Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specific antibodies.     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase

Summary The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP₂·Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG₁ and IgG_2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin. This study demonstrates some of the potential advantages of using an epitope specific monoclonal bi-specific developing reagent like McC10 in an immunobased technique like ICC. Its potential use in a variety of other immunobased procedures is discussed.     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase. Applications in immunoassays

The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP2.Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG1 and IgG2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a bi-specific monoclonal antibody for simultaneous detection of rabbit IgG and horseradish peroxidase: use as a general reagent in immunocytochemistry and enzyme-linked immunosorbent assay

We describe the development of bi-specific monoclonal antibodies (MAb) capable of simultaneous recognition of rabbit immunoglobulin G (IgG) and horseradish peroxidase (HRP) for use in a variety of immunobased techniques. This bi-specific antibody, named McC8, was produced by fusion of the aminopterin-sensitive mouse hybridoma MAP.Ag.1, which secretes MAb against HRP, and splenocytes from a mouse previously immunized with whole rabbit IgG. The resultant hybrid-hybridoma co-dominantly expresses and secretes the immunoglobulin chains, i.e., IgG1 and IgG2b, of its respective parents, as determined by radial immunodiffusion. The binding sites on rabbit IgG for McC8 were determined on Western blots and in competition solid-phase enzymatic immunoassays with the use of allotype-specific rabbit sera. Both these techniques demonstrated that McC8 recognizes the light chain of the rabbit IgG molecule with preferential binding to the B4 kappa light-chain allotype. McC8 was successfully used in two-step immunocytochemistry for localization of calcitonin gene-related peptide (CGRP) in fibers of the superficial layers of the spinal trigeminal nucleus of the rat, as well as for localization of glial fibrillary acidic protein (GFAP)-immunoreactive sites in primary rat septal cell cultures, thus demonstrating its potential as a general developing reagent in conventional immunocytochemistry. McC8 compared favorably with peroxidase-antiperoxidase immunocytochemistry with respect to sensitivity. However, the bi-specific developing reagent proved superior to the conventional peroxidase-antiperoxidase procedure when both were employed in a similar fashion in tissues prone to display high background staining. Finally, McC8 was also employed as a developing reagent in a competitive ELISA designed for quantitation of CGRP with the use of a rabbit anti-CGRP primary antibody. The sensitivity of this quantitative ELISA (190 pg or 50 fmol CGRP per well) renders this bi-specific antibody suitable for use in quantitative immunoassays for detection of relevant peptides in biological systems.     

The production of a “universal developer” for the immunological detection of human IgG and its application in immunodiagnostics

Summary This study describes the development of a bispecific monoclonal antibody capable of the simultaneous recognition of horseradish peroxidase (HRP) and human IgG. This antibody, coded McC2, has been applied in a novel manner as a universal developing reagent for the detection of human IgG. McC2 cross-reacts with all human IgG subtypes and was found to recognise an epitope on the Fe portion of human IgG. McC2 does not cross-react with human IgM or IgA. This bi-specific antibody belongs to the mouse IgG₁ subclass. McC2 was used for the detection of human IgG in a simple one step enzyme-linked immunosorbant assay (ELISA). Use of this bi-specific antibody in this assay resulted in an excellent signal to noise ratio with background in negative control wells virtually nonexistent. McC2 was also applied in a clinical diagnostic test for the detection of auto anti-nuclear antibodies in patient sera. McC2 was substituted, in a blind study, for a HRP-conjugated second antibody supplied with the test kit. All sera were tested both with the kit's second antibody and McC2. When using McC2, we obtained no false positive results whereas five false positives were obtained when using the kit's second antibody. However, one false negative result was obtained with the use of McC2 as a developing reagent while none were noted with the use of the kit's second antibody.This study demonstrates the potential use of bi-specific universal developers in a wide variety of immunobased techniques as well as the potential advantages for the production of a complete panel of bi-specific developing monoclonal antibodies against IgGs from a number of different species.     

Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Summary Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specifie antibodies.     

A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free Reagents,Immunofluorescence, and Multiple Antibody Staining

Immunohistochemistry on mouse tissue utilizing mouse monoclonal antibodies presents a challenge. Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue. This can lead to significant spurious staining. Therefore, a “mouse-on-mouse” staining strategy is needed to yield credible data. This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system. The mouse primary antibody is first combined with an Fab fragment of an anti-mouse antibody in a tube and allowed sufficient time to form an antibody complex. Any non-complexed secondary antibody is bound up with mouse serum. The mixture is then applied to the tissue. The flexibility of this method is confirmed with the use of different anti-mouse antibodies followed by a variety of detection reagents. These techniques can be used for immunohistochemistry (IHC), immunofluorescence (IF), as well as staining with multiple primary antibodies. This method has also been adapted to other models, such as using human antibodies on human tissue and using multiple rabbit antibodies in dual immunofluorescence.     

Monoclonal antibodies, act two: new molecules for new challenges

After early difficulties due in part to their mouse origin and questionable selection criteria, monoclonal antibodies have become major therapeutic tools thanks to more and more sophisticated molecular engineering. They are now used in a growing number of therapeutic areas. Molecular engineering has focused on the improvement of antibody affinity, the reduction of immunogenicity due to the murine origin of the first generation of monoclonal antibodies and on the increase of antibody effector properties, initially limited by their murine origin. The current success of antibodies raises new challenges that the scientific and medical communities are taking up: design of antibodies with optimized functional properties, with lower side effects, design of new molecular formats (drug-coupled antibodies, bi-specific antibodies, antibodies with optimized half-lives), detection and selection of "responder" patients. As a new antibody generation is quickly emerging, the future of antibodies is already at sight: development of oligoclonal strategies where cocktails of monoclonal antibodies are used, rationale selection of eligible patients, bulk production at lower costs. To date, twenty-three monoclonal antibodies have received an approval in the United States and/or in Europe and more than two hundred and fifty are currently evaluated in clinical trials. A new wave is coming...     

Bromodeoxyuridine and lododeoxyuridine Double Immunostaining for Epoxy Resin Sections

To establish bromodeoxyuridine (BrdUrd)/iododeoxyuridine (IdUrd) double immunostaining for thick sections of epoxy resin-embedded tissues, young hamsters received intra-peritoneal injections of IdUrd and BrdUrd 3 hr and 1 hr before sacrifice, respectively. The intestines were fixed with phosphate-buffered 4% paraformaldehyde and embedded in an Epon-Araldite mixture. The epoxy resin was removed completely by a sodium methoxide/benzene/methanol solution. This epoxy resin removal method was effective for BrdUrd/IdUrd immunostaining using a mono-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd and IdUrd (IU-4), followed by the ABC complex method. Epoxy sections stained with these antibodies showed clear localization of nuclei incorporating the two thymidine analogues with precise morphology of labeled cells. Furthermore, ultrastructural observation of thin sections adjacent to thick sections immunostained for BrdUrd/IdUrd confirmed the cell type and ultrastructural features of cells labeled with these thymidine analogues.     

Use of tetrameric antibody complexes to stain cells for flow cytometry

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.     

A proteomics-based approach for monoclonal antibody characterization

The determination of monoclonal antibody specificity is dependent upon the availability of purified antigen. Such material is not always available and this has proven to be one of the rate-limiting steps in monoclonal antibody production. The aim of the present study was to develop a generic approach to defining antibody specificity that bypassed the need for pure antigens through the use of proteomics. The scheme and its application to several biological mixtures are described. The results demonstrate the ability of the approach to identify antibodies against both the major components and the minor contaminants of a protein mixture. This approach should markedly enhance the characterization of antibodies to complex antigen mixtures.     

Chronic delivery of antibody fragments using immunoisolated cell implants as a passive vaccination tool

Background

Monoclonal antibodies and antibody fragments are powerful biotherapeutics for various debilitating diseases. However, high production costs, functional limitations such as inadequate pharmacokinetics and tissue accessibility are the current principal disadvantages for broadening their use in clinic.

Methodology and Principal Findings

We report a novel method for the long-term delivery of antibody fragments. We designed an allogenous immunoisolated implant consisting of polymer encapsulated myoblasts engineered to chronically release scFv antibodies targeted against the N-terminus of the Aβ peptide. Following a 6-month intracerebral therapy we observed a significant reduction of the production and aggregation of the Aβ peptide in the APP23 transgenic mouse model of Alzheimer''s disease. In addition, functional assessment showed prevention of behavioral deficits related to anxiety and memory traits.

Conclusions and Significance

The chronic local release of antibodies using immunoisolated polymer cell implants represents an alternative passive vaccination strategy in Alzheimer''s disease. This novel technique could potentially benefit other diseases presently treated by local and systemic antibody administration.     

Production and purification of IgY antibodies from chicken egg yolk

The isolation of polyclonal antibodies from the serum of immunized mammals has significantly contributed to scientific research and diagnosis. The fact that recent technologies allow the production of antibodies in the yolk of eggs laid by hens, has led to the development of an alternative method for antibody generation that is less stressful to animals. As hens are kept under almost all their natural conditions, antibodies are isolated from the collected eggs; this technology is expected to become an interesting alternative to the conventionally serum-based techniques that eventually require to sacrifice the animal.Here we present a modified protocol for the isolation of IgY antibodies from immunized chickens and provide comparison between two chicken breeds in relative to IgY yield per egg. Our results show the possibility of generating large quantities of highly pure IgY from chicken eggs and also show large differences in the yield of IgY production between the two studied breeds. The results of this work indicate that IgY technology can be used for the production of primary antibodies for immunological work and disease diagnosis.     

The in vitro antibody response to cell surface antigens. II. Monoclonal antibodies to human leukemia cells.

A method has been developed for the production of monoclonal mouse antibody responses in vitro against human cell surface antigens. Limiting numbers of immune spleen cells were transferred to syngeneic, irradiated recipients whose spleen fragments were then cultured in vitro and stimulated to produce antibody. The majority of the antibody from any one fragment culture was likely to be the product of a single donor B cell and thus monoclonal. Evidence for this included a linear relationship between donor cell transferred and spleen fragments producing antibody, extremely restricted isoelectric focusing patterns of the individual antibody products, and unique reactivity patterns of these antibodies against a panel of human lymphoid cells. Different human B leukemia cells were seen as immunogenically distinct by the mouse. By using the monoclonal mouse antibodies as probes, a fine analysis of cell surface antigens is jow possible.     

Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments

An efficient method of formulating serum-free medium (SFM) for production of therapeutic antibodies by recombinant CHO (rCHO) cells was developed using two rCHO cell lines producing a therapeutic antibody. In this method, ten kinds of SFM were prepared by supplementing the basal SFM with statistically designed mixtures (total 5 g L^?1) of three non-animal-derived hydrolysates: yeastolate, soy hydrolysate, and wheat gluten hydrolysate. When the two rCHO cell lines were cultivated, the mixtures of soy hydrolysate and wheat gluten hydrolysate showed a positive effect on cell growth. On the other hand, the mixtures including a high portion of yeastolate significantly enhanced specific antibody productivity. To reconstitute the mixture ratios of the three hydrolysates for high growth and antibody production, the effect of each medium was analyzed by the statistical program Design-Expert®. The resulting medium gave a 1.9–3.3-fold increase in the maximum antibody concentration, compared to the basal SFM. Taken together, the supplementation of hydrolysates to the basal SFM with the help of statistical analysis is an efficient means of developing SFM for therapeutic antibody production by rCHO cells.     

Establishment and validation of an immunoenzymatic assay to determine mouse IgG levels based on a rat monoclonal antibody

In this paper we describe an immunoenzymatic assay based on a rat monoclonal antibody (Ram kappa) developed to determine mouse IgG concentration, which is widely used for samples obtained on purification processes, like supernatant waste and the content of IgG in the vaccine (rHBsAg). This assay involves the use of a rat antibody-horseradish peroxidase-conjugated for the revealing of the antigen-antibody reaction. The rat antibody was produced in cell culture using a dialysis tube (DT). The immunoassay was standardized following several concepts, such as specificity, precision, and linearity. The result obtained permitted us to replace the use of polyclonal antibodies to determine the kappa light chain mouse antibodies by a rat monoclonal antibody of high sensibility and reproducibility. The assay permitted a reliable measurement of murine kappa Ig up to 0.68 ng/ml and was capable of quantifying 6.25 ng/ml. Due to the high frequency of the kappa light chain in mouse antibodies this system acquires a great application.     

The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide-biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs.     

A general method for studying the secretion of macromolecules by single cells. I. Detection of immunoglobulin-secreting cells.

A general method for detecting single cells secreting macromolecules has been developed and applied to the detection of mouse cells secreting antibodies. Secreted Ig molecules were precipitated in the immediate vicinity of an active cell with rabbit anti-mouse Ig antibody. Rapid removal of excess antibody not incorporated into immunoprecipitates was achieved with an electrophoresis technique. The immuno-precipitate surrounding the active cell was then stained with sheep anti-rabbit Ig antibody labeled with horseradish peroxidase. The use of enzyme markers has made the procedure much more convenient and rapid than previous precipitin assays for cell secretion that used radioiodine for detecting immunoprecipitates. Moreover, the availability of several enzyme markers makes possible the detection of cells secreting more than one molecular species. Experiments were also run in which cells producing antibodies specific for horseradish peroxidase (HRP) could be identified among the population of cells producing other immunoglobulins. Presumably, HRP-labeled antigens could be used to identify cells producing other specific antibodies. The generality of this procedure suggests that it may be useful for detecting single T-cells releasing regulatory molecules, since specific antisera are already available for several of these molecules.     

Comparison of GK1.5 and chimeric rat/mouse GK1.5 anti-CD4 antibodies for prolongation of skin allograft survival and suppression of alloantibody production in mice.

GK1.5 is a rat mAb that recognizes the mouse CD4 Ag. It has been shown to deplete CD4+ cells in vivo and to be immunosuppressive. To evaluate the effect of the C region of this antibody in achieving cell depletion, chimeric antibodies, each having the rat GK1.5 V regions and one of the four mouse IgG C region isotypes, were compared with the native rat antibody. The chimeric antibodies and the native antibody were tested for their ability to mediate in vitro C-dependent cytotoxicity, in vivo cell depletion, and prolongation of allogeneic skin graft survival and suppression of alloantibody production. In vitro C-dependent cytotoxicity assays revealed that rat IgG2b and the chimeric antibodies containing mouse IgG2a, mouse IgG2b, and mouse IgG3 were effective in lysing CD4+ lymphocytes whereas mouse IgG1 was ineffective. In vivo studies of CD4+ cell depletion showed that mouse IgG2a, rat IgG2b, and mouse IgG2b were effective isotypes, mouse IgG1 was less effective, and mouse IgG3 did not deplete CD4+ cells. A correlation was found between the ability of an isotype to deplete CD4+ cells in vivo and its ability to prolong the survival of skin allografts and to suppress alloantibody production. The nondepleting mouse IgG3 was ineffective in these assays. Overall the most effective mouse isotype was IgG2a which was as effective as rat IgG2b. These results indicate 1) that syngeneic isotypes of mAb can cause cell depletion and consequently the prolongation of allograft rejection and suppression of alloantibody production; 2) that not all isotypes are equally effective; and 3) that the ability of a given isotype to deplete cells in vivo does not correlate with its ability to mediate C-dependent lysis in vitro. Our results are consistent with the hypothesis that in vivo depletion of cells is mediated by opsonization and binding through the FcR.     

Comparison of the production of a human monoclonal antibody against HIV-1 by heterohybridoma cells and recombinant CHO cells: A flow cytometric study

The production of human monoclonal antibodies for therapeutic use is of increasing importance for treatment of viral infections such as AIDS. As human x mouse heterohybridomas rarely reach the growth rates and cell specific production rates of mouse hybridomas the transfection of standard cell lines, such as CHO or BHK, is a promising alternative. This has the additional advantage that the IgG subtype can be changed to suit the desired application. However, the use of a cell line that has not originally developed to produce antibodies, as lymphocytes and myeloma cells have, might have unrecognised drawbacks. This will be especially significant in the case of antibodies as each molecule consists of 4 chains linked by disulphide bonds which require specific intracellular factors to be properly folded and processed (Heavy chain binding protein, Protein Disulfide Isomerase a.o.). In this study we have therefore compared two cell lines: a human x mouse heterohybridoma producing IAM-2F5, a human IgG₃ antibody specific for HIV-1 with neutralising properties and a Chinese Hamster Ovary cell transfected with dihydrofolate reductase and with the heavy and light chain genes of IAM-2F5 modified to IgG₁. From each cell line three subclones were selected with low, medium and high specific production rates. Batch cultures were performed and the following cellular parameters analysed by flow cytometry; 1) total RNA content (translational activity); 2) total protein content; 3) cell cycle phase distribution; 4) concentration of light and heavy chains; 5) concentration of helper proteins such as BiP and PDI. The production rate of heterohybridoma cells was best reflected in the intracellular concentration of kappa chain, while the gamma chain concentration was comparable for all three subclones. In the CHO cells the gamma chain expression and thus gene copy number appeared to be the limiting factor. The GRP78/BiP concentration in CHO remained unchanged in spite of a 5-fold higher concentration of gamma chain in the high producing subclone. The PDI concentration in CHO cells was much lower compared to the heterohybridoma cells, irrespective of production rates.Abbreviations PDI protein disulfide isomerase - GRP78/BiP Glucose regulated protein; Heavy chain binding protein