Immunoglobulin bound to platelets as immune complexes or specific antibody in specimens from acquired immunodeficiency syndrome and immune thrombocytopenic purpura.

Acquired immunodeficiency syndrome (AIDS), lymphadenopathy syndrome (LAS), and immune thrombocytopenic purpura (ITP) specimens were tested by an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig) bound to platelets. All specimen evaluations were performed with General Diagnostic's newly developed kit procedure. The test measured but did not distinguish immune complex (IC) binding with platelet Fc receptor sites from platelet-specific antibody (PAb) binding with platelet antigen Fab-binding sites. Alkaline phosphatase-labeled antihuman IgG as conjugate detected IgG as low as 2.0 ng/ml or platelet-adsorbed, heat-aggregated IgG, simulating IC, at 2-10 ng/ml. There was a high prevalence of platelet-bound Ig in AIDS specimens (25/25) compared with normals (2/15), detected primarily by the indirect ELISA (p less than 0.001), and a preponderance of PAb in ITP specimens (5/5) compared with normals (6/22), by the direct ELISA (p less than 0.01). AIDS specimens had a geometric mean titer (GMT) of 173 ng of IgG bound/10(7) platelets, compared with the Ig from ITP, which had a GMT of 20 (p less than .0002). Monoclonal antibody to human receptors for IgG Fc fragment (anti Fc gamma R) inhibited 69% of specimens tested as having platelet-bindable antibody. Thus, the ELISA procedure would be useful in assessing but not in differentiating platelet-bound Ig in patients with AIDS and ITP and certain other clinical groups tested.

Cancer therapy with chemically modified enzymes. II. The therapeutic effectiveness of arginase, and arginase modified by the covalent attachment of polyethylene glycol, on the taper liver tumor and the L5178Y murine leukemia.

Monomethoxypolyethylene glycol (PEG) was attached covalently to arginase. PEG-arginase was effective in prolonging the survival times of mice injected with the Taper liver tumor, whereas unmodified arginase was ineffective. PEG-arginase was more effective than arginase in the in vitro destruction of L5178Y mouse leukemia. However, neither PEG-arginase nor arginase inhibited the in vivo growth of this tumor.

Induction of tolerance in mice by uricase and monomethoxypolyethylene glycol-modified uricase.

The ability to induce tolerance to uricase by the administration of native uricase, and uricase modified by the covalent attachment of monomethoxypolyethylene glycol (PEG) was examined. Uricase, and uricase with PEG attached to 35% (PEG-uricase 35%) and 70% (PEG-uricase 70%) of available amino groups were found to induce tolerance in mice not previously sensitized to uricase. There was a dampening of the IgG, IgE and IgM antibody response to uricase which persisted even after a second sensitizing dose of uricase was administered to these animals. PEG-uricases were found to have little or no immunogenicity when injected into mice and a reduced immunogenicity and antigenicity when tested in rabbits. Native uricase, however, was found to be immunogenic and antigenic in mice and rabbits. Mice sensitized to native uricase were injected with uricase, PEG-uricase 35% or 70% to induce tolerance. After a second sensitizing injection of uricase, circulating levels of IgE, IgG and IgM were measured. All three enzymes induced tolerance in the IgE class of antibody but there was no significant change in the hemagglutinating antibody levels of the mice. Mice injected with 1 mg of uricase died from anaphylaxis. PEG-uricase 35% was found to induce the most effective tolerance in both unsensitized and sensitized mice.

Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to bovine liver arginase using 2,4,6-trichloro-s-triazine as the coupling agent. The conjugate (PEG-arginase), with PEG attached to 53% of the amino groups, retained 65% of its original enzymatic activity. Mice were injected intravenously with arginase or PEG-arginase for periods of one to three months. The blood-circulating life of PEG-arginase was greatly extended over that of arginase. The half-life of injected arginase at day 30 was less than 1 h, whereas that of the PEG-enzyme was 12 h. Antisera from mice injected with native arginase reacted against arginase but not against PEG-arginase when tested by immunodiffusion. Antisera from animals injected with PEG-arginase reacted neither with native arginase nor PEG-arginase. The data indicate that arginase modified by PEG has been rendered both non-immunogenic and non-antigenic when tested in mice. The injection of PEG-arginase into mice did not induce tolerance toward the native enzyme. Injected PEG-arginase, in the presence of precipitating antibody directed against native arginase, circulated at the same level as in virgin animals. The attachment of PEG to arginase altered its kinetic properties.