An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0·8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO± lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.Some metalloenzyme properties of the phosphatase were also studied and it was demonstrated that both Mg2+ and Zn 2 + ions are necessary for hydrolytic activity.Treatment with neuraminidase retarded the anodal migration of the enzyme during electrophoresis on cellulose acetate membranes but did not influence its activity or catalytic properties. These results suggest that the uterine alkaline phosphatase is a sialoglycoprotein. The sialic acid residues, however, do not appear to constitute part of the active centre of the enzyme.In addition, the optimum pH for activity depended on substrate concentration and decreased with decreasing substrate concentration. Apparent Km values also depended on variations in pH and decreased with decreasing pH. Plots of pKm versus pH revealed a functional group with a pK value of 9 ·45. The enzyme also hydrolysed a variety of compounds having either phosphomonoester or pyrophosphate linkages and was inactivated after heating at 60°C for 15 min. The activation energy, determined from a linear Arrhenius plot, was 50·1 kJmol-'.

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody C017-1A (Abl) developed anti-idiotypic antibodies (Ab2) to the Abl. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Abl-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Abl and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with AbM and were Abl-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.We have previously demonstrated that gastrointestinal cancer patients treated with anti-colorectal carcinoma (CRC) monoclonal antibody (mAb) C017-1A, referred to hereafter as Abl, raised anti-idiotypic antibodies (Ab2) to the administered Abl. The Ab2 functionally mimicked in vitro the tumor antigen (Ag) defined by AbM (1-3). We had originally postulated that Ab2 induced in the patients anti-anti-idiotypic antibodies (Ab3) with Abl-like binding specificities for tumor cells (1). We had further postulated that Ab3 responses may underlie delayed clinical responses observed in some of the Abl-treated patients (1, 4). In the initial studies, Ab3 could not be demonstrated in patients' sera, presumably because they were bound to circulating Ab2 or to the patients' tumor cells. Therefore, in the present study, peripheral blood mononuclear cells (PBMCs) of Abl-treated patients were stimulated in culture with the Ag defined by AbM or with heterologous Ab2 that functionally mimics the Ag (5) and immunologically cross reacts with the patients' Ab2. The secreted human antibodies were characterized. Our results demonstrate idiotypic cascades in cancer patients treated with Abl. MATERIALS AND METHODSPatients. The clinical history and treatment plan of the five patients included in the present study have been described in detail (6). Briefly, five patients with advanced adenocarcinoma of the pancreas were treated with a single infusion of 400 mg of AbM mixed with autologous leukocytes. Two untreated patients (patients 7 and 8) with metastatic CRC also were included.Tumor Cells. Human CRC cells SW 1116 (7) and melanoma cells were used as target cells in mixed hemadsorption assay.Antibodies. Anti-CRC mAb C017-1A (IgG2a), referred to as Abl, has been described (7). As controls, anti-CRC mAb C029 (IgG1), anti-breast carcinoma mAb BR-140 (IgG2a), and anti-lung carcinoma mAb LU-16B13 (IgG2a) were u...

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A group of 29 re-anastomosed men were examined with respect to semen quality, anti-sperm antibody titres in serum and seminal plasma, presence of anti-sperm antibodies on sperm, and success rate in inducing pregnancy. Results indicated no association between pre-reversal serum anti-sperm antibody titres and post-reversal semen quality, but a pregnancy induction rate of zero was associated with serum anti-sperm antibody titres greater than 160. It is recommended that men considering reversal, with anti-sperm antibody titres of this level, should receive counselling about the possibility of post-reversal infertility.

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