20-alpha-Hydroxysteroid dehydrogenase from pseudopregnant rat ovary: obtention and characterization of a monoclonal antibody against the enzyme activity.

The enzyme 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD) was purified from pseudopregnant rat ovaries and used as antigen for the development of a monoclonal antibody by the hybridoma technique. Spleen cells of BALB/c mice immunized with purified 20-alpha-HSD were fused with SP2/0 mouse myeloma cells. Among the colonies of hybrid cells, one (designated mAb-HSD 11) was found to be secreting antibodies (IgM) able to inhibit 20-alpha-HSD activity. The antibody-secreting hybridome was amplified by ascitic fluid production and the monoclonal antibody purified by Bakerbond ABx procedure. Purified mAb-HSD 11 was able to inhibit 20-alpha-HSD activity in a dose-dependent manner. Studies of Michaelis constants of 20-alpha-HSD indicate that this monoclonal antibody increases the Km for 20-alpha-dihydroprogesterone and decreases the Vmax.     

Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm

Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgGlK) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be “shielded” from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when viewed with an epifluorescence microscope. A lipophilic counterstain (red fluorescence) was used to insure that all sperm were visualized. Sperm in fresh-extended or frozen-thawed semen were incubated with hybridoma supernatant containing monoclonal antibody for 30 min at 37°C, then a second antibody (rabbit anti-mouse IgG-FITC) was added for 30 min at 37°C. Unbound antibody was removed by dilution and centrifugation. Sperm were resuspended in phosphate-buffered saline containing Evan's blue as a counterstain. All sperm fluoresced bright red, regardless of the status of cell membranes, except that in cells with damaged plasma-acrosomal membranes, the green fluorescence associated with antibody was overriding for the rostral portion. By counting fluorescent and nonflourescent “acrosomes”, the percentage of sperm with intact plasma-acrosomal membranes was easily determined. Evaluation of five mixtures of undamaged and damaged sperm by this procedure gave a correlation of 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome. Intra- and interassay coefficients of variability were < 6%.     

Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium-induced root nodules of pea cross-react with plasma membranes and Golgi bodies

Three rat hybridoma lines that produced monoclonal antibodies reacting with the peribacteroid membrane from Pisum sativum were isolated, and these all appeared to recognize the same antigenic structure. Using one of these monoclonal antibodies, AFRC MAC 64, electron microscopy of immunogold-stained thin sections of nodule tissue revealed that the antigen, present in the peribacteroid membrane, was also found in the plant plasma membranes and in the Golgi bodies, but not in the endoplasmic reticulum. When peribacteroid membrane proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electro-blotting, it was found that MAC 64 bound to a series of protease-sensitive bands that migrated in the mol. wt. range 50-85 K. The epitope was sensitive to periodate oxidation and its structure may therefore involve the carbohydrate component of a membrane glycoprotein. We suggest that this structure originates in the Golgi apparatus and is subsequently transferred to the peribacteroid membranes and plasma membranes. The monoclonal antibody also reacted with peribacteroid membranes from nodules of Vicia and lupin, and with plasma membranes and Golgi membranes from uninfected plant cells, including root tip cells from onion (Allium cepa), indicating that the antigen is highly conserved in the plasma membranes of plant cells.     

囊素与杂交瘤细胞的结合及结合肽的鉴定

【目的】囊素(BS)是禽类和哺乳动物中具有重要免疫调节功能的多肽,能有效促进杂交瘤细胞抗体的分泌,为探讨杂交瘤细胞是否有BS受体分子的表达。【方法和结果】本研究采用荧光显微镜、激光扫描共聚焦显微镜和流式细胞仪分析的方法,检测BS与杂交瘤细胞的结合特性。实验结果证实BS与杂交瘤细胞的结合具有特异性、趋饱和性和可逆性。为进一步分析BS与杂交瘤细胞的结合位点,实验中以BS分子作为靶标,对噬菌体随机12肽库进行了4轮亲和筛选,ELISA和竞争抑制试验显示2个噬菌体克隆能特异性与BS结合。对阳性噬菌体克隆进行序列测定分析表明,其插入的12肽分别为:ACTKHLCLLQPL、MSCNDTLCLLPN,保守序列为LCLL。体外实验表明,2个人工合成的12均都能在一定程度上抑制BS与杂交瘤细胞的特异性结合。【结论】本研究表明杂交瘤细胞具有BS结合的受体,这为进一步研究BS促杂交瘤细胞抗体分泌的信号传导通路奠定了基础。     

Effect of temperature on hybridoma cell cycle and MAb production

The kinetics of growth and antibody formation of an anti-interleukin-2 producing hybridoma line were studied in suspension culture at temperatures ranging from 34 degrees C to 39 degrees C. Flow cytometry was used to determine the effect of temperature on the cell cycle. Maximum cell density and monoclonal antibody yield were observed at 37 degrees C. The specific monoclonal antibody production rate was approximately constant throughout each batch experiment. Lower temperatures caused cells to stay longer in the G(1)-phase of the cell cycle, but temperature had only a marginal effect on the specific antibody production rate. Arresting of cells in the G(1)-phase by means of temperature was, therefore, not suited for enhanced monoclonal antibody production. Rather, antibody production for this hybridoma was directly linked to viable cell concentration. (c) 1992 John Wiley & Sons, Inc.     

Effect of shear rate on hybridoma cell metabolism

The effect of shear rate on cell growth and monoclonal antibody production of hybridoma cells was studied. The dependence of agitation rate on antibody production is discussed by measuring the amount of monoclonal antibody in cells cultured by a spinner vessel. The effect of shear rate is also studied by exposing a homogeneous shear flow to hybridoma cells in a cone-and-plate viscometer. The dependence of shear rate on hybridoma cells was observed and the increase of antibody production was arised from the increase of secretion from cells.     

Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators

Growth of a hybridoma culture, along with production of monoclonal antibody, was demonstrated over extended periods in polysulfone hollow fiber membrane modules. The molecular weight cutoffs of the membranes were 70,000, 50,000, and 100,000 daltons. The hybridoma cell line, designated 65/26, produced IgG (2b/kappa) directed at mouse thymus cell surface antigen, TL.1. Cell growth occurred in the shell space of the reactor, using supplemented RPMI 1640 (20% fetal bovine serum) supplied from a separate reservoir vessel through the hollow fiber lumen. The reservoir contained 125 mL media, which was changed every 4 days. Concentrations of immunoglobulin were determined by an enzyme immunoassay (using protein A and alkaline phosphatase-labeled antibody conjugate). For the 10K, 50K, and 100K hollow fiber membrane modules, the maximum IgG concentrations detected in the 2.5-mL shell space were 47.5-80, 510, and 740 mug/mL, respectively. In the 125-mL reservoir for the 100K hollow fiber membrane module, the IgG concentration was measured at 260 mug/mL These values compare with an IgG concentration of 1 mug/mL when grown in a standard tissue culture flask and 3.2-7.6 mug/mL when grown in 100 ml media in a spinner flask. In addition, 10K and 50K hollow fiber membrane modules were run in a mode that decreased the fetal bovine serum supplement with time. Differences between these systems suggest that it is possible to obtain high IgG accumulation rates, both during and after the exponential growth phase of the hybridoma population.     

Quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging

Surface plasmon resonance imaging (SPRi) is most frequently used for the label-free measurement of biomolecular interactions. Here we explore the potential of SPRi to measure antibody production of individual hybridoma cells. As a model system, cells from a hybridoma, producing monoclonal antibodies recognizing epithelial cell adhesion molecule (EpCAM), were used. Recombinant human EpCAM protein was immobilized on an SPR sensor and hybridoma cells were introduced into an IBIS MX96 SPR imager and the SPRi response was followed for 10 h. SPRi responses were detected on the spots of the sensor only where ligands of the produced antibody were present. By measuring the SPRi signals on individual cells the antibody production of the individual cells was measured and production rates were calculated. For 53 single EpCAM hybridoma cells the production ranged from 0.16 to 11.95 pg (mean 2.96 pg per cell, SD 2.51) over a period of 10 h. Antibody excretion per cell per hour ranged from 0.02 to 1.19 pg (mean 0.30, SD 0.25). Here we demonstrate for the first time that antibody production of individual cells can be measured and quantified by SPRi, opening a new avenue for measuring excretion products of individual cells.     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Production of monoclonal antibodies by hybridoma cells in a flat sheet membrane bioreactor.

The purpose of this study was to investigate hybridoma growth and monoclonal antibody formation in a flat sheet membrane bioreactor. The effects of several different molecular weight cutoff membranes on growth and antibody formation were investigated. Nutrient and toxic product diffusion through the membranes were quantified, and the kinetic and physical constants of the system were determined.     

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.     

The production and characterization of monoclonal antibodies to a human colonic antigen associated with ulcerative colitis: cellular localization of the antigen by using the monoclonal antibody

We detected in human colon extracts a 40 kDa protein(s) that specifically reacts with tissue-bound IgG obtained from the colon of patients with ulcerative colitis or CCA-IgG. Using the hybridoma technology, we developed monoclonal antibodies to this 40 kDa protein. The specific immunoreactivity of one of the monoclonal antibodies (7E12H12, IgM isotype) against the 40 kDa protein was demonstrated both by ELISA and by immunotransblot. Competitive binding experiments showed that CCA-IgG inhibits the binding of 7E12H12 to the 40 kDa protein, suggesting the recognition of common epitope(s) on the 40 kDa protein by the monoclonal antibody and CCA-IgG. 7E12H12 was used to determine cellular localization of the 40 kDa protein. Biopsy tissue specimens from colon, esophagus, stomach, duodenum, jejunum, ileum, liver, pancreas, lungs, kidneys, salivary, and mammary glands were obtained. Tissue specimens were fixed in 4% paraformaldehyde or in 10% formalin. Sections were sequentially incubated with the hybridoma supernatant, biotinylated anti-mouse IgM, avidin-biotin-peroxidase complex, and 3,3'-diaminobenzidine. An unrelated hybridoma supernatant was used as control. The monoclonal antibody exclusively recognized colonic epithelial cells both in the crypt and on the luminal surface. Immunoreactivity was present on the plasma membrane chiefly along the basolateral areas of the cells. Plasma membrane localization of the 40 kDa protein was confirmed by immunoelectron microscopy. All colonic mucosal biopsy specimens from both adult and fetal colon reacted with the monoclonal antibody. None of the biopsy specimens from stomach, duodenum, jejunum, ileum, liver, pancreas, or non-gastrointestinal tissue reacted with the antibody, confirming the organ specificity of the 40 kDa protein. The interaction between this colonic epithelial membrane protein and the CCA-IgG may play an important role in the pathogenesis of ulcerative colitis.     

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand

Background

The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF) irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water) produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production.

Results

In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts proliferate slower in NPD water.

Conclusion

Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion. Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.     

Electrical stimulation of hybridoma cells producing monoclonal antibody to cAMP

Electrical stimulation was applied to hybridoma cells in order to activate metabolic activities and increase the monoclonal antibody production. Hybridoma cells that produce monoclonal antibody to adenosine 3':5'-cyclic monophosphate were placed on a transparent glass electrode immersed in medium and subjected to electric pulses (pulse shape, alternating rectangular; field strength, 4 X 10(3) V X m-1; frequency, 5 kHz; pulse mode, 0.5 min application and 4.5 min pause). After 48 h of incubation, the concentration of lactic acid in the medium reached 8.4 mM, approx. 30% higher than that obtained without electric stimulation. Similarly, cell growth rate was promoted by the electric stimulation, reaching a maximum stimulation after 40 h. When the hybridoma was cultured for 48 h with electrical stimulation, the antibody concentration in the medium reached 22.3 microgram X ml-1, approx. 10% higher than the control, with a concomitant 16% increase in cell concentration. Longer periods of electric pulse application, however, caused an inhibitory effect on the hybridoma growth. The most probable cause of the inhibition are reactive oxygen species such as superoxide and hydrogen peroxide, which are inevitably generated by electrolysis. The presence of superoxide dismutase (EC 1.15.1.1) reduced the inhibitory effects. In conclusion, metabolic activities including monoclonal antibody production were activated by the electrical stimulation.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

人IL17-RD胞外区真核表达及其单克隆抗体的制备

为了进一步研究白介素17受体D (IL-17RD) 在IL-17信号的调节作用,探索是否可以通过单克隆抗体阻断IL-17RD介导的IL-17信号通路而缓解自身免疫疾病,利用昆虫表达载体从Sf9细胞中表达纯化人IL-17RD-ECD蛋白,免疫Balb/C小鼠30 d,取小鼠脾脏细胞并与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株能稳定分泌抗IL-17RD-ECD的杂交瘤细胞株1F8。经过初步鉴定,该细胞株分泌的抗体类型为IgG1+kappa类,经过Western blot     

Filtration-based perfusion of hybridoma cultures in protein-free medium: Reduction of membrane fouling by medium supplementation with DNase I

In this study, a filtration-based perfusion process was developed for the production of monoclonal antibodies (IgM) by suspended hybridoma cells grown in protein-free medium. It was found that the use of protein-free medium for perfusion culture generated the formation of numerous visible suspended particles consisting of dead cells and cellular debris aggregated into fibrous material. Surprisingly high apparent viabilities were observed in such protein-free cultures. In addition, membrane fouling occurred more rapidly in protein-free medium than in conventional serum-supplemented medium. By the addition of deoxyribonuclease I (DNase I) to the protein-free medium, it was possible to prevent the formation of aggregates and to follow the evolution of the total cell population more accurately. Moreover, DNase I significantly reduced the fouling of filtration membranes, and that, for two different types of separation systems (cross-flow and vortex-flow filtration) and two different types of membranes (polycarbonate and hydrophilized polysultone). From these results, it is clear that the presence of DNA fragments liberated following cellular death is playing an important role in membrane fouling. Longevity of filtration membranes was found to be considerably greater using a vortex-flow filtration module than with a static plate-and-frame cross-flow filtration module. The use of vortex-flow filtration of conjuction with DNase I allowed maintenance of perfusion cultures for more than 1 month without membrane fouling or antibody retention and with a constant permeate IgM concentration of 250 mg/L. Hybridomacells appeared to gradually adapt to increasing rotational speed in the vortex-flow filtration module.     

Modulation of hybridoma cell growth and antibody production by coating cell culture material with extracellular matrix proteins

The influence of coating polystyrene tissue culture plates with different proteins on murine hybridoma cell growth and antibody production was investigated. Fibronectin, collagen I, bovine serum albumin and laminin were used to coat NUNC^® and COSTAR^® cell culture plates. Cell number and antibody concentration in culture fluids were quantified as indicators for cell viability, proliferation and productivity. Adhesive behaviour, morphology, expression of surface receptors of hybridoma cells and the presence of tyrosine-phosphorylated proteins in cell lysates were characterized by cell adhesion experiments, microscopy, flow cytometry and Western Blot analysis.It was shown that coatings with fibronectin (0.2 μg/ml) lead to a substantial improvement of cell growth by 50–70% and an increase of monoclonal antibody production by 100–120%.Collagen I coatings showed an improvement in cell growth by 30–70% and by 60% for the production of monoclonal antibodies. Coatings with BSA and laminin had minor effects on these parameters. It was found that the hybridoma cell lines used in this study did not express the α₂-chain of the α₂β₁-integrin, which is responsible for binding to collagen and laminin.However, the presence of β₁-integrin on the cell surface was shown, which should enable hybridoma cells to bind fibronectin. We propose, therefore, that fibronectin adsorption to cell culture materials may be a promising approach to enhance the production of monoclonal antibodies by cultivated hybridoma cells.     

Effects of two different nutrition supply methods on improving hybridoma cell production

Hybridoma cells are featured by the effective utilization of both B lymphocytes and immortalized myeloma cells, allowing for the continuous generation of monoclonal antibodies specific to antigens. With regard to conventional hybridoma technology, B lymphocytes must be fused with myeloma cells using various methods to generate hybridoma cells. Nutrition plays an important role in hybridoma cell survival and amplification, which determines the fusion effect and antibody production. Here we compared the growth and survival rates of hybridoma in a commonly used peritoneal macrophage feeder layer (PMFL) nutrition supply system with a commercial hybridoma feeder additive (HFA) nutrition supply system at the post fusion stage and discussed the titer of monoclonal antibodies by enzyme linked immunosorbent assay (ELISA). Our results indicate that commercially available HFA promotes the survival and amplification of hybridoma clones and improves the titer of monoclonal antibodies indirectly.