On-line analysis and control are critical for the optimization of product yields in animal cell culture. The close monitor of viable cell number helps to gain a better insight into the metabolism and to refine culture strategy. In this study, we use the oxygen uptake rate (OUR) to estimate the number of viable cell and the OUR-based feed-back control strategy for nutrients feeding to improve the efficiency of cell culture. A hybridoma cell line (HAb18) was cultured in fed-batch and perfusion model using serum free medium in 5L CelliGen Plus bioreactor (NBS Co., American) and 5L Biostat B bioreactor (Braun Co., Germany). The system and the method for online monitoring OUR in bioreactors, based on the dynamic measurement of dissolved oxygen (DO), were developed. The method of on-line cell concentration estimation was established based on the relationship between the growth of the hybridoma and the uptake rate of oxygen. This method was then used to determine OUR and the concentrations of cell, antibody, glucose, lactate, glutamine and ammonia in the bioreactors at given times. The relationship between OUR and nutrients metabolism was studied and OUR-based feed-back control strategy, which used the state deltaOUR = 0 as the regulation point, was established and used to control the rates of nutrients or medium feeding rate in the perfusion culture. The results showed that there was close relationship between OUR, concentration of live cells, productivity of antibody and consumption of glutamine. The sudden decrease in OUR may be caused by glutamine depletion, and with different delay times, the viable cell concentration and antibody productivity also decreased. The further analysis revealed the linear relationship between OUR and the density of live cells in the exponential growth phase as qOUR = (0.103 +/- 0.028) x 10(-12) mol/cell/h. These findings can be applied to the on-line detection of live cell density. Our study also indicated that by adjusting the perfusion rate with OUR-based feed-back control strategy, it is feasible to continuously increase in viable cell density and antibody concentration in the perfusion culture.
Bioreactors ; Cell Culture Techniques ; methods ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Hybridomas ; metabolism ; Oxygen ; metabolism

Monoclonal antibody producted by continuous perfusion culture was recovered and purified by expended bed adsorption chromatography. A packed bed column XK16/20 was used for method scouting with Streamline SP adsorbent. Two expended bed columns Streamline-25 and -50 were used for method optimization and pilot scale experiment, respectively. The recovery yield of monoclonal antibody was above 90% in a 5 - 7 fold enhanced purity and 10 fold increased concentration. According to the different concentration of monoclonal antibody in cell culture broth, about 18 - 50L fluid can be treated in a single cycle. MAb purification from lab scale (400mg per cycle) to a small pilot scale (2g per cycle) has been achieved. Compared with packed bed adsorption, the preparation cycle was half shortened, and the cost of production and the complexity of process were decreased markedly. It has been proven that a purification process based on expended bed adsorption technique is simple, efficient and economical.
Adsorption ; Antibodies, Monoclonal ; biosynthesis ; isolation & purification ; Bioreactors ; Cell Culture Techniques ; methods ; Chromatography ; methods ; Hybridomas ; cytology ; metabolism

With the application of monoclonal antibody technology more and more widely, its production technology is becoming more and more perfect. Small molecule monoclonal antibody technology is becoming a hot research topic for people. The application of traditional Chinese medicine small molecule monoclonal antibody technology has been more and more widely, the technology for effective Chinese medicine component knockout provide strong technical support. The preparation of monoclonal antibodies and small molecule knockout technology are reviewed in this paper. The preparation of several steps, such as: in the process of preparation of antigen, hapten carrier coupling, coupling ratio determination and identification of artificial antigen and establishment of animal immunization and hybridoma cell lines of monoclonal antibody, the large-scale preparation; small molecule monoclonal antibody on Immune in affinity chromatography column method is discussed in detail. The author believes that this technology will make the traditional Chinese medicine research on a higher level, and improve the level of internationalization of Chinese medicine research.
Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; immunology ; Humans ; Hybridomas ; metabolism ; Immunologic Techniques ; methods ; trends

Hybridoma cells were cultured by continuous perfusion in Fibra-Cel of 5L packed-bed bioreactor for 22 days in low serum or serum-free media. The corresponded amino acids were fed and serum concentration was decreased by analyzing glucose concentration, oxygen uptake rate, secretary antibody amount and amino acids concentration in culture supernatant. Comparing with continuous perfusion culture that amino acids were not fed, antibody amount of production was increased about 2-3 times. The inoculated cell density was 2.5 x 10(5) cells/mL, while the final cell density was 8.79 x 10(8) cells/mL. Antibody production was reached 295 mg/L/d at average level, and the highest level was reached 532 mg/L/d. These results provided a primary mode of enlarge culture for monoclonal antibody industralization.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cell Division ; Cells, Cultured ; Glucose ; pharmacology ; Hybridomas ; metabolism ; Mice ; Mice, Inbred BALB C ; Oxygen Consumption ; Perfusion

OBJECTIVETo prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration.

METHODSMcAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb.

RESULTSMcAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively.

CONCLUSIONThe McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.

Antibodies, Monoclonal ; biosynthesis ; immunology ; isolation & purification ; CD3 Complex ; immunology ; Cell Line ; Chromatography, Affinity ; Humans ; Hybridomas ; metabolism ; Jurkat Cells ; K562 Cells

UNLABELLEDTo reduce the huge labor-cost in the screening in traditional monoclonal antibody generation, We established a new system for monoclonal antibody generation integrating with protein array. BALB/c mice were immunized by eight recombinant proteins respectively, and the positive hybridoma cells were obtained by cell fusion and ELISA screening. All the eight kinds of positive hybridoma cells were mixed, cloned, screened by protein array, and definite dilution cloned.

RESULTS175 single cell clones were obtained by complex cloning, and 119 of those were positive clones. Then 8 positive cell lines were generated by the following 2 rounds definite dilution cloning. By comparing with the traditional method, we got 8 monoclonal antibodies using the combined protein array screening and multiplex cloning method in 1 cycle, and fewer amounts of antigens were used. As a result, the combined protein array and multiplex cloning method could be used as an economical, rapid and simple tool applying in high throughput monoclonal antibody generation.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; isolation & purification ; Female ; Humans ; Hybridomas ; metabolism ; Mice ; Mice, Inbred BALB C ; Protein Array Analysis

The study of antibodies has been a focal point in modern biology and medicine since the early 1900s. However, progress in therapeutic antibody development was slow and intermittent until recently. The first antibody therapy, murine-derived murononab OKT3 for acute organ rejection, was approved by the US Food and Drug Administration (FDA) in 1986, more than a decade after César Milstein and Georges Köhler developed methods for the isolation of mouse monoclonal antibodies from hybridoma cells in 1975. As a result of the scientific, technological, and clinical breakthroughs in the 1980s and 1990s, the pace of therapeutic antibody discovery and development accelerated. Antibodies are becoming a major drug modality with more than two dozen therapeutic antibodies in the clinic and hundreds more in development. Despite the progress, need for improvement exists at every level. Antibody therapeutics provides fertile ground for protein scientists to fulfill the dream of personalized medicine through basic scientific discovery and technological innovation.
Animals ; Antibodies, Monoclonal ; therapeutic use ; Biotechnology ; methods ; Humans ; Hybridomas ; metabolism ; Immunotherapy ; methods ; United States ; United States Food and Drug Administration

Animals ; Antibodies, Monoclonal ; Cell Line ; Fibroblast Growth Factor 7 ; immunology ; Humans ; Hybridomas ; Keratinocytes ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred Strains ; Recombinant Proteins ; immunology

The environmental pollution by heavy metals such as mercury, cadmium and lead has become a worldwide public health hazard. To rapidly and inexpensively monitor environmental heavy metals is a prerequisite for minimizing human and animal exposure. The development of immunoassays to detect mercury ion residues has been a promising trend with the advantage of rapid and cheap operation. We reported the isolation and characterization of mercury-specific monoclonal antibodies. Because Hg2+ ions are too small to elicit an immune response, the metal was coupled to protein carrier (keyhole limpet, KLH) using a chelator (diethylenetriamine pentaacetic acid, DTPA). After the synthesis of antigen and characterization, monoclonal antibodies against mercury ions were generated by immunizing BALB/c mice with mercury conjugated antigen (Hg-DTPA-KLH). The stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. The hybridoma cells were subcloned by the limiting dilution and screened by ELISA, two hybridoma cell lines producing stably specific monoclonal antibodies (MAbs) against mercury ions were obtained, named H2H5 and H1H8. The ascites fluid was produced in BABL/c mice by intraperitoneal injection of 1 x 10(7) H2H5 and H1H8 cells, respectively. The titers of ascites were all above 1:51 200. The isotyping of secrete antibodies from two hybridoma cell lines was IgG1, kappa type. These data laid a potency of establishing immunoassays methods of determining Hg2+ ion residues and had the realistic significance for improving the efficiency and quality of risk assessment.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Chelating Agents ; chemistry ; Environmental Pollutants ; analysis ; immunology ; Female ; Hybridomas ; metabolism ; Immunoassay ; methods ; Mercury ; analysis ; immunology ; Mice ; Mice, Inbred BALB C

AIMTo obtain Clenbuterol monoclonal antibodies.

METHODSClenbuterol complete antigen was prepared with diazotization method. BALB/c mice was immunized with subtractive immunization, Clenbuterol monoclonal antibody was prepared with rule hybridoma technique.

RESULTSThe mice obtained tolerance to BSA by subtractive immunization. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%, and the specific clenbuterol monoclonal antibody was obtained at last.

CONCLUSIONMonoclonal antibodies to micromolecule contaminant be prepared by subtractive immunization, could decrease the workload in the bolting of monoclonal antibodies, and increase the chance to obtain the antibody of expected.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Clenbuterol ; immunology ; Female ; Hybridomas ; metabolism ; Immunization ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Serum Albumin, Bovine ; immunology