Selection And Identification Of Human Phage Single Chain Antibody Variable Fragment

The use of monoclonal antibodies (mAbs) by hybridma fusion as therapeutic agents is gaining importance in the prevention, diagnosis and treatment of various diseases. But it is severely limited in their clinical applications. Phage display technology , which is an important tool for the high affinity Antibody, can make humanization antibody without immunization. This kind of antibody can avoid the side-effect caused by human antimouse antibody (HAMA). New selection system must be adopted in the antigens can not be purified or the unknown tumor antigens. The ability of phage displaying antibody to undergo receptor-mediated internalization indicates that phage antibody libraries might be selected not only for their cell binding but also for their internalization into mammalian cells. So internalization selection strategy had beenadopted in the study. Objective:During the selection of phage antibody, many times of PCR reaction will be used to identify the existence of the insert genes. The bacterium colonies PCR methods were adopted in the screening of phage antibody, and its values were studied. The binding vivid of the selected phage antibody needs to be identified by whole cell ELISA. The reaction conditions between the antigens and the antibody in the ELISA should be optimized to built steady cell ELISA in the phage antibody selection. The selection conditions including washing solution, elution solution, blocking solution, selection volume and the cell state were optimized to reduce the non-specific phage antibody, and to avoid the loss of the specific phage antibody. The technology of recombinant in cells was used to exploit a second library to increase the number and diversity of phage antibody. To select the internalizing antibody, internalization selection strategy had been adopted in the study. Fingerprint analysis of the phage DNA were studied. The specificity of the ScFv to hepatocellular cells and its site of the antigen binding ScFv were also studied. Methods:This paper were divided into 5 parts.1 Bacterium colonies PCR methods applied to the screening of phage antibody libraryFive positive monoclonal bacterium were acted as templates in PCR after they were suspended in double distilled H20 or suspendedin 0.1% Triton X-100 and then boiled. Contemporarily their DNAs were extracted and digested by two enzymes.2 Whole cell ELISA in the selection of the phage antibodyDifferent numbers of SMMC-7721 cells about 1X10% 2X104, 3 X104and 5X104were planted in each well to study the effect on the ELISA. The polycolonalScFvs diluted in l:10> l:20> l:40> l:80> 1:100 were used in reaction to determined the effect of the dilution rate on ELISA. The 3 kinds of reaction conditions including A: 37°C, lh, B: room temperature, 3h and C: 4°C overnight of the first antibody were tested to study the effect on the ELISA. The second antibody diluted in different rate were tested to study the effect on the ELISA.3 Optimising the selection of ScFv on cells and the pre-selection of the phage libraryThe phage antibodies preparing freshly infected the bacteria TGI grown at OD6Oonm 0. 5 to study the effect of preserving condition on the infecting vivid with those preserved in 4°C,-20°C respectively. The blocking buffer of 10%NCS> 1%BSA and 4% no-fat milk powder were used in the selection precess respectively to study blocking value on non-specific phage antibody. The phage antibodies preparing freshly of 1 mL(1012cfu) were selected in different volumes of 5mL> IOitiLn 15mL to study the effect of selection volume on the results. The cells were selected in fixed state or suspension respectively to study the effect of cell state on the selection results. ScFv were selected to hepatocellular cells for four rounds, then bacterium colonies PCR and enzymes digestiondetermining the inserted genes. The positive colones by PCR were detected by ELISA to determine the combining vivid.4 The selection of ScFv to hepatocellular cells by recombination in vivo.The phage antibodies from primary library preparing freshly of 8X 1012cfu were added in lOOmL BS1365 bacteria grown at OD60o? 0. 5. Phagemid were left for lh without shaking at 37 "C to allow infection. Ampicillin were added, recombination was allowed to continue by shaking overnight growth at 30°C. Human phage ScFvs were selected by internalization using hepatocellular cells. In the first 2 rounds, SMMC-7721 were used directly in selection to avoid the loss of rare ScFvs, and from the third round, the normal human fifroblast cell line and liver cell line were used to absorb the non-specific phage antibody, then the SMMC-7721 were used by internalization. Bacterium colonies PCR and enzymes digestion determined the inserted genes. The positive colones by PCR were processed by ELISA to detect the combining vivid. Cell ELISA were processed once more to detect the specificity to hepatocellular cells using human liver cell line L-02 as antigen. Fingerprint analysis of the colones gained ScFvs were detected .5 Sequencing the DNA of the ScFv and analysis of the specificity to hepatocellular cellsThe VH and VL sequence of the ScFv D5 were compared with the Kabat debase using the software of BLAST, DNA club and DNAssist. The genes were searched in the Ig debase. Cells of SMMC—7721 and L —02 were collected respectively. Then they were detected by FCMafter the ScFv and the antibody of sheep anti-M13 and the antibody of FITC conjugated anti-sheep were added. Cells of SMMC-7721 were detected by Immunofluorescence assay and Laser confocal scanning microscope after they were reacted with antibody above. Results:1 In the PCR reaction the bacterium were acted as templates in PCR. The time of initial denaturation was prolonged to 5 mins. This made the bacteria crack sufficiently, and the DNA plasmid were released and denatured. The results were ideal. The identification of PCR productand enaymes digest proved the right results. The positive clones were contemporarily banked and preserved while the PCR temples were made.2 The experiment of cell ELISA determined the most appropriate cell number is 2 X 10" in each well of a 96-well plates. The polycolonal antibodies diluted 1:40 were proper. The reaction conditions of the first antibody were 4°C overnight. The second antibody diluted by 1:2500 is the most appropriate.3 The phage antibody preserved in 4°C had lower infecting vivid, while the phage antibody freshly prepared had the highest infecting vivid. The infecting vivid of the phage antibody preserved in -20 °C had no obviously decreasing. Several other factors proved that 4% no-fat milk powder can reduce non-specific phage antibody obviously, a relatively small volume had the best selection results, the fixed cells is better to the suspended cells as antigen. The pre-selection by the optimized selection conditions proved that the specific phage antibody had been enriched. Identification byPCR and enzymes digest found no loss of insert clones.4 The technology of recombinant in cells was used to exploit a second library which had 6. 7X1011 phage antibody number. The rate of phage with insert genes increased to 100% from 80% before selection. The analysis by ELISA to the positive clones by PCR found that 92% clones can bind to SMMC—7721. The three clones named D5.D23, D27 Can bind to SMMC—7721, but not to normal human liver cell line L-02. So these 3 clones can be recognized as phage antibody specific to hepatocellular cells. Fingerprint analysis of the phage DNA suggest that Clone D5 have a similar sequence to the D23, and the Clone D27 had another sequence. The clone D5 having high affinity to hepatocellular will be studied continually.5 The sequence analysis showed that the genes of VH and VL were right. The putative amino acid sequence included four reliable frames and 3 complementarity determinant regions. The VH and the VL also included the sequence of half-cystine. FCM showed that 41. 2% hepatocellular cells can combine to the ScFv while the normal human lilver cell acted as control. Immunofluorescence assay showed that the ScFv can specifically bind to the SMMC-7721, but not bind to the normal liver cell L-02. Laser confocal scanning microscope show that the binding site was on the membrane of the hepatocellular cells.Conclusion:1 The bacteria colony PCR method, which is simple, quick and effective, is valuably extended in largely screening positive recombinant bacterium. The positive clones were contemporarily...