| Hematopoietic malignancy is one of the refractory diseases in the world. Human leukocyte cluster differentiation (CD) antigen analysis will facilitate the diagnosis and treatment of this tumor. Production of monoclonal antibody (McAb) against certain unknown antigens on the surface of the tumor cells might explore the application of a new area or even a new therapy. ZCH-2B8a (IgG2aK) is a novel McAb generated in our laboratory recently using human myeloblastic leukemia cell line KG la as immunogen. This antibody has been submitted to the 8th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the feedback results showed that the antibody recognized an unknown molecule on the surface of some blood cells, belonging to a new CD molecule. Illustration of the biological features of the antigen recognized by the antibody and the cloning and sequencing of the encoding gene may confer an important significance and application potential.1 The preparation and charaterzation of fluorescein isothiocyanateconjugated 2B8a antibody (2B8a-FITC)In order to further study the function of the 2B8aAg analysed by using flow cytometry (FCM), the directly labeled antibody of 2B8a i.e. FITC-2B8a, is the first and the important step. Antibody source was derived from the ascites generated in the Balb/c mice by inoculating 1 x 10~6 2B8a hybridoma cells into peritoneum. The antibody was purified by SPA-affinity colume and the quality of the antibody purified was characterized by SDS-PAGE showing 99% purity of the antibody. The purified 2B8aAb was directly conjugated with FITC dye according to the modified Marsshall method. The free FITC was removed by dialysis with large quantity of phosphate buffered saline (PBS). The quality of the fluorescent antibody 2B8a-FITC was determined by the optical density (OD) absorbance ratio of A295/A280 with a spectrophotometer. The OD ratio determined was 0.56, within the normal range of standard reagent. FCM analysis showed that the reactivity of 2B8a-FITC by direct staining with Raji cells was similar to that of 2B8a by indirectstainging with a second antibody of FITC conjugated goat-anti-mouse immunoglobulin (GAM-FITC). The shape of histograms looked similar and the percentage of positive cells was close (98.11 % vs 90.77%). The reactive intensity in mean fluorescence intensity (MFI, 62.88) of 2B8aAb by direct staining was slightly weaker than that by indirect staining via GAM-FITC (MFI = 103.55), which was as usual. Those results indicated that the fluorescent antibody 2B8a-FITC was successfully prepared.2 The characteristics of antigen recognized by antibody ZCH-2B8aIn order to demonstrate the function and its clinical application potential of the antibody 2B8a, the preliminary biological characteristics of the antigen recognized by the antibody was investigated. (1) The reactivities of the antibody 2B8a with normal and malignant cells:Using a multi-parameter flow cytometry (FCM), the reactivities of the antibody ZCH-2B8a were investigated with normal blood cell components including T, B, natural killer (NK), neutrophils, monocytes, dendritic cells (DC), red blood cells (RBC), platelet (Pit) and the hematopoietic stem/progenitor cells derived from either bone marrow or G-CSF mobilized peripheral blood CD34+ cells, malignant cell lines including 14 hematopoietic, 5 neuroblastoma, 1 colon cancer and 1 amniotic epithelium cell lines, and the cells from 100 newly diagnosed leukemia patients. 2B8a antibody reacted with 3/3 specimens of blood B cells with a positive rate of 26.29% and 2/3 specimens of monocytes with an average positive rate of 59.84%. 2B8a was weakly reactive with neutrophils (23.72%) and negative for T cells, NK cells, DC, RBC and Pit. The antibody reacted with the CD34+ cells from 3 marrow samples with an average positive rate of 39.33% while it was negative for the CD34+ cells derived from 3 samples of G-CSF mobilized CD34+ peripheral blood stem/progenitor cells (PBSC, 1.25%). Cell line analysis showed that the antibody was brightly reactive with 3/4 B lineage cell lines (Raji, SMS-SB, Nalm-6) with the positive rates of 98.78%, 98.61% and 94.93%, respectively, and negative for NALL-1, which was only 5.68%. It reacted with monoblastic cell line (U937, 67.78%) while it was only weakly positive or negative for other myeloid leukemia cell lines including MegOl (33.40%), HL60 (29.70%), K562 (28.19%), KGla (16.23 %) and HEL92.1.7 (8.02%). Among 4 T lineage leukemia, 5 neuroblastoma and 1 colon cancer cell lines tested, only Molt-3 was found weakly positive (31.40%) for 2B8a, while the remaining 3 T cell lines (Molt4, JM and CCRF-CEM), 5 neuroblastoma cell lines (LA-N1, KCNR, BE, SK-N-SH,SK-N-AS) and the colon cancer cell line (HR8348) tested were negative. An amniotic epithelium cell line (FL) was showed positive for the antibody (45.03%). ZCH-2B8a primarily reacted with lymphoid leukemias, of which acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL) accounted for 9/31 and 2/5, respectively, no significant difference was found statistically between ALL and CLL (P = 0.8456 > 0.05);2B8a did not react with myeloid leukemias in most cases, with only 3/57 of acute myeloid leukemia (AML) and 0/5 of chronic myelogeneous leukemia (CML) cases, in which no significant difference -between AML and CML, however, significant difference between myeloid and lymphoid leukemias (yf =10.2907, P<0.01) was found;In ALL, 2B8a only reacted with B lineage (9/28), while not with T and NK lineage leukemias. In B lineage leukemia, early Pre-B ALL was the most reactive type of disease, positive in 3/5 of cases, significantly higher than cALL cases (5/21, P=0.0173<0.05) with declining expression pattern with cell maturation. There was no reactivity in mature B-ALL. Conclusions: 2B8a antibody primarily reacts with B cells, neutrophils and monocytes in peripheral blood, and hematopoietic stem/progenitor cells derived from bone marrow CD34+ cells. B lineage and monocytic lineage malignant cells are positive for the antibody, which indicates its clinical application potentials.(2) Trypsin digestion analysis of the antigen:In order to understand the antigen property, the trypsin digestion analyis was performed. The expression of 2B8aAg on Raji cells was determined by FCM before and after digestion with 0.25% trypsin. The expression of 2B8a antigen before trypsin treatment (83.57%) on Raji cells was significantly reduced after trypsin treatment (26.99%), which indicated that 2B8aAb recognized a protein antigen sensitive to trypsin digestion.(3) Influence of 2B8aAb block on Nalm-6 cell growth:After Nalm-6 cells were blocked by appropriate 2B8a antibody (culture supernatant) , they were cultured in RPMI 1640 supplemented with 20% of heat-inactivated calf serum and the the cell numbers were enumerated on 24h, 48h, 72h and 96h of culture. The results showed that no significant difference was found in comparison with the control group, which indicated that no influence of 2B8aAb on Nalm-6 cell growth could be found.(4) Comparison of 2B8a expression between multidrug-resistent (MDR) cell lines and their non-MDR parental cell lines:In order to illustrate the expression difference between MDR and non-MDR cell lines, two groups of MDR leukemia cell lines including HL60 and its vincristine-resistentcounterpart HL60/VCR, K562 and its MDR counterparts including K562/ADR (adrimycin resiostent), K562/HHT (homoharringtonine resistent), K562/VCR (vincristine-resistent) were investigated by FCM. The result showed: in the group of HL60 and HL60/VCR, 2B8aAb not only reacted with HL60 (29.64%), but also with HL60/VCR (47.70 %).There was no signification^=0.343>0.05) between HL60 and HL60/VCR by nonparametric test;But in the other group of MDR cell lines, 2B8aAg expressed on K562 (43.06%) cells while hardly express on its MDR counterparts(K562/HHT 17.76%,K562/ADR 3.53 %and K562/VCR 4.44%). There were notable signification between K562 and K562/ADR(/> = 0.029<0.05) . K562 and K562/VCR(/> =0.029<0.05)B\A no signification existed between K562 and K562/HHT(P =0.114>0.05). These results indicated that 2B8aAg perhaps had relation with MDR.But further study was needed for its mechanism.3 Cloning and sequencing of the coding gene of 2B8a antigenThe preliminary studies have shown that 2B8aAg was a presumble protein. A gene encoding the protein antigen must exist. In order to clone the target gene, a T7 phage display system was used to establish a phage display library using whole Raji cell cDNA since 2B8aAb reacted with more than 98% of Raji cells. The establishment of the phage display library was carried out according to the method described by the manufacturing company. Briefly, 1x10 growing Raji cells in logarithmic growth state were collected. The total RNA was extracted using TRIZOL reagent. Then mRNA was puried with PolyATtract? mRNA Isolation System perchased from Promega company according to the method provided by the manufacturing company. cDNA was synthesized by MMLV reverse transcriptase (RT) provided in the kit. The concentration and purity of the nucleic acid were determined by spectrophotometer and 1% agarose gel electrophoresis analysis, respectively. The cDNA was then treated with T4 DNA polymerase to modify the ends of the cDNAs and ligated with directional EcoR I/Hind III Linkers. Following linker ligation, the cDNAs were digested sequentially with Hind III and EcoR I. The cDNAs were then passed through a small gel filtration column to remove excess linkers and cDNA products smaller than 300 bps, which were then ready for insertion into EcoR I/Hind III digested vector arms. After ligation of synthesized cDNA inserts and T7 phage vector arms, the ligation products was added directly to T7 Packaging Extracts for in vitro packaging. The establisment of the cDNA library was accomplished after in vitro packaging. The primary capacity of the library was 6x10 determined by plaque assay. In order to characterize thequality of library established, the inserted genes from 10 randomized plaques were analysed by picking monoplaque and PCR amplification using the primers provided by the kit. 6/10 plaques analysed were found to have inserts = 500bps, which implied that the establishement of the library was successful. Duo to the low titeration of the primary library, a further amplification of the library was performed. The titeration increased to 2.5><1O10 pfu/ml after amplification. Screening of the library by biopanning using McAb 2B8a was performed. After five rounds of biopanning, the maximum enrichment for phages that bind to the McAb 2B8a was reached. Then a"scrape" from an individual plaque yields phage DNA for PCR. Another 10 plaques were analysed after biopanning, of . which 6 positive clones were obtained by PCR amplification. All the 6 positive bands were sent for sequencing and the sequences obtained were analysed through BLASTn in the gene bank. Five inserted gene sequences were found to be in highly homologous, most probably was the hypothetical gene (BC094882) in the gene bank. This gene expresses in normal germinal center B cells of lymph nodes. The size of the gene is 2436bp and the open read frame is on 61~2280bps encoding a protein of 739aa. The researchers from the Mammalian Gene Collection Program Team recently submitted the gene to the gene bank on May 6, 2005 when they finished more than 15,000 full-length human and mouse cDNA sequences. After the gene sequences of the five inserts were converted into the amino acid sequences by DNASIS software, the amino acid sequence of our presumable antigen gene was exactly the same as the 228aa sequence started from the stop codon of the carboxylic end of the protein encoded by the hypothetical gene. The expected sequence from the 229th aa - 236th aa from the carboxylic end sequenced by this study was entirely different. In conclusion, the 2B8a antigen-coding gene is probably the BC094882-like gene, yet it needs a further confirmation.
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