Combined use of111In-labeled pentetreotide and three-step immunoscintigraphy with antichromogranin a monoclonal antibody in the diagnosis of pituitary adenomas

For various pituitary adenomas, it has been demonstrated that somatostatin receptor can be present. Pilot studies have shown that radio-indium labeled pentetreotide allows very good scintigraphic localization of somatostatin receptor-bearing cell masses. Recently, the presence of CgA in pituitary adenomas has also been demonstrated. MAb A11, raised against CgA, has been successfully used with a three-step ISG for the diagnosis of neuroendocrine tumors. Therefore the combined use of three-step ISG with MAb A11 and radiolabeled somatostatin can be useful in the diagnosis of pituitary adenomas. Twelve patients, 5 secreting (group A) and 7 nonsecreting (group B) pituitary adenomas, were enrolled in the study. All patients underwent three-step ISG, and, 2 wk later, scintigraphy with¹¹¹In-labeled pentetreotide (Octreoscan). Three-step ISG consisted of iv injection of 1 mg of biotinylated MAb A11 (first step), followed by 10 mg of avidin (second step) and [^99mTc]PnAO-biotin (third step). Tomographic imaging were acquired for three-step ISG and Octreoscan at 2 and 4 h after radiotracer injection, respectively. The results are the following: 2 patients of group A (secreting tumors) had a positive three-step ISG, whereas all the patients but one of the same group had a positive pentetreotide study; all the patients of group B (nonsecreting tumors) had a positive three-step ISG and 4 had a positive pentetreotide scintigraphy. These data suggest the utility of the combined use of these techniques for a better diagnosis of pituitary adenomas.     

Stage-specific Monoclonal Antibodies to the Potato Cyst Nematode Globodera pallida Stone (Behrens)

Using standard hybridoma technology and hierarchical screening, monoclonal antibodies (MAbs) were obtained with specific reactivity against two developmental stages of Globodera pallida. The procedure was based on enzyme-linked immunosorbent assay (ELISA) with homogenates prepared from second-stage juveniles, young adult females, and potato roots. Hybridomas were formed by fusing myelomas with splenocytes derived from mice immunized with either infective juveniles or females of G. pallida. About 600 hybridoma lines were screened from the fusion involving the mouse immunized with juveniles. Two MAbs (LJMAbl &2) were identified with high reactivity toward second-stage juveniles but no reactivity with either potato roots or females of G. pallida. A total of 630 cell lines was screened from the corresponding fusion involving the spleen of a mouse receiving immunogens from adult female nematodes. One MAb (LFMAbl) was obtained with the required specificity against only adult female G. pallida. This work extends the application of monoclonal antibodies in nematology from valuable probes for research and species identification to recognition of developmental stages. These specific MAbs have potential value in plant breeding programs for screening for resistant lines unable to support nematode development.     

Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate

Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 x 10(5) cells/mL to 31 x 10(5) cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 x 10(5) cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, q(Mab), continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. (c) 1993 John Wiley & Sons, Inc.     

Cell retention-chemostat studies of hybridoma cells-analysis of hybridoma growth and metabolism in continuous suspension culture in serum-free medium

The steady-state metabolic parameters for a hybridoma cell line have been determined in continuous suspension-perfusion culture over a wide range of perfusion rates and cell bleed rates. Significant increases in viable cell concentrations and volumetric productivities were achieved at high perfusion rates and low cell bleed rates. At the low growth rates examined in this study, cellular metabolism shifted to become more oxidative, and as a result, the fraction of consumed substrate converted to inhibitory metabolic by-products was reduced. Specific antibody productivity was found to be non-growth associated. (c) 1993 John Wiley & Sons, Inc.     

Loss of antibody productivity is highly reproducible in multiple hybridoma subclones

An immunoglobulin G (IgG(2b)) producing hybridoma cell line (S3H5/gamma2bA2) was cloned and subcloned. Twenty subclones were grown in parallel while being adapted in a stepwise fashion to serum-free medium. Following adaptation to serum-free medium, it was found that 16 of the 20 subclones remained at a relatively constant proportion of nonproducing cells. Three of the remaining subclones transiently deviated from this balance but eventually returned toward this population composition. One subclone continued to lose productivity. A population balance was reached at approximately 8% of the population being nonproducers. The loss of antibody productivity was thus highly reproducible. (c) 1993 John Wiley & Sons, Inc.     

Nerve growth factor antibodies recognize neurotrophin-3

The immunological properties of the neurotrophins NGF, BDNF, and NT-3 were compared using polyclonal and monoclonal antibodies against the subunit of mouse NGF. Affinity-purified anti-NGF IgG consistently recognized NGF and NT-3 on Western blots, and inhibited the trophic activity of NGF and NT-3 but not BDNF. In contrast, anti-NGF monoclonal antibodies did not block the trophic activity of either NT-3 or BDNF. These results are consistent with the greater structural overlap between NGF and NT-3 than between NGF and BDNF.     

In vitro study of the proteolytic activity of rumen anaerobic fungi

Abstract To better define the antigenic structure of the outer cell membranes of Legionellae, a panel of 6 monoclonal antibodies was raised against partially purified outer membranes of Legionella pneumophila serogroup 1, Corby strain. This study describes the purification and characterization of one of these monoclonal antibodies reacting with a 135-kDa protein, which was shown to be common to all 14 serogroups of Legionella pneumophila . It shows no cross-reactivity with 20 other Legionella species, or 9 other Gram-negative species tested by SDS-PAGE and Western blotting procedures. The epitope would appear to be predominantly surface exposed and, from preliminary detergent extraction studies, not peptidoglycan-associated.     

Mouse blastocyst immunosurgery with commercial antiserum to mouse erythrocytes

Summary Immunosurgery is a useful technique for the isolation of inner cell masses from murine blastocysts. Conventionally, rabbit antisera made ad hoc against murine splenic or fetal cells or fibroblasts have been used as antibody sources. We investigated the feasibility of using commercially available rabbit antiserum to murine erythrocytes (anti-RBC) and compared it with rabbit antiserum generated ad hoc to murine L-cells (anti-L-cell). Our results indicate that anti-RBC is at least as effective as anti-L-cell serum for the immunosurgical isolation of inner cell masses, which became either miniblastocysts (later forming outgrowths) or embryoid bodies (undergoing ectoderm-endodermlike differentiation within 48 h). Because anti-RBC is commercially available, the technical modification described herein increases the accessibility of the immunosurgical protocol for the isolation of murine inner cell masses.     

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.     

Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4

Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.