Yoshihiro Komada

A monoclonal antibody (SJ-9A4) to P24 present on common alls, neuroblastomas and platelets - I. Characterization and development of a unique radioimmunometric assay.

We report the development and characterization of SJ-9A4, a monoclonal antibody (MoAb) produced against common acute lymphoblastic leukemia (C-ALL) cell lines. SJ-9A4 reacted with C-ALL, B-cell chronic lymphocytic leukemia (B-CLL), platelets and C-ALL neuroblastoma (NB) and the K562 cell lines. It had no significant reactivity with erythrocytes, granulocytes, circulating T or B lymphocytes, monocytes, granulocytic cell lines or a Ewings sarcoma cell line. SJ-9A4 was shown to recognize the same region as two other MoAb to the p24 antigen, BA-2 and DU-ALL-1, as demonstrated by their ability to inhibit the binding of labeled SJ-9A4 to NALM-1 and NB cells. Other MoAb: J5, PI 153/3 and monoclonal anti-HLA-DR antibodies gave no inhibition. A solid phase indirect radioimmunometric assay (IRA) was developed which enabled the detection of P24 from C-ALL cells, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) or wheat germ agglutinin (WGA) and SJ-9A4 simultaneously. When BA-2 and DU-ALL-1 were used in place of SJ-9A4, similar IRA results were obtained. Using the RCA1/SJ-9A4-IRA, P24 from as few as 1.6 X 10(4) cells of a C-ALL cell line could be detected; however, similar extracts of NB cell lines were negative despite high levels of SJ-9A4 binding to intact cells. The presence of P24 in NB extracts was demonstrated by (1) preincubation of NB extracts with SJ-9A4 which blocked MoAb binding to P24 and (2) immunoadsorption of P24 from solubilized membranes of 35S-methionine (met) labeled NB cells. Treatment of NB cells with neuraminidase did not result in IRA binding when either RCA1 or WGA were used as the solid phase lectin indicating that the differences in lectin affinity are not due to over sialation of NB membrane glycoproteins. These findings demonstrate a difference in the glycosylation of P24 from C-ALL and NB cells.

Shedding of the common acute lymphoblastic leukemia antigen (calla) by lymphoblastoid cell lines

The release of common acute lymphoblastic leukemia antigen (CALLA) into culture medium by lymphoblastoid cell lines was studied. CALLA release was detected using a solid phase indirect radioimmunometric assay which combines the specificity of lectins and monoclonal antibodies and using immunoadsorption of labeled CALLA in spent medium from cells incubated with 35S-methionine. No CALLA was present in the medium of cells pulse-labeled at 37 degrees C, when they were placed at 4 degrees C, thus this is an active process. Sequential analysis of soluble CALLA revealed shedding of CALLA to be associated with cell growth.

A monoclonal antibody (SJ-9A4) to P24 present on common ALLS, neuroblastomas and platelets — II. Characterization of P24 and shedding in vitro and in vivo

The release of soluble P24 antigen into culture medium by common acute lymphoblastic leukemia (C-ALL) and neuroblastoma (NB) cell lines was studied. P24 release by C-ALL cells was detected using a solid phase indirect radioimmunometric assay (IRA) which combines the specificity of lectins and monoclonal antibodies (MoAb) and using immunoadsorption of labeled P24 in spent medium from cells incubated with 35S-methionine (met). No P24 was present in the medium of cells pulse labeled at 37 degrees C when they were placed at 4 degrees C, thus this is an active process. P24 release by NB cells could not be detected by IRA, but could be detected by immunoadsorption of spent medium of metabolically-labeled cells. The absence of IRA activity of P24 from NB spent medium was due to decreased glycosylation and thus no binding to the lectins employed in the IRA was observed. This was confirmed by lectin affinity chromatography which showed that P24 in the spent medium from C-ALL cells bound Ricinus communis agglutinin (RCA1), wheat germ agglutinin (WGA), concanavalin A (Con A), and lentil lectin (LcH), but not peanut agglutinin (PNA). P24 from NB cell spent medium did not bind to any of these lectins. The lectin affinity of P24 derived from lymphoblasts is consistent with the presence of N-linked oligosaccharide chains having N-acetyl glucosamine residues, a mannose core, and a terminal D-galactose. P24 from C-ALL cell spent medium was present in the 35-45% fraction of a saturated ammonium sulfate (SAS) partition of spent medium. The P24 antigen was detected in the fractionated plasma of five patients with C-ALL at the time of diagnosis and was undetectable when the patients had achieved a complete remission. Plasma from 2 patients with P24 negative ALL, normal human plasma, and normal human serum had no detectable activity.