Individual Hybridoma Research Articles

High-throughput single-cell antibody secretion quantification and enrichment using droplet microfluidics-based FRET assay

SummaryHigh-throughput screening and enrichment of antibody-producing cells have many important applications. Herein, we present a droplet microfluidic approach for high-throughput screening and sorting of antibody-secreting cells using a Förster resonance electron transfer (FRET)-based assay. The FRET signal is mediated by the specific binding of the secreted antibody to two fluorescently labeled probes supplied within a droplet. Functional hybridoma cells expressing either membrane-bound or secreted monoclonal antibodies (mAbs), or both, were efficiently differentiated in less than 30 min. The antibody secretion rate by individual hybridoma cells was recorded in the range of 14,000 Abs/min, while the density of membrane-bound fraction was approximately 100 Abs/μm2. Combining the FRET assay with droplet-based single-cell sorting, an 800-fold enrichment of antigen-specific cells was achieved after one round of sorting. The presented system overcomes several key limitations observed in conventional FACS-based screening methods and should be applicable to assaying various other secreted proteins.

Nanodisc technology facilitates identification of monoclonal antibodies targeting multi-pass membrane proteins

Multi-pass membrane proteins are important targets of biologic medicines. Given the inherent difficulties in working with membrane proteins, we sought to investigate the utility of membrane scaffold protein nanodiscs as a means of solubilizing membrane proteins to aid antibody discovery. Using a model multi-pass membrane protein, we demonstrate how incorporation of a multi-pass membrane protein into nanodiscs can be used in flow cytometry to identify antigen-specific hybridoma. The use of target protein-loaded nanodiscs to sort individual hybridoma early in the screening process can reduce the time required to identify antibodies against multi-pass membrane proteins.

Is cancer progression caused by gradual or simultaneous acquisitions of new chromosomes?

BackgroundFoulds defined, “Tumor progression (as a) permanent, irreversible qualitative change in one or more of its characters” (Cancer Res. 1954). Accordingly progressions, such as metastases and acquired drug-resistance, were since found to be subspecies of cancers with conserved and numerous new chromosomes. Here we ask whether cancers acquire numerous new chromosomes gradually or simultaneously in progressions. The currently prevailing theory of Nowell (Science, 1976) holds that unexplained “genetic instability” generates “variant sublines (with) changes in chromosome number” and that “clonal” progressions arise by “stepwise selection of more aggressive sublines”. The literature, however, contains many examples of “immediate” selections of progressions with numerous new chromosomes - notably experimentally initiated fusions between cancers and heterologous cells. Furthermore, the stepwise progression theory predicts intermediate sublines of cancers with multiple non-clonal additions of new chromosomes. However, the literature does not describe such intermediates.ResultsIn view of these inconsistencies with stepwise progression we test here a saltational theory, in which the inherent variability of cancer-specific aneuploidy generates “immediate” progressions with individual clonal karyotypes, transcriptomes and phenotypes in single steps. Using cell fusion as an established controllable model of “immediate” progression, we generated seven immortal murine hybridomas by fusing immortal murine myeloma cells and normal antibody-producing B-cells with polyethylene glycol within a few minutes. These immortal hybridomas contained individual sets of 71 to 105 clonal chromosomes, compared to the 52 chromosomes of the parental myeloma. Thus the myeloma had gained 19 to 53 new clonal chromosomes in seven individual hybridomas in a single step. Furthermore, no stable intermediates were found, as would be predicted by a saltational process.ConclusionsWe conclude that random fusions between myelomas and normal B-cells generate clonal hybridomas with multiple, individual chromosomes in single steps. Similar single-step mechanisms may also generate the “late” clonal progressions of cancers with gains of numerous new chromosomes and thus explain the absence of intermediates. Latency would reflect the low probability of rare stochastic progressions. In conclusion, the karyotypic clonality of hybridomas and spontaneous progressions suggests karyotypic alterations as proximate causes of neoplastic progressions. Since cancer-specific aneuploidy catalyzes karyotypic variation, the degree of aneuploidy predicts the clinical risk of neoplastic progression, confirming classical predictions based on DNA content.

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 10h. 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 11.95pg (mean 2.96pg per cell, SD 2.51) over a period of 10h. Antibody excretion per cell per hour ranged from 0.02 to 1.19pg (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.

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity.

Quantitative Imaging of Protein Secretions from Single Cells in Real Time

Cell-to-cell signaling often involves the secretion of proteins, which creates spatial and temporal concentration profiles in the extra-cellular environment for the receiving cells to detect and interpret. Real-time measurements of secreted protein concentrations at the single cell level are thus fundamental to understanding these communications pathways but have proven difficult to realize in practice. Here we present a label-free technique based upon nanoplasmonic imaging and high-affinity binding which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from individual hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell. The technique is incorporated on to a wide-field microscope which enables real-time transmitted light and fluorescence imaging of the cells as well. We anticipate this technique will be broadly applicable to the real-time characterization of both paracrine and autocrine signaling pathways.

Functional single-cell hybridoma screening using droplet-based microfluidics

Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1:10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.

Screening individual hybridomas by microengraving to discover monoclonal antibodies

The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (approximately 10(5) cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1-3 d (9-12 d including the steps for preparing arrays of microwells).

Errata

Microbiology and ImmunologyVolume 53, Issue 1 p. 58-58 Free Access Errata This article corrects the following: Variable gene family usage of protective and non-protective anti-Vibrio cholerae O1 LPS antibody heavy chains Terri K. Wade, William F. Wade, Volume 52Issue 12Microbiology and Immunology pages: 611-620 First Published online: December 3, 2008 First published: 15 January 2009 https://doi.org/10.1111/j.1348-0421.2008.00110.xCitations: 2AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Microbiology and Immunology, the footnotes for Tables 2, 3 and 4 were incorrect.1 The correct table legends and their footnotes should be as follows: Table 2 Hybridomas investigated in the present study No footnote. Table 3 O1 LPS binding and vibriocidal capacity of the anti-O1 LPS mAbs ELISA (OD) and vibriocidal data were generated using a standard volume from supernatants of individual hybridomas that contained different concentrations of mAb. The effective concentration of mAb was calculated by dividing the concentration of mAb (μg/mL) by the reciprocal of the end-point dilution. The lack of vibriocidal activity was because IgA does not fix complement. Other isotypes (e.g. IgM, IgG1) of mAbs that fix complement that were not vibriocidal were also denoted as negative (neg). Table 4 Variable heavy chain gene usage (family member) of the anti-O1 LPS mAbs The V family usage was determined from the DNA sequences of the mAbs using the Ig BLAST program from NCBI (http://www.ncbi.nlm.nih.gov/igblast). The cDNA sequences for all the mAbs are available upon request. The specific family member for individual mAb that was assigned and the percent identity to that member are: mAb 22 (Vh9.1, 99.0%), mAb 23 (Vh5, 7183.a15.24, 99.3%), mAb 24 (Vh1, J558.8, 99.7%), mAb 26 (Vh5, 7183.a47.7, 96.2%), mAb 35 (Vh1, J558.47, 100%), mAb 36 (Vh1, J558.45, 99.7%), mAb 40 (Vh6, J606.4.82, 99.7%), mAb 47 (Vh5, 7183.a47.76, 99.0%), mAb 55 (Vh3, 36–60, 96.5%), mAb 58 (Vh1, J558.8, 99.7%), mAb 1 (Vh1, J558.29, 95.2%), mAb 6 (Vh11, 100%), mAb 9 (Vh5, 7183.a33.55, 95.6%), mAb 11 (Vh5, 7183.a37.59, 96.6%), mAb 16 (Vh9.1, 100%), mAb 18 (Vh3, 36–60, 92.5%) and mAb 19 (Vh5, 7183.a47.24, 96%). Reference 1 Wade T.K., Wade W.F. (2008) Variable gene family usage of protective and non-protective anti-Vibrio cholerae O1 LPS antibody heavy chains. Microbiol Immunol 52: 611– 20. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Citing Literature Volume53, Issue1January 2009Pages 58-58 ReferencesRelatedInformation

Antibody discovery via multiplexed single cell characterization

The secreted immunoglobulin footprint of single hybridoma cells, containing ~ 10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cell's product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted antibody footprint allowed rank ordering by affinity in the same primary screen. As more criteria were added to the selection process, the frequency of positive cells went down; in some cases, the favorable cell was present at < 1/50,000. Recovery of the cell of interest was accomplished by plating the cells in a viscous medium on top of a membrane. After collecting the antibody footprint on a capture surface beneath the membrane, the immobilized cells were transferred to an incubator while the footprints were analyzed to locate the hybridoma cells of interest. The desired cells were then cloned by picking them from the corresponding locations on the membrane.

Optimization of the surfaces used to capture antibodies from single hybridomas reduces the time required for microengraving

The most common method for the generation of monoclonal antibodies involves the identification and isolation of hybridomas from polyclonal populations. The discovery of new antibodies for biochemical and immunohistochemical assays in a rapid and efficient manner, however, remains a challenge. Here, a series of experiments are described that realize significant improvements to an approach for screening large numbers of single cells to identify antigen-specific monoclonal antibodies in a high-throughput manner (105–106 cells in less than 12 h). The soft lithographic process called microengraving yields microarrays of monoclonal antibodies that can be correlated to individual hybridomas; the cells can then be retrieved and expanded to establish new cell lines. The factors examined here included the glass slide used for the microarray, the buffer used to deposit capture antibodies onto the glass, the type of polyclonal antibodies used to capture the secreted antibodies, and the time required for microengraving. Compared to earlier reports of this method, these studies resulted in increased signal-to-noise ratios for individual elements in the microarrays produced, and a considerable decrease in the time required to produce one microarray from a set of cells (from 2–4 h to 3–10 min). These technical advances will improve the throughput and reduce the costs for this alternative to traditional screening by limiting serial dilution.

Synergistic Pathogenic Effects of Combined Mouse Monoclonal Anti-Desmoglein 3 IgG Antibodies on Pemphigus Vulgaris Blister Formation

Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by anti-desmoglein 3 (Dsg3) IgG antibodies. Previously, we generated an active mouse model for PV by adoptive transfer of splenocytes from immunized or naive Dsg3(-/-) mice. In this study, we isolated 10 anti-Dsg3 IgG mAbs (NAK-series) from PV model mice generated by transfer of naive Dsg3(-/-) splenocytes. We characterized their epitopes using domain-swapped and point-mutated Dsg1/Dsg3 molecules and examined their pathogenic activities in blister formation in three different assays. In a passive transfer model using neonatal mice, eight of 10 NAK mAbs showed pathogenic activity when injected together with half the minimum pathogenic dose of anti-Dsg1 IgG autoantibodies from pemphigus foliaceus (PF) patients. None of the mAbs could induce the PV phenotype when individual hybridoma clones were inoculated by peritoneal injection into adult Rag2(-/-) mice. NAK mAbs displayed a range of potency in an in vitro dissociation assay using primary cultured mouse keratinocytes. Interestingly, when multiple hybridoma clones recognizing different epitopes were inoculated in combination, recipient mice developed the PV phenotype. In vitro dissociation assays confirmed that combined NAK mAbs had synergistic pathogenic effects. These findings indicate that although an individual anti-Dsg3 IgG is not sufficient to cause blistering in adult mice, several together can induce the PV phenotype. These mAbs will provide a valuable tool to investigate the molecular mechanisms of blister formation, mimicking the effects of the polyclonal IgG antibodies found in patients.

A microengraving method for rapid selection of single cells producing antigen-specific antibodies

Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.

Recent advances in idiotype vaccination for the treatment of non-Hodgkin's lymphoma

RECENT ADVANCES IN IDIOTYPE VACCINATION FOR THE TREATIVl]ENT OF NON-HODGKIN'S LYMPHOMA. Hendrik Veelken, Stanford University Medical Center. The hypervariable regions of the antigen receptors (idiotypes, ld) expressed by non-Hodgkin's lymphoma (NHL) cells constitute individual tumor-specific antigens and hence attractive target molecules for induction of specific anti-tumor immune responses. In two notable phase II trials, vaccination of follicular lymphoma patients in clinical remitdon with Id (produced fiom an individual lymphoma-derived hybridoma cell line) was associated with disappearance of minimal residual disease, and superior disease-flee and overall survival in patients who developed a measurable anti-idiotype immune response. However, the vast effort to produce an individual Id vaccine by hybridoma technology has hitherto limited large-seale clinical testing. Therefore, three recombinant approaches for ld vaccine production are currently being evaluated in early clinical trials: Transfection of a cell line with expres~on vectors encoding lymphoma-derived IgH and lgL chains, production of Id scFv fragments in tobacco plants by a hybrid TMV vector, and expression of functionally folded Id Fab fragments in the periplasma orE. coil The current experience with the bscterinl expression system indicates the potential to produce a vaccine within 3 weeks of obtaining a lymphoma biopsy, and yields of up to 20 nag highly purified Fab fragment in a 10 1 fermentation system under GMP criteria. In vitro stimulation experiments with recombinant Id Fab proved the existence of Id-specific CTL in the blood of NHL patients. Immunizations with a mixture ofld Fab and a lipid-based adjuvant i.d., combined with s.c. GMCSF, appear to be safe and well tolerated. Naked DNA and viral vectors Its tumor vaccines Gabriele Pecher Charit6 Humboldt-University Berl~ Dept. of Oncology/Hematology, AG Molecular G~meand Innmmotherapy, Hessische Str. 3-4, 10115 Berlin, Germany

T cell recognition of the immunodominant Sendai virus NP324-332/Kb epitope is focused on the center of the peptide.

The CTL response to Sendai virus in C57BL/6 mice is directed almost exclusively to a single H-2Kb-restricted epitope derived from the virus nucleoprotein, NP324-332 (FAPGNYPAL). We have previously shown that the repertoire of T cells elicited by this epitope following primary Sendai virus infection is very diverse. The current experiments were undertaken to determine how a diverse array of TCR are able to interact with a single class I epitope. Crystallographic analysis of NP324-332 bound to Kb has shown that the side chains of peptide residues F1, G4, N5, and A8 protrude toward the solvent and are potentially available for recognition by the TCR. Notably, the N5 residue protrudes prominently from the peptide-binding site due to its localization on a bulge in the center of NP324-332. To determine the importance of these residues for T cell recognition, we analyzed the response of a large panel of hybridomas to NP324-332 analogues substituted at these four positions. The data suggested that there is dominant recognition of the central G4 and N5 residues at the center of the peptide. However, individual hybridomas exhibited distinct patterns of fine specificity for residues F1 and A8, in that they were dependent on one, both, or neither of these residues for recognition of NP324-332. These data are consistent with a critical role for the G4 and N5 residues in governing NP324-332/Kb recognition by T cells and may have implications for T cell recognition of class-I restricted epitopes in general.

Production of monoclonal antibodies using a secretion capture report web.

We describe a novel method for the production of monoclonal antibodies using a secretion capture report web (SCRW). Following HAT selection in bulk culture, individual hybridomas are encapsulated in biotinylated agarose drops. Antibody secreted by the hybridoma is captured within the agarose drop using an avidin bridge and biotinylated anti-mouse immunoglobulin. The secreted antibody is detected by a fluorescent reporter which can be either a second anti-mouse antibody or an antigen. The binding of the reporter can be quantitated and the desired hybridoma directly cloned by flow cytometry. Multiparameter (i.e., two-color) reporter analysis can also be used to selectively enrich and clone rare hybridomas secreting antibodies directed to unique epitopes. The method allows the characterization of thousands of clones per second and the isolation of hundreds of clones per day.

Contribution of the TCR alpha-chain to the differential recognition of bacterial and retroviral superantigens.

The TCR alpha-chain has been implicated in superantigen recognition by some T cells. In this study, recognition of a retroviral (minor lymphocyte stimulating Ag-1, Mls-1) and a bacterial (staphylococcal enterotoxin B, SEB) superantigen by T cells expressing an identical (transgenic) V beta 8.1 beta-chain and either a V alpha 2+ or V alpha 11+ alpha-chain have been compared. Individual hybridomas were found to vary in their recognition of the two superantigens. Whereas some T cell hybridomas recognized both superantigens comparably, other hybridomas preferentially recognized either Mls-1 or SEB, indicating that the contribution of the TCR alpha-chain to recognition of these superantigens is not equivalent. The degree of heterogeneity in recognition varied between the V alpha 2+ and V alpha 11+ hybridomas, although there were individual hybridomas within each group with distinct patterns of reactivity that varied only in CDR3 alpha. These data are consistent with recent evidence that recognition of Mls-1 and SEB involves distinct TCR/MHC interactions.

Secretion capture and report web: use of affinity derivatized agarose microdroplets for the selection of hybridoma cells

A novel assay is described which allows the entrapment and detection of the immunoglobulin secreted from individual viable hybridoma cells using a secretion capture and reporter web (SCRW). By encapsulating the cells in agarose microdroplets which have been derivatized to create a fluorescent antigen-specific sandwich assay, flow cytometry can be used to identify and sort productive cells from a heterogeneous population. Using agarase, the cells can be recovered from the microdroplets and clonally expanded after selection. The assay has been used to reclone rare secretors from hybridoma cultures and to enhance the production of cultures with poor producers. The assay is easily generalized for the detection of any secreted protein for which specific antibodies or other ligands are available.

Use of mutagens to increase rate of immunoglobulin isotype switching of hybridoma cells.

Isotype switching of hybridoma clones may be essential when the class of the antibody produced does not suit the task for which it was generated. In those instances immunoglobulin (Ig) switch variants can be isolated in vitro but the success of isolating these rare variants primarily depends on the frequency of switching of each individual hybridoma. Variations in the frequency are noted not only between hybridomas secreting different classes but also between fresh clones isolated from the same hybridoma. Immunoglobulin switch variants may be identified and isolated using the sib selection and the ELISA spot assay; however, when the frequency of switching is low, this may be extremely difficult and sometimes impossible. In the present article we demonstrate that ICR191 may increase the frequency of switching and that these Ig switch antibodies maintain the same antigen specificity and normal-sized heavy chain.

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescence-activated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouse-IgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell deposition provides positive attributes of several traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures.