protein family consists of four members:JAK1, JAK2, JAK3 and TYK2. JAK proteins family has 7 homology domains in structure (JAK homology domain, JH), respectively domain JH1-JH7. Each domain has a different function, JH1 domain is the kinase region, domain JH2 is the false kinase, and the domains JH6 and JH7 are receptor regions. JAK family is an important signal sensor of cytokines, growth factors and interferon, takes part in the development of many diseases in human. The main pathway is, through the corresponding receptors recongnite cytokines, growth factors and other factors, then JAK was activated, the activated JAK promoted the activation of downstream signals, thereby generating a series of reactions. Different members of the JAK family involved in different types of cytokine signal tanceductin. JAK1 is one of the important members of the JAK family. It is protein molecular weight is of about 130kD, is widely expressed in tissues cells.The present study has demonstrated that many signal transductions of cytokines in the body need JAK1 protein. JAK1 gene mutations and its excess expressing in cells plays an important role in the medical field, particularly in tumors. According to the research results we can find that in many cytokines signal transduction in the body need to be involved in protein JAK1 JAK1 gene mutations and its excess expressing in cells plays an important role in the medical field, particularly in tumors. JAK-STAT pathway, most importantly, involved in a large number of cytokines and growth factor signaling. Including GH (growth hormone), GM-CSF (granulocyte/ macrophage colony stimulating factor), EGF (epidermal growth factor), and so on. These cytokines and growth factors in the membrane have corresponding receptors, these receptors t themselves do no have kinase activity, through JAK binding sites in vivo., enabling signals from the extracellular to intracellular. via JAK activation Meanwhile JAK-STAT pathway is involved in vivo cell proliferation, cell differentiation, apoptosis, cell survival and immune regulation process, the pathway is one of the ubiquitous signaling pathway. Therefore, the detection of JAK protein and the interactions in protein pathway are especially important.At present, the technology of yeast two-hybrid, phage display, GST pull down, tandem affinity purification, co-immunoprecipitation has developed for protein-protein interaction. Those traditional testing technology has a lot of insufficient place in the application. Yeast two-hybrid technology can’t detect the interaction, which happen in the cytoplasm, the cell membrane and extracellular proteins; GST pull-down cannot be used for a large-scale screening for protein-protein interaction, and it may have a false positive easily by interference of endogenous proteins; The label of tandem affinity purification technology will impact the structure of proteins sometimes, and expression of fusion proteins may lead to false positives; Co-immunoprecipitation, with a low sensitivity, can’t detect the interaction with a low affinity and momentary interaction of proteins, besides, the preparation of antibody is not easy.Tag immunology, combining immunology and marking technology for the analysis of the determination, is a subject with concentration of experimental technology, clinical application, and foundational medicine. The fundamental of tag immunology is that using highly specific reaction of antigen-antibody and the high sensitive of markers to check concentration and activity of various bioactive substances inside the body. With the continuous development of molecular biological and tags technology, tag immunology has been widely used in various fields. Nowadays, technologies of tag immunology include:enzyme immunoassay (EIA), fluorescence immunity analysis (FIA), chemiluminescence immunoassay (CLIA) and radioimmunity analysis (RIA), colloidal gold immunoassay (GICA), time-resolved fluorescence immunoassay (TRFIA), etc. There are some disadvantages in the application for those traditional tag technologies. EIA, with easy deactivation of enzyme and impacting spatial structure of marks by enzyme, can’t improve its sensitivity effectively; TRFIA has a complex operation, and it is easy to cause the pollution of environment and the test samples, which may lead to the background interference of measurement; CLIA, with a short light-emitting time and expensive cost, is susceptible to be interfered by operating environment, and the sample is just tested only once instead of duplicate detection; Additionally, these technologies mostly use microwell plate as reaction carrier, which need to be washed repeatedly to separate the free molecular and targeted molecular. The disadvantages of tedious operation process, long reaction time, high consumption of the samples and easy pollution also can’t be ignored. As a result, a technique with a large-scale use, high throughput, general type, quickly detection and accurately identification of the authenticity is desperately needed.Amplified luminescent proximity homogeneous immunoassay (AlphaLISA) technology as a new immunoassay technology is introduced. The luminescent oxygen channeling assay (LOCI) is a homogeneous immunoassay that uses an excited and reactive form of singlet oxygen, which is produced from ambient O2 in solution, for energy and as a signal transmitter. And relies on PerkinElmer’s exclusive AlphaScreen(?). It has singlet oxygen nano-materials, laser technology and short half-life and other advantages, to achieve the operation in a homogeneous reaction. Also it overcomes the drawback of requiring washing and isolating the free labeled binding tag. AlphaLISA has many advantages, for example, low background signal, non-radioactive pollution, simple operation, good reproducibility detected, without washing to get separation of bound and free label tag, high sensitivity, high throughput, ease of automation and so on.To construct a eukaryotic expression vector for nonreceptor tyrosine kinase-JAKl and STAT3 gene and express in 293 T cells. The study optimized the system of AlphaLISA technology. Finally AlphaLISA detected the interactions of JAK1 protein and protein were detected path, which laid a foundation of further research on protein-protein interaction of JAK1.Method:Total RNA was extracted from Hela cells, with which JAK1 gene was amplified by RT-PCR and inserted into eukaryotic expression vector pcDNA4.0-His.293T cells were transfected with the constructed recombinant plasmid penter-JAKl, observed by fluorescent microscopy 48h later, and determined for expression level of JAK1 by Western blot 24h,36h and 48h later.2 In this study, AlphaLISA kits were developed for detecting AFP using antibody-sandwich method.2.1 Calibrations:The calibrator series were carried out by diluting AFP antigen in the buffer. The desired standard concentrations for AFP were 0,5,20,125,250 and 500 U/L. The standards prepared were lyophilized according to 1 mL aliquot and stored at 4℃ until used.2.2 Coupling of antibody to microspheres:0.2 mg of AFP antibody was added to microspincolumn, and was centrifuged for 6 min at 8000g. Then, the antibody was washed about six times using 0.1 mol/L MES buffer (pH 5.0). The solution was added to 1 mg acceptor microspheres, and than added 1OμL of 25 mg/mL NaBH3CN (confected of MES) and 1.25μL of 10% Tween-20.The solution incubated at 37℃ for 48 hours in dark. The total volume of the reaction solution was 200μL. The fresh carboxy-methoxyl amine (CMO) solution (65 mg/mL) was confected in a 0.8 M NaOH to block nonconjugated sites.10 μL of CMO solution was added to the reaction incubated for one hour at 37℃ in dark. The reaction solution was centrifuged and the supernatant was removed. After the last centrifugation, the microspheres were keep in storage buffer (200 μL of PBS+0.05% Proclin-300) at the concentration of 5 mg/mL.2.3 Coupling of antibody to Biotiny:1 mg of antibody was added to microspincolumn, and was centrifuged for 6 min at 8000 g. Then, the antibody was washed about six times using 0.1 mol/L, pH 9.5 Na2CO3/NaHCCO3 repeatedly. NHS-D-Biotin was dissolved in DMSO immediately to use in dark place at a concentration of 22 mg/mL. A volume equal to 10% of the total volume of the AFP antibody solution was added into the Biotin solution in portions to the AFP antibody solution with gentle stirring, and than incubated at room temperature for 4 hours. The unreacted Biotin was filtered with microspincolumn. The biotinylated AFP antibody was stored (0.5 mg/mL) at-20℃.2.4 The optimization of the reation time of AlphaLISA.The reaction time is set gradient 1 Omin,15min,30min to establish the best reaction time2.5 The optimization of the coverage of antibody and beads.Holding the microspheres manual dosage 1mg, connect the AFP antibody prepared a series of dosage 200μg,150μg,125μg, 100μg. Twenty-five microliters of diluted biotin-QPs suspension and 175μL of the streptavidin-coated donor-beads were added into the wells in the dark. Additionally, wells were prepared with phthalocyanine (3 ng/well) in place of the donor-beads. The plates were incubated for 15 min with shaking at 37℃ in the dark. The fluorescence intensity was measured on the EnSpire(?)multimode plate reader. Keeping the coverage of AFP antibody in an amount of 125μg, the receptor microsphere preparation into a series of dosage lmg,800μg, 600μg,400μg respectively, which is in accordance receptor antibody microsphere method to connect. Microspheres and the AFP antibodies while the amount down to 1/5 of before, that the coverage of the microspheres is 200μg, and the AFP antibody dosage is 25μg. Also set the AFP antibody coverage of 30μg as a control group.2.6 The optimization of the coverage of antibody to biotin.In order to reduce the coverage of antibodies connection biotin, biotinylated antibody we will simultaneously decrease the coverage of the antibody and beads to establish an optimum coverage of biotinylated antibody.2.7 The optimization of reacte bufferIn order to choose the best reaction liquid environment, we comparied with PBS and analyse buffer to establish the optimum reaction buffer.3 The detection of protein interactionUnder the optimum conditions of AlphaLISA system JAK1 antibodies are connected with the photosensitive microspheres, while the GHR, IL4R, CSF3R, IL10RA, IL2RB, ABL1 antibodies attached to the light emitting microspheres. Check the interaction of protein JAK1 and protein GHR, IL4R, CSF3R, IL10RA, IL2RB, ABL1. JAK1 antibodies are connected with the photosensitive microspheres, while the STAT3 antibody attached to the light microspheres to detect interacting proteins of JAK1 and STAT3 protein.JAK2 antibody attached to biotin, check the protein interaction detection of JAK2 and STAT3 protein.Result:1 Restriction analysis and sequencing proved that penter-JAK1 and penter-STAT3 was a correct recombinant plasmid. Green fluorescence was observed in 293T cells 48h after transfection with the plasmid. The target protein band by Western blot. Best transfection reagent is used in an amount transfection reagents is 3:1, and the best transfection time is 48h.2 AFP target is used to establish AlphaLISA system and optimized.The results showed that 15min reaction time means the strongest fluorescence; The fluorescence of the coverage of antibody linked microspheres down to 25μg connected 200μg receptor microspheres is similary with the fluorescence of the 200μg antibodies linked by lmg microspheres; the coverage of antibody linked biotin down to 25μg.The fluorescence of analysis buffer as reaction buffer was significantly higher than PBS.3 Under the optimum conditions of AlphaLISA system. We can check the interaction of protein JAK1 and protein GHR, IL4R, CSF3R, IL10RA, IL2RB, ABL1.And to detect interacting proteins of JAK1 and STAT3 protein.Also check the protein interaction detection of JAK2 and STAT3 protein.Conclusion:Eukaryotic expression vector penter-JAK1 and penter-STAT3 was successfully constructed and expressed effectively in 293T cells, which laid a foundation of further research on protein-protein interaction of JAK1. Best transfection reagent is used in an amount transfection reagents is 3:1, and the best transfection time is 48h. It is successful to use AFP target to establish AlphaLISA system, and than that 15min is the optimized reaction time. Using 25μg antibody linked 200μg microspheres was successfully. The optimized coverage of antibody linked biotin was 25μg. The analysis buffer was the optimized reaction buffer. AlphaLISA could used check the interaction of protein JAK1 and protein STAT3, GHR, IL4R, CSF3R, IL10RA, IL2RB, ABL1. Also check the interaction of protein STAT3 and protein JAK2.