A semi-automated fluorescent (SAF) assay using viable, whole cells for screening hybridoma supernatants

In the production of monoclonal antibodies, a rapid, sensitive, accurate assay is needed for the critical step of screening. We report the modification of an assay using viable whole cells for screening hybridoma supernatants. The modified assay uses fluorescent second antibodies for detection and has been adapted to an instrument capable of automating a number of assay steps. The modified assay is compared to a dot radioimmunoassay developed and used in our laboratory. The fluorescence assay is highly sensitive but shows more background effect, especially in samples with high protein content, such as ascites. The automated fluorescence assay is very rapid, capable of completing an assay in less than 90 min, and can be performed with minimal operator involvement. The assay was performed successfully with several different antibodies and cell types. This screening procedure should be especially useful for laboratories with large numbers of fusions to evaluate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26678/1/0000222.pd

High-dose, unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to “blockade” nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 μg of 131 I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of 125 I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before 131 I tumor-specific monoclonal antibody, will not enhance tumor imaging.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46853/1/262_2004_Article_BF00205633.pd

Radiolabeled antibodies, albumin and antimony sulfide colloid: A comparison as lymphoscintigraphic agents

The kinetics of lymph node and systemic uptake of members of three different classes of lymphoscintigraphic agents were studied in normal laboratory rats. 99mTc antimony trisulfide colloid (TcSbC), 99mTc human serum albumin (TcHSA), 125I 5G6.4 (a murine IgG2ak monoclonal antibody), 125I 763.24T (a murine IgG1), and 125I FT166 (a murine IgM monoclonal) all current or potential lymphoscintigraphic agents, were injected subcutaneously into the hind foot pads of healthy rats. Ipsilateral and contralateral popliteal lymph nodes were sampled up to 4 h post-injection. Subcutaneous injection resulted in far higher nodal uptake for all agents than i.v. delivery with ipsilateral popliteal node/blood ratios 1 h postsubcutaneous injection of: for TcSbC (1900)>125I IgM (497)>TcHSA (72)>125I Intact IgG2a or 125I IgG1 at approximately 10. Thus, while all agents achieve popliteal node/blood ratios far greater than unity, TcSbc has the greatest absolute and relative nodal accumulation, greater than the 125I IgM monoclonal antibody and TcHSA. Uptake of the intact 125I IgG antibodies is lowest. These data suggest that TcSbC in particular, as well as TcHSA and IgM may be most useful as non-specific nodel imaging agents, while the lower background activity of the IgGs may make targeting specific antigen in nodes more feasible.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27517/1/0000561.pd

Monoclonal antibody (5G6.4) against ovarian carcinoma shows inhibition of in vitro colony formation

Monoclonal antibodies (MAbs) have the potential for diagnosis and therapy of cancer. 5G6.4 is a MAb of the IgG2a class which was produced by immunization of BALB/c mice with human ovarian carcinoma (Ov Ca) cells. To further characterize 5G6.4, its effect on cell growth was tested using a human Ov Ca cell line established in our laboratory. A clonogenic assay was set up in 48-well plates in a double agar system. The cells were plated and 5G6.4 was added at different concentrations. Control plates consisted of cells with media without MAb. Negative control plates were also prepared using the same concentrations of an isotype-matched antimelanoma MAb, 225.28s. Colony formation (CF) was reduced to 50% or less of control with increasing amounts of 5G6.4 up to 50 [mu]g/ml. Although CF was still depressed at concentrations above 50 [mu]g/ml, the inhibition did not follow a directly proportional line; instead, it followed a bell-shaped curve. Plates with the control MAb, 225.28s, did not show this response. Similar results were obtained with cells from malignant Ov Ca ascites in the same clonogenic assay. Our study suggests that in the evaluation of the in vitro effect of MAb on growth, the concentration of MAb is crucial and may not show a linear response and that 5G6.4 may have a direct therapeutic effect by blocking the growth of Ov Ca cells. 5G6.4 is presently under study for therapy in an animal model.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26666/1/0000210.pd

The intraperitoneal delivery of radiolabeled monoclonal antibodies: studies on the regional delivery advantage

The i.p. delivery of murine monoclonal antibody was compared with i.v. delivery in normal mice and rats, in normal nude mice and in those with i.p. human ovarian carcinoma xenografts. In normal rats, all classes of antibodies and antibody fragments evaluated were cleared from the peritoneal cavity at comparable rates. The regional delivery (Rd 1 ) advantage to the peritoneal cavity following i.p. delivery was thus most dependent on the rate of clearance of the antibody or fragment from the blood stream. Determining the exact i.p. delivery advantage was problematic due to the difficulty in reliably obtaining peritoneal fluid later than 9–10 h after i.p. injection in normal animals. During the first 9 h following i.p. injection, the Rd(0–9/0–9) was, for a murine IgG2ak Fab>F(ab′) 2 >IgG (at 13.6>10>7.9). Two murine IgMs evaluated differed in Rd(0–9) at 27.1 and 9.2 respectively. When blood levels were extrapolated to infinity, these Rd (0–9/∞) values were considerably lower with the Fab having the highest Rd at 4.67. The i.p. Rd advantage was almost solely due to the i.p. antibody levels seen in the first 24 h after injection, as after that time, blood levels become comparable to those seen following i.v. injection. Normal tissues obtained at sacrifice 5–7 days after i.p. injection. Normal tissues obtained at sacrifice 5–7 days after i.p. or i.v. injection in rats showed comparable levels of radioantibody activity, whether the injection was i.p. or i.v. (except for higher diaphragmatic levels following i.p. delivery). In nude mice with i.p. human-derived ovarian tumors, intact IgG clearance from the peritoneal cavity to the blood was considerably slower than in normal animals, and early i.p. tumor uptake of specific antibody was significantly higher than that following i.v. antibody delivery. With higher early tumor uptake and lower systemic exposure, early tumor/nontumor ratios were significantly greater than those for i.v. delivery, though not beyond 48 h after i.p. injection. This study demonstrates the pharmacokinetic rationale for i.p. monoclonal antibody delivery, especially for agents cleared rapidly from the blood, such as antibody fragments. In addition, definite i.p. delivery benefit for antibody specific to i.p. tumors in the i.p. ovarian cancer system was shown soon after injection. These data regarding i.p. antibody delivery should be useful in rationally planning diagnostic and therapeutic studies involving the i.p. delivery of unmodified and immunoconjugated monoclonal antibodies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46854/1/262_2004_Article_BF00199929.pd

Interleukin-8 is essential for normal urothelial cell survival

Interleukin-8 (IL-8; CXCL8) has been shown to play a role in multiple cellular processes. Here, we report an additional role of IL-8 as a growth and essential survival factor for normal human urothelial cells. Supplementing exogenous recombinant human IL-8 to normal urothelial cells promoted cell growth through the Akt pathway. Inhibition of IL-8 expression by small inhibitory RNA (siRNA) caused normal urothelial cells to die. Addition of recombinant human IL-8 rescued the normal urothelial cells treated with IL-8 siRNA. This rescue effect could be blocked by antibodies to the IL-8 receptor CXCR1 but not by CXCR2, suggesting that normal urothelial cells normally have IL-8 autocrine or paracrine activity for survival and growth mediated by CXCR1. IL-8 mRNA levels were lower in samples from patients with interstitial cystitis, a urinary bladder disorder associated with urothelial cell dysfunction and/or loss. Taken together, these results suggest that IL-8 is an important normal urothelial growth factor and is necessary for normal urothelial cell survival in vitro and in vivo. Lower IL-8 expression levels in the urinary bladder may contribute to pathophysiology of interstitial cystitis

Immunologic Detection of the Cellular Receptor for Urokinase Plasminogen Activator

The cellular receptor for urokinase plasminogen activator (uPA-R) is a monomeric phosphatidylinositol-linked glycoprotein (gp40-65) that may contribute to the invasive capacity of tumor and inflammatory cells by focusing the activity of urokinase (uPA) in converting plasminogen to plasmin, a serine protease capable of degrading extracellular matrix proteins. The further characterization of uPA-R has been facilitated by our recent development of a monoclonal antibody, anti-Mo3f, specific for uPA-R. This mAb bound to uPA-R expressed by phorbol myristate acetate-stimulated U-937 cells and by NIH-3T3 cells permanently transfected with uPA-R cDNA. In competitive binding assays, anti-Mo3f inhibited the binding of fluorescein-conjugated uPA ligand to uPA-R expressed by U-937 cells and uPA-R transfectants; conversely, preexposure of cells to saturating quantities of exogenous uPA partially blocked the subsequent binding of anti-Mo3f mAb to uPA-R. Anti-Mo3f mAb was employed as the capture reagent in an ELISA for the quantitation of soluble forms of uPA-R (derived from U-937 cells and recombinant uPA-R) which had a sensitivity of approximately 4-12 ng/ml. Anti-Mo3f mAb was also applied as a serologic probe for the detection of uPA-R expressed by human tumor tissues. By immunoperoxidase staining, anti-Mo3f demonstrated positive tumor cell staining in 4 of 16 breast and 7 of 31 prostate carcinomas in formalin-fixed, paraffin-embedded specimens. These data indicate that the anti-M03f mAb detects an epitope proximate to or within the ligand binding domain (domain 1) of uPA-R and may be useful as a tool for the serologic detection of uPA-R in soluble form or associated with human tumors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31653/1/0000587.pd