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The immune response of 56 colorectal cancer patients to a single infusion of 1 mg of radiolabeled (111In) mouse B72.3-GYK-DTPA immunoconjugate was examined using a double-antigen radiometric assay system. The incidence of antibody response was 48% to polyclonal mouse IgG, 71% to mouse B72.3, and 62% to chimeric B72.3. Twelve patients (23%) had an antibody response to B72.3 V region in the absence of binding to polyclonal mouse IgG. An antiidiotype response was demonstrated in sera from 36% of 25 patients examined and correlated well with chimeric B72.3-GYK-DTPA immunoconjugate binding (r = 0.72, moderately well with mouse B72.3 binding (r = 0.56), and not at all with polyclonal mouse IgG binding (r = 0.28). The peak antibody response occurred most frequently 2 weeks postinfusion, although a "delayed" peak response to chimeric B72.2 occurred in 29% of patients. This study suggests that mouse B72.3 causes an immune response in the majority of patients and that antibody response to the V region is common. Understanding the physiological significance of these antibody responses will require correlation with the kinetics and tumor localization of repeat infusions of such immunoconjugates.

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Studies with goat and rabbit anti-mouse antibody, as models of human anti-mouse antibody (HAMA), have shown that several of the commonly used two -site immunoassays (e.g., for CA-125, carcinoembryonic antigen, choriogonadotropin, lutropin, hepatitis B surface antigen, thyrotropin) are susceptible to interference by this type of bridging heterophile antibody. In most cases the interference can be blocked by incubation with mouse IgG. We studied HAMA interference in an assay of hepatitis B surface antigen by using HAMA-positive sera from a transplant patient given OKT3 and from a cancer patient given CYT-103 (modified antibody B72.3). The HAMA interference attributable to the OKT3 could be blocked by incubation with mouse IgG at room temperature. In contrast, the interference caused by the B72.3-induced HAMA could be blocked by prolonged incubation with high concentrations of the B72.3 antibody at 4 degrees C. A limited survey of 50 hospital patients selected without conscious bias revealed two HAMA-positive patients, only one of whom was known to have been exposed to mouse immunoglobulin. HAMA interferences are currently a minor problem in routine laboratory medicine, but the increasing use of diagnostic and therapeutic products involving mouse-origin monoclonal antibodies will make the detection and elimination of HAMA interferences an important part of laboratory practice in the future.

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This highly sensitive immunoenzymometric method involves monoclonal antibodies, a common-capture microsphere, and a rapid, membrane-filtration separation step. The common-capture solid phase is monoclonal anti-fluorescein antibody convalently attached to 6.5 micron-diameter latex particles. In sandwich-type assays for large-molecule analytes, the capture antibody is conjugated with fluorescein isothiocyanate and the probe antibody is conjugated with beta-galactosidase (EC 3.2.1.23). In competitive assays for small analytes, the analyte-beta-galactosidase conjugate competes with the analyte in the clinical samples for the fluoresceinated capture antibody. After simultaneous incubation of the reagents for 2 h, the bound and unbound reagents are separated by filtration through the bottom of each well of a 96-well plate. Substrate (4-methylumbelliferyl-beta-D-galactopyranoside) is then added to the wells, and the rate of product formation is determined kinetically for 12 min. The rate is proportional to the concentration of analyte in the sandwich assays and inversely proportional in the competitive assays. The assay results for choriogonadotropin, thyrotropin, digoxin, and thyroxin show the assay to be sensitive, rapid, and applicable to any size analyte. With this system, several different sandwich and (or) competitive-type assays can be performed simultaneously on the same plate.

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The variable region sequences of light and heavy chains of three hybridoma antibodies to alpha (1----6) dextran, two from BALB/c and one from C57BL/6 mice, were determined by cloning and sequencing their cDNA. The three kappa-light chains are identical in nucleotide and amino acid sequences, except for the use of different J by BALB/c and C57BL/6; all three had the germ-line sequence of antibodies to 2-phenyloxazolone (20). Nevertheless, 2-phenyloxazolone BSA did not cross-react in gel with antidextrans, nor did dextran react with anti-2-phenyloxazolone ascitic fluids. The heavy chains differed, the BALB/c hybridomas having only three amino acid differences in CDR2 and two in CDR3; the C57BL/6 hybridoma differed throughout the variable region. All three VH are members of the J558 family. The three identical V kappa sequences suggest a significant role in dextran binding, with the differences in CDR of VH and the various J mini-genes of VL and VH being responsible for only fine differences in specificity. Alternatively, the role of V kappa might be minor, with most of the complementarity ascribable to VH. Additional sequences are needed to evaluate whether these data are typical of the repertoire of anti-alpha (1----6) dextran-combining sites.

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Rabbit antibody produced in response to the purified mitogenic glycoprotein lectin from Wistaria floribunda seeds (WFM) contains anti-carbohydrate antibody. This antibody, which represents 25% of the total antibody precipitated by the homologous antigen cross-reacts with the glycoprotein hemagglutinating lectins from Sophora japonica (SJL), W. floribunda (WFA) and the glycoprotein bromelain, but not the protein lectin from Maclura pomifera seeds. The cross-reactive reaction is totally abolished by the presence of glycopeptides obtained from SJL. Utilization of a fluorometric binding assay employing fluorescein derivatized glycopeptides from SJL, bromelain, fetuin and ovalbumin, it was found that the total anti-carbohydrate antibody population best reacts with the following carbohydrate structure: MAN alpha 1 leads to 6 MAN alpha 1 leads to 6 MAN beta 1 leads to 4 GLCNAC beta 1 leads to 4 GLCNAC beta 1 leads to Asn. Substitution of the beta-mannosyl moiety at position 3 results in structures not capable of binding to the anti-carbohydrate antibody. This antibody appears to distinguish between those glycan moieties of glycoproteins commonly found in animals from those lacking 3-O-substitution of the beta-mannosyl residue as found in some plant glycoproteins.

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