| Medical immune analysis consists of humoral immunity and cellular immunity analysis of two most analysis. Humoral immune analysis is a toxic substance, free in the host extracellular antigen and its determination in body fluids, such as viruses, bacteria; cell immune analysis is the determination of the cell membrane surface specific protein or invading into the host cell, the virus intracellular bacteria or foreign organizations, cancerous cells. Immune analysis requires high affinity, high specificity and high sensitivity detection. In order to achieve this goal, this paper study on the preparation of monoclonal antibodies with high affinity and specificity for cell fusion technique, marking with fluorescent nanoparticles with high quantum yield, and fluorescence microscopy imaging; laser induced fluorescence array analysis and chemiluminescence array analysis in detecting. Nanoparticle immune sensor for the above research preparation has been successfully used for analysis of cell immune colon cancer,7721liver cancer cells and HHCC tumor cells, for sensitive detection of these cancer cells, with the early diagnosis of cancer cell flow cytometry. The specific protein with two different sites identification of high affinity monoclonal antibody was prepared by the cell fusion technique. The high sensitivity of Staphylococcus aureus enterotoxin C1and cystatin C and high specificity immune analysis were devoloped. Specifically described as follows:(1) A new kind of ultrabright fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle (FCMSN) is reported. A luminescent dye, Rhodamine6G or tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), is doped inside nanochannels of a silica matrix. The hydrophobic groups in the silica matrix avoid the leakage of dye from open channels. The amines groups on the surface of the FCMSN improve the modification performance of the nanoparticle. Because the nanochannels are isolated by a network skeleton of silica, fluorescence quenching based on the inner filter effect of the fluorescent dyes immobilized in nanochannels is weakened effectively. The Quantum Yield of obtained90nm silica particles was about61%. Compared with the fluorescent core-shell nanoparticle, the chemiluminescence reagents can freely enter the nanoparticles to react with fluorescent dyes to create chemiluminescence. The results show that the FCMSN are both fluorescent labels and chemiluminescent labels. In biological applications, the NaIO4oxidation method was proven to be superior to the glutaraldehyde method. The amount of amino could affect the specificity of the FCMSN. The fluorescence microscopy imaging demonstrated that the FCMSN is viable for biological applications.(2) A novel method for sensitive detection of liver cancer cells using anti-CD155and anti-CD112monoclonal antibodies conjugated ultrabright fluorescent mesoporous silica nanoparticle (FCMSN) encapsulating Rhodamine6G and fluorescein has been developed. The diameter of obtained nanoparticle was90nm. The quantum yield of FCMSN was69%. Because the emission of fluorescein has a high degree of overlap with excitation of Rhodamine6G, and the two kinds of dyes are close enough in the nanoparticle, the fluorescence resonance energy transfer occurs between two kinds of dyes. It not only maintains the original feature which the nanochannels and network skeleton of silica weaken the inner filter of dye, but also makes the excitation peak wider and increases useful load amount of dye. Because the wider stokes shift weaken the interference of excitation, the detecting sensitivity is enhanced at the same time. The NaIO4oxidation method, which didn’t use cross-linker, was used covalent immobilization of monoclonal antibodies on the FCMSN. This method can maintain the activity of the monoclonal antibodies more easily than the glutaraldehyde method. These advantages ensure that nanosensor has high sensitivity and specificity for detecting liver cancer SMMC-7721cell and HHCC cell.(3) A sensitive and selective sensor for detecting colon cancer cells based on nanoparticle covalent modified anti-human epithelial cell adhesion molecule (EpCAM) antibody is developed. The transmission electron microscope (TEM) images showed that the nanoparticle and functionalized nanoparticle had good decentrality for application. The NaIO4oxidation method, which was used oxidizing antibody for immobilization of conjugating antibody on the silica-coated fluorescent nanoparticles, maintained the activities of antibodies very well. The fluorescence microscopy imaging and flow cytometer (FCM) experiments demonstrated that the nanosensor could increase the signal intensity obviously and distinguish three kinds of target cells (colo205, sw480and NCM460) well. The membrane and nuclear staining showed the distribution and abundance of EpCAM in cells’ membrane. It also provides a possibility to quantify special membrane proteins on different regions of cells’surface. At the end, the result of detecting a simple sample proved that colo205cells were selected by anti-EpCAM antibody nanosensors in this environment, and made a good foundation for subsequent research.(4) Sensitive chemiluminescence immunoassay and laser induced fluorescence immunoassay for staphylococcal enterotoxin C1(SEC1) based on the functionalized dye-encapsulated mesoporous silica nanoparticle (FCMSN) as label has been developed. Because of hydrophobic interaction between the dyes and the hydrophobic groups, the dyes are fixed in the MSN’s matrix effectively. The amines groups on the surface of the FCMSN improve the labeling properties of the nanoparticles. In the sandwich immunoassay, the capturing monoclonal antibody (G8) and the detecting monoclonal antibody (C4) were used. In chemiluminescence detection steps, TCPO system was adopted. The results showed that the functionalized MSN played the role of signal amplification effectively. The range of detecting SEC1was0.025-2ng/mL with a detection limit of0.019ng/ml (3σ). The regression equation of the working curve is I=3O27.5[SEC1](ng/mL)+1804.6(R2=0.9958). The relative standard deviation (R.S.D.) for11parallel measurements of1ng/ml SEC1was4.6%. The performance of laser induced fluorescence immunoassay was0.05-2ng/mL,0.042ng/mL,I=302.5[SEC1](ng/mL)+672.6(R2=0.9908). R.S.D. was5.3%after detecting lng/mL SEC1(N=11). Furthermore, fully automated chemiluminescence analyzer improves the detection efficiency. The results demonstrated that these methods offer potential advantages of sensitivity, simplicity and rapidity for detecting SEC1.(5) Cystatin C is a significant cysteine protease inhibitor in human bodies, and is proposed as a fascinating novel marker of glomerular filtration rate for kidney injury detection. In this article, we report an ultra-sensitive, simple, rapid chemiluminescence immunoassays method for cystatin C detection using functionalized MSN. After a three step hydrolysis, the amino-functionalized MSN encapsulating dye has hydrophobic environment for fixing dye and amino groups for biologic modification. The NaIO4immobilization method maintains the activity of antibody very well. The sandwich immunoassay using two monoclonal antibodies was chosen for its selectivity. The analysis showed a linear response within the range of0.0025-0.5ng/mL (R2=0.9936). The relative standard deviation (R.S.D.) for11parallel measurements of0.25ng/mL CysC was4.2%. The automated chemiluminescence analyzer can detect96well continuously. The results proved that this method is ultra-sensitive, simple, and rapid for detecting cystatin C... |
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