Particle concentration fluorescence immunoassay (PCFIA): a new, rapid immunoassay technique with high sensitivity

Jolley, Michael E.; Wang, Chao Huei J; Ekenberg, Steven J.; Zuelke, Mark S.; Kelso, David M.

doi:10.1016/0022-1759(84)90082-6

Cited by 129 publications

(43 citation statements)

References 49 publications

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“…Supernatants from in vitro cultured plasmacytoma cells were assayed for the presence of immunoglobulin using a Pandex machine (Baxter Healthcare, Deerfield, IL) (14).…”

Section: Methodsmentioning

confidence: 99%

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

Degrassi

Hilbert

Rudikoff

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma ceUls, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmaicytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion.

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“…Supernatants from in vitro cultured plasmacytoma cells were assayed for the presence of immunoglobulin using a Pandex machine (Baxter Healthcare, Deerfield, IL) (14).…”

Section: Methodsmentioning

confidence: 99%

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

Degrassi

Hilbert

Rudikoff

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…The antibodies were purified by protein A chromatography (6). Solid-phase immunoassays were performed on a screen machine (Pandex) by using a protocol similar to that described by Jolly et al (16). Antigens were adsorbed directly onto polystyrene particles (diameter, 0.8 ,um) in a 0.25% working solution of 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) in 0.9% pyrogen-free saline at pH 7.2.…”

mentioning

confidence: 99%

Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12

Rocque¹,

Coughlin²,

McGroarty³

1987

J Bacteriol

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Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies. Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype). Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000. After heating to 100°C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000. The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydratespecific silver stain, while the heated monomer band showed no staining. In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer. Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE. LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies. Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer. Thus, some but not aHl of the LPS could be released from trimer complexes by boiling in SDS. The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.The structure and interactions of the outer membrane porin proteins OmpF and OmpC from Escherichia coli are very similar. They have similar molecular weights and amino acid sequences (22), the native proteins are very high in beta structure (25, 32), both are tightly associated with the peptidoglycan (9, 19, 32), and they are very tightly associated with lipopolysaccharide (LPS) (10,12,36,40). Porins are known to form water-filled channels (2, 33) and are unusually stable to a wide variety of perturbants such as sodium dodecyl sulfate (SDS) detergent (24, 25, 41), pH extremes from 2 to 12 (8), and temperatures up to 70°C (32). Porins are also resistant to many proteases, perhaps because of their tight association with LPS (12); the proteins are thought to be functional only in the presence of LPS (12,20,33). Nakae et al. (24) have suggested that only the lipid component of LPS is essential for porin function. In contrast, Parr and co-workers (30) have reported that porin is functional in the absence of LPS. Thus, the importance of bound LPS for porin structure and function has yet to be resolved.The OmpF porin exists as a trimeric complex (24, 29) with three identical subunits with approximate molecular weights of 36,000 (13, 32). Low SDS binding coupled with the tight, compact shape of the trimer results in more rapid migration on polyacrylamide gels than the aggregate molecular weight would suggest. Investigator...

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“…The pACYC184 plasmid confers chloramphenicol resistance and the pBR plasmid confers ampicillin resistance; it is therefore possible to transform E. coli with both L and H chain plasmids and select cells expressing dual drug resistance phenotypes. To trans- IgG, /xg/ml was 20 Ag/ml as measured by particle concentration fluorescence immunoassay (13). The SG3/7 cells, which also showed resistance to both G418 and mycophenolic acid, produced and secreted only the L chain proteins (Fig.…”

mentioning

confidence: 99%

Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-1A.

Sun¹,

Curtis²,

Rakowicz-Szulczynska³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

109

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We have cloned the genomic DNA fragments encoding the heavy and light chain variable regions of monoclonal antibody 17-1A, and we have inserted them into mammalian expression vectors containing genomic DNA segments encoding human y3 and K constant regions. The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2/0 mouse myeloma cells resulted in the production of functional IgG that retained the specific binding to the surface antigen 17-lA expressed on colorectal carcinoma cells.Monoclonal antibodies (mAbs) are highly specific wellcharacterized reagents. They have found wide applications in vitro for immunochemical characterization and quantitation of antigens. Increasingly they are being used in vivo for both diagnosis and therapy (1). Their in vivo application is limited because in most cases human mAbs of the desired specificity are difficult to prepare (2). Most available mAbs are derived from mouse hybridomas, and their inherent immunogenicity in patients precludes their long-term administration (1). In an attempt to circumvent this problem, chimeric antibodies in which the antigen-specific variable (V) regions of the mouse antibodies are joined to the constant (C) regions of human antibodies have been produced (3, 4). These molecules, which are largely human in composition, should be much less immunogenic and hence should be more suitable for application in vivo.The mouse mAb 17-1A was raised against SW1083 colorectal carcinoma cells and appears to recognize a cancerassociated surface antigen expressed on these cells (5). Here we describe the construction of immunoglobulin genes in which the DNA segments encoding the V regions from the heavy (H) and light (L) chains of this mouse hybridoma were joined to the DNA segments encoding human -y3 and K C regions. Transfection of expression vectors containing these chimeric immunoglobulin genes into mouse myeloma cells resulted in the production of functional chimeric IgG with the same binding specificity as the original hybridoma antibody.MATERIALS AND METHODS cDNA Library Construction. Cytoplasmic RNA was extracted from 17-1A cells (the hybridoma cells that produce mAb 17-1A), and poly(A)+ RNA was prepared by oligo(dT)-cellulose chromatography. Double-stranded cDNA was synthesized with poly(A)+ RNA as a template, using avian myeloblastosis virus reverse transcriptase and Escherichia coli DNA polymerase I. Double-stranded cDNA was treated with S1 nuclease, elongated with deoxycytidine residues, and annealed with oligo(dG)-tailed pUC8 previously cut with Pst I (6). The recombinant plasmids were used to transform E. coli DH1, and colonies were screened according to the general method described by Maniatis et al. (7).Genomic Library Construction. A genomic DNA library for 17-1A cells was constructed in X phage vector EMBL3A. High molecular weight DNA was partially digested with restriction endonuclease Sau3A and size-fractionated on a 10-40% sucrose density gradient. DNA fragments 18-23 kilobases (kb) long were ...

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Particle concentration fluorescence immunoassay (PCFIA): a new, rapid immunoassay technique with high sensitivity

Cited by 129 publications

References 49 publications

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12

Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-1A.

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