An ICP-MS platform for metal content assessment of cell culture media and evaluation of spikes in metal concentration on the quality of an IgG3:κ monoclonal antibody during production.

Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced.

Assessment of humoral immune responses to blood-stage malaria antigens following ChAd63-MVA immunization, controlled human malaria infection and natural exposure.

The development of protective vaccines against many difficult infectious pathogens will necessitate the induction of effective antibody responses. Here we assess humoral immune responses against two antigens from the blood-stage merozoite of the Plasmodium falciparum human malaria parasite--MSP1 and AMA1. These antigens were delivered to healthy malaria-naïve adult volunteers in Phase Ia clinical trials using recombinant replication-deficient viral vectors--ChAd63 to prime the immune response and MVA to boost. In subsequent Phase IIa clinical trials, immunized volunteers underwent controlled human malaria infection (CHMI) with P. falciparum to assess vaccine efficacy, whereby all but one volunteer developed low-density blood-stage parasitemia. Here we assess serum antibody responses against both the MSP1 and AMA1 antigens following i) ChAd63-MVA immunization, ii) immunization and CHMI, and iii) primary malaria exposure in the context of CHMI in unimmunized control volunteers. Responses were also assessed in a cohort of naturally-immune Kenyan adults to provide comparison with those induced by a lifetime of natural malaria exposure. Serum antibody responses against MSP1 and AMA1 were characterized in terms of i) total IgG responses before and after CHMI, ii) responses to allelic variants of MSP1 and AMA1, iii) functional growth inhibitory activity (GIA), iv) IgG avidity, and v) isotype responses (IgG1-4, IgA and IgM). These data provide the first in-depth assessment of the quality of adenovirus-MVA vaccine-induced antibody responses in humans, along with assessment of how these responses are modulated by subsequent low-density parasite exposure. Notable differences were observed in qualitative aspects of the human antibody responses against these malaria antigens depending on the means of their induction and/or exposure of the host to the malaria parasite. Given the continued clinical development of viral vectored vaccines for malaria and a range of other diseases targets, these data should help to guide further immuno-monitoring studies of vaccine-induced human antibody responses.

Quantitative immunohistochemical assessment of IgA, IgM, IgG and antigen-specific immunoglobulin secreting plasma cells in pig small intestinal lamina propria.

Intestinal immune response plays an important defensive role for pathogens, particularly for those transmitted by the oro-faecal route or for foecal shedding modulation. This work examined three parts of intestine from twelve gilts experimentally infected with PCV2-spiked semen, six vaccinated (V group) and six unvaccinated (NV group) against PCV2, 29 and 53 days post infection (DPI). An immunohistochemical investigation for IgA-, IgG- and IgM-antibody bearing plasma cells (PCs) was run on intestinal samples coupled with a sandwich immunohistochemical method to reveal anti-PCV2 antibody-secreting PCs. Plasma cell density was compared in the two groups of animals at 29 and 53 DPI. The IgA, IgG and IgM PC density did not differ between groups but displayed an increase from the upper (villus) to the lower part of the crypts while a decreasing trend in PC density was identified from duodenum to ileum. In the NV group, no increase in anti-PCV2 PC density was demonstrable in the two sampling moment: the amounts of lamina propria PCV2-specific antibody-producing PCs remained constant, 10.55 ± 4.24 and 10.06 ± 5.01 at 29 DPI and 53 DPI, respectively. In the V group a significant increase in PCV2-specific antibody-producing PCs was observed over time. The amounts of PCV2-specific antibody-producing PCs increased from 9.37 ± 13.36 at 29 DPI to 18.76 ± 15.83 at 53 DPI. The data on IgA, IgM and IgG PC counts can be considered reference values in a population of adult pigs. The sandwich method can be proposed as a technique able to identify specific antibody-secreting PCs in formalin-fixed paraffin-embedded tissues. A practical application of the sandwich method is the demonstration of a "booster-like" response of the lamina propria in vaccinated compared to unvaccinated animals. After virus challenge, vaccination induced an increase in the number of PCs containing specific anti-PCV2 antibodies at the level of intestinal mucosa.

Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections.

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.

Protective effects of Mentha haplocalyx ethanol extract (MH) in a mouse model of allergic asthma.

Mentha haplocalyx Briq., a commonly used herb in traditional Oriental medicine, has a variety of known pharmacological properties. However, neither the protective effects of Mentha haplocalyx ethanol extract (MH) against inflammation of the airway in an asthmatic model nor the mechanisms involved, have previously been reported. In the present study, an ovalbumin (OVA)-induced mouse model of allergic asthma was used to investigate whether MH was effective against the disease through regulation of airway inflammation. The MH treatment significantly inhibited increases in immunoglobulin (Ig) E and T-helper 2 (Th2)-type cytokines such as IL-4 and IL-5 in bronchoalveolar lavage fluid (BALF) and lung tissue. Inflammatory cell infiltration of the airway in mice treated with MH was effectively alleviated when compared with infiltration seen in the OVA-induced group. These data indicated that decreased cytokine levels are the result of the decreased number of invaded leukocytes. Also, the generation of reactive oxygen species (ROS) in BALF was diminished by MH treatment. Taken together, these findings indicate that the administration of MH may have potential therapeutic value in the treatment of inflammatory disease.

Novel micro-bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed-batch CHO cultures.

With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench-top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high-throughput (HT) technology for process development. One such high-throughput system is the SimCell platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell micro-bioreactor technology for fed-batch cultivation of a GS-CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas-permeable chambers based on a micro-fluidic design, with six micro-bioreactors (MBs) per micro-bioreactor array (MBA). Online, non-invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3- and 100-L bioreactor scales. The results of the study demonstrate that the SimCell platform operated under fed-batch conditions could support viable cell concentrations up to least 12 x 10(6) cells/mL. In addition, both intra-MB (MB to MB) as well as intra-MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra-MB and -MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) x 100. The % CV values for most intra-MB and intra-MBA measurements were generally under 10% and the intra-MBA values were slightly lower than those for intra-MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench-top, and pilot scale bioreactor cultivations and found to be within +/-20% of the historical averages.

Rapid production of recombinant human IgG With improved ADCC effector function in a transient expression system.

Rapid production of recombinant human IgG with improved antibody dependent cell-mediated cytotoxicity (ADCC) effector function is presented. The technique employs transient expression of IgG in suspension growing HEK-293F cells in the presence of the glycosidase inhibitor kifunensine. The procedure takes approximately 7 days, provided that expression plasmids encoding the IgG of interest are available. Kifunensine inhibits the N-linked glycosylation pathway of HEK-293F cells in the endoplasmatic reticulum, resulting in IgG with oligomannose type glycans lacking core-fucose. IgG1 transiently produced in kifunensine- treated HEK-293F cells has improved affinity for the FcgammaRIIIA molecule as measured in an ELISA based assay, and almost eightfold enhanced ADCC using primary peripheral blood mononuclear effector cells.

4th European Antibody Congress 2008: December 1-3, 2008, Geneva, Switzerland.

The Fourth European Antibody meeting, organized by Terrapin Ltd., was held in Geneva, a center of the European biopharmaceutical industry. Merck-Serono, NovImmune, Pierre Fabre and Therapeomic are located nearby, as are R&D centers of Boehringer-Ingelheim, Novartis, Roche and Sanofi-Aventis. Over 40 speakers and more than 200 delegates attended the event. Companies represented included Abbott, Ablynx, Adnexus/ BMS, Astra-Zeneca/ CAT/ Medimmune, BiogenIdec, BioRad, Centocor (Johnson & Johnson), Crucell/ DSM, Domantis, Dyax, Genmab, Genzyme, Glycart/ Roche, Haptogen, Immunogen, Kyowa-Kirin, LFB, Medarex, Merck-Serono, Micromet, Novartis, Pierre Fabre Laboratories, Roche, Sanofi-Aventis, Seattle-Genetics, Transgene, UCB Celltech and Wyeth. Other attendees included those based in academe or government (University of Amsterdam, University of Zurich, University Hospital-Lyon, Ecole Polytechnique Federale de Lausanne, INSERM, Tufts University, US National Institutes of Health), consultants, and patent attorneys (Edwards, Angell, Palmer & Dodge). The meeting was very interactive and included exchanges during the many scheduled networking times (exhibitions, speed-networking, lunches and evening receptions). The first day of the three day conference was dedicated to advances in understanding antibody structure-function relationships. Challenges and opportunities in antibody development were the focus of the second day and the third day featured discussion of innovative antibodies and antibody alternatives.

Comparison of mouse and rabbit model for the assessment of strong PGM-containing oil-based adjuvants.

Peptidoglycan monomer (PGM) is an adjuvant active molecule with potential for use in human and veterinary vaccine. PGM's action is short-lived in mammals hence its effects might be limited. Novel PGM-containing oil-based formulations have been developed recently by incorporation of PGM into Montanide ISA720 and ISA206 adjuvants with the aim to prolong and improve immunostimulating activities of PGM. In the present work we studied the efficacy of such novel adjuvant formulations using two different antigens, ovalbumin and snake venom, respectively. Novel formulations were also tested in two experimental models, mice and rabbits. In rabbits the incorporation of PGM into oil-based adjuvants led to overall improvement of antigen-specific IgG response. However, in the mouse model, under experimental conditions used, it was not possible to distinguish differences in antigen-specific IgG response among several strong oil-based adjuvant formulations.

Immunogenicity, safety and tolerability of meningococcal C CRM197 conjugate vaccine administered 3, 5 and 11 months post-natally to pre- and full-term infants.

A total of 79 pre-term infants with a gestational age > or =32 weeks and 74 full-term infants were studied in order to evaluate the immunogenicity, safety and tolerability of meningococcal C (MenC)-CRM(197) conjugate vaccine administered 3, 5 and 11 months post-natally. The evoked immune response seemed to be substantially similar in the pre- and full-term infants, and there were only clinically marginal differences in safety and tolerability between the groups. The results support the use of two doses of MenC-CRM(197) vaccine at 3 and 5 months of age for primary immunisation, with a booster dose being given at about 1 year. In addition to reducing costs, this scheme seems to assure global immunogenicity and potential efficacy that is better than that offered by the accelerated scheme of administration with only three doses of vaccine in the first months of life, and similar to that observed with a fourth dose used as booster after the first year.

Tsetse fly saliva biases the immune response to Th2 and induces anti-vector antibodies that are a useful tool for exposure assessment.

Tsetse flies (Glossina sp.) are blood-feeding dipteran insects that transmit African trypanosomes, parasites that are responsible for human sleeping sickness and veterinary infections. Increasing attention is being paid to the effects of tsetse fly saliva deposited at the feeding site, which enables the blood-feeding process and putatively promotes parasite transmission. Here we demonstrate that saliva induces strong humoral responses against the major 43-45 kDa protein fraction (tsetse salivary gland proteins 1 and 2 - Tsal1 and Tsal2) in mice and humans and suppresses murine T and B cell responses to heterologous antigen. The saliva-induced immune response is associated with a Th2-biased cytokine profile and the production of mainly IgG1 and IgE antibody isotypes. Functionally, the antibodies raised in mice exposed to tsetse fly bites or induced after experimental saliva immunisation do not affect the fly's blood-feeding efficiency nor its survival. We propose that anti-saliva as well as anti-Tsal1/2 antibody responses can be used in epidemiological studies as a tool to analyze human exposure to tsetse flies.

IGIV: contents, properties, and methods of industrial production--evolving closer to a more physiologic product.

Is the process the product? Immune globulin intravenous (IGIV) is not manufactured, but is purified (fractionated) from human plasma. Machines can only damage what Mother Nature makes; they cannot improve it. Therefore, fractionators of biologic molecules must strive to ensure what is taken from a human body is exactly the same when it is returned to the human body for optimal tolerability and safety. The processes of purification have the potential to adversely affect the product. Four primary purification processes exist for commercial IGIV. The Cohn-Oncley process is 1940s technology, which has been modified through the decades, but the basic process remains unchanged. The Kistler-Nitschmann process was developed in the 1950s by the Central Laboratory of the Swiss Red Cross (ZLB, today known as ZLB-Behring, a subsidiary of CSL Limited). Various attempts have been made to utilize chromatography as the sole separation technology without much success. Most recently, Bayer HealthCare (Talecris Biotherapeutics acquired the contributed assets of the worldwide plasma business of Bayer Biological Products and became operational April 1, 2005; all plasma-based products, including Gamunex, Prolastin, the hyperimmune line (Fraction II), Plasbumin (Bayer Albumin), Koate DVI, and Thrombate III were included) introduced a new product into the United States and Canada that utilizes caprylate and chromatography for high purity, better yields, and integration of safety and efficacy. This is the first new IGIV purification technology in over 20 years.

Humoral heart rejection (severe allograft dysfunction with no signs of cellular rejection or ischemia): incidence, management, and the value of C4d for diagnosis.

Severe allograft dysfunction after heart transplant (HT), without ischemia or evidence of cellular rejection upon endomyocardial biopsy (EMB), is a rare but potentially fatal condition that suggests humoral rejection (HR). Its incidence, and the methods of choice for its diagnosis and management, remain uncertain. We retrospectively studied 445 HT patients (April 1991-December 2003) to determine incidence of HR diagnosed by clinical and conventional histopathological criteria. We used immunofluorescence (IF) techniques to test archived frozen EMB issue for IgM, IgG, C1q, C3, fibrin and C4d. Twelve patients (2.7%) fulfilled the criteria for HR after a mean time post-HT of 21.3 +/- 24.7 months (range: 2-72 months). Patients were treated with high doses of steroids and plasmapheresis, with successful recovery in 11 cases. IF studies using classical markers were mainly negative for the six patients with enough EMB tissue for testing. All six patients showed positivity for C4d during the HR episode but not before or after. Humoral rejection was observed in less than 3% of HT patients. Plasmapheresis treatment was highly effective. Classical IF tests were not useful for diagnosis, but C4d appears to be useful both for confirmation of diagnosis and for monitoring response to treatment.

The cost of a specific immune response in young guinea pigs.

The specific immune system is a protective mechanism that detects infection and fights it by production of antibodies. Newborns are especially susceptible to infections because their immune system is not yet as fully developed as that of adults. This has been well established in altricial mammals. Fighting infection is associated with costs (metabolic rate, protein synthesis) potentially affecting other developmental processes. We investigated the specific immune response in a precocial mammal, by testing the response of 3 and 7 day old young guinea pigs (Cavia aperea f. porcellus) against a non-pathogenic antigen (KLH) and determined the effect of the immune response on growth and metabolic rate. Challenged young produced a substantial specific immune response (IgG). The efficiency of the immune response was almost identical in 3 and 7 day old young, but lower than in adult females. Antibody titres achieved by actively immunised young pups were as high as titres transferred transplacentally by mothers immunised on day 40 and 47 of pregnancy. In comparison to a control group, the immune response did not influence growth and metabolic rate measured on day 4 after each immune challenge and was not reflected by changes in hematocrit value. We discuss whether the weaker immune response of pups is caused by reduced allocation of limited resources in growing young or by the immature immune system of young animals.

Immunological tools for the assessment of both humoral and cellular immune responses in Foxes (Vulpes vulpes) using ovalbumin and cholera toxin B as an antigenic model.

The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterized, and specific immunological tools are needed. To investigate both the humoral and cellular immune response, we used ovalbumin (OVA) and cholera toxin B (CTB) as an antigenic model to set-up ELISA and ELISPOT antibodies secreting cells (ASC) assays in the fox model. Identification of antibodies that cross-react with fox immunoglobulin was performed by Western blot, and their use was adapted for both the ELISA and ELISPOT ASC assay. The humoral and cellular specific immune responses were assessed after intra-muscular or intra-nasal immunization. Intra-muscular immunization resulted in the development of both cellular and humoral anti-OVA and anti-CTB responses in peripheral blood mononuclear cells (PBMCs). Immunization via the intra-nasal route resulted in the development of a cellular and humoral response against CTB in PBMCs. This immune response was confirmed using splenocytes from immunized animals by ELISPOT assay at euthanasia. Females immunized via the intra-nasal route developed specific anti-CTB IgM, IgA and IgG in vaginal fluids after the initial boost (day 26) showing that mucosal immunization produces a vaginal immune response in foxes. These immunological tools developed here are now available to be adapted to other antigenic models to facilitate further immune studies in foxes.

Evaluation of intrathecal synthesis of specific IgG antibodies against Toxoplasma gondii in the diagnosis assessment of presumptive toxoplasma encephalitis in AIDS patients.

The diagnosis of neurotoxoplasmosis in patients with acquired immunodeficiency syndrome is mainly based on tomographic or magnetic resonance findings and on the response to specific treatment. We studied 55 patients with AIDS and neurotoxoplasmosis according to these diagnostic criteria (group 1), 37 patients with AIDS and neurological involvement of other etiology (group 2), and 16 anti-HIV-negative individuals with neurological manifestations (group 3). Serum and cerebrospinal fluid were examined for the presence of anti-T. gondii IgG, by indirect immunofluorescence. In 72 of them, the total amounts of these antibodies were determined in order to assess local production of anti-T. gondii antibodies in the central nervous system and to correlate their titers with infection activity in patients with AIDS and neurotoxoplasmosis. IgG titers > or = 1/64 in cerebrospinal fluid reached 100% specificity for the diagnosis of neurotoxoplasmosis in AIDS. Evidence of local synthesis of these antibodies was detected in 42.8% of patients of group 1, in 29.1% of patients of group 2 and in no patient of group 3. The test showed 70.8% specificity and therefore was not useful in our study for the differential diagnosis of neurotoxoplasmosis in patients with AIDS.

Assessment of the inherent allergenic potential of proteins in mice.

There is considerable interest in the design of approaches that will permit the accurate identification and characterization of proteins that have the inherent potential to induce sensitization and cause food allergy. Among the methods used currently as part of such assessments are consideration of structural similarity to, or amino acid sequence homology with, known human allergens; whether there exists immunologic cross-reactivity with known allergens; and measurement of resistance to proteolytic digestion in a simulated gastric fluid. Although such approaches provide information that will contribute to a safety assessment, they do not--either individually or collectively--provide a direct evaluation of the ability of a novel protein to cause allergic sensitization. For this reason, work is in progress to design and evaluate suitable animal models that will provide a more holistic assessment of allergenic potential. In this laboratory, the approach we have taken has been to examine the characteristics of immune responses induced in mice following parenteral (intraperitoneal) exposure to test proteins. The basis of this method is to determine simultaneously the overall immunogenic potential of proteins [measured as a function of immunoglobulin (Ig) G antibody responses] and to compare this with their ability to provoke IgE antibody production, IgE being the antibody that effects allergic sensitization. Although this approach has not yet been evaluated fully, the results available to date suggest that it will be possible to distinguish proteins that have the inherent potential to induce allergic sensitization from those that do not. In this article we summarize progress to date in the context of the scientific background against which such methods are being developed.

Assessment of safety and the immune response to the CD4 "internal antigen" mouse anti-idiotypic Mab 16D7 in four patients with SLE.

We have analyzed the immune response elicited with the human CD4 internal antigen anti-idiotypic monoclonal antibody 16D7 in four patients with active systemic lupus erythematosus and assessed the safety of the treatment. Patients I and 2 received three 2-mg 16D7 subcutaneous (SC) injections at 3-week intervals and mainly developed IgGI, whereas IgG1, IgG3, and IgG4 were detected in the sera of the other two patients (3 and 4) who received the same amount of 16D7 on days 0, 28, and 70. 16D7-F(ab')2 isotypic determinant-specific antibodies levels, evaluated by sera reactivity with the 16D7-isotype matched anti-idiotypic monoclonal antibody 14D6-F(ab')2, were low or undetectable in patient 1 and became detectable following the first and second boosters in patient 3 and patients 2 and 4, respectively. The highest level was seen in patients 3 and 4. The focusing pattern (spectrotype) of 16D7 idiotypic-specific antibodies suggested that multiple V genes are involved or many somatic mutations occur. Once established, each patient's spectrotype remained stable. Although spectrotype were individually distinct, all four patients produced CD4-specific antibodies, indicating that this response crosses the genetic barrier. Disease relapsed after 11 (patient 2), 40 (patients 3 and 4), and 125 (patient 1) weeks. The lack of side effects and the prolonged periods of disease control (33 and 103 weeks after the last booster) warrants an extension of this study.

Assessment of CD10 in the diagnosis of small B-cell lymphomas: a multiparameter flow cytometric study.

We evaluated the usefulness of multiparameter flow cytometry with cluster analysis in the diagnosis of a series of 100 well-characterized small B-cell lymphomas (SBCLs). The histologic diagnoses in the 100 cases were follicular lymphoma (FL) in 58, marginal zone lymphoma (MZL) in 17, small lymphocytic lymphoma in 15, and mantle cell lymphoma (MCL) in 10. Of the 58 FLs, 57 were CD10 positive (98% sensitivity). The 1 negative case was unusual in that it occurred in the small intestine. However; architectural, cytologic, and immunohistochemicalfeatures were diagnostic of FL. Of 42 other SBCLs, 2 were CD10+ (95% specificity); 1 was a CD5+/cyclin D1 + MCL, and the other was an extranodal MZL. We found that assessment of CD10 expression using multiparameter flow cytometry with cluster analysis is highly sensitive and specific for the diagnosis of FL, validating its usefulness in situations in which adequate tissue is not available for definitive histologic diagnosis.

In vitro production of immunoglobulins of various classes and subclasses by cord blood B cells in African neonates: modeling and assessment of determination.

Cord blood B cells obtained from neonates of healthy Senegalese mothers were assayed in vitro for their capacity to fully differentiate and secrete immunoglobulins (Ig) of various classes and subclasses. Stimulation of mononuclear cells with SAC particles or anti-micro antibodies in the presence of IL-4, or with IL-2 and IL-10 induced a strong production of IgG, provided that an additional CD40/CD40L signal was present, in contrast to adult cell cultures. Cord blood mononuclear cells differentially stimulated with various cytokines in order to lead to Ig heavy chain switching and production of the various classes/subclasses consistently produced IgG1, IgG3, IgG4, IgE and IgA. This system has been applied to immune cells from African neonates that have not been extensively studied previously. Estimation of Ig production as OD ratios could be applied to cultures where cord blood B cells are stimulated with defined antigens of human pathogens to which the fetus immune system was primed in utero.