The clinical status of immunoscintigraphy with monoclonal antibodies in ocular melanoma

A summary is given of data on the clinical experience with the anti-melanoma antibody 225.28S in ophthalmology and the tissue expression of the high molecular weight-melanoma associated antigen in choroidal melanoma. Results are discussed in relation to other detection methods for ocular melanoma currently employed by the ophthalmologist.     

Protein assays for monoclonal antibodies

Several techniques were evaluated for the quantitation of the total protein content of an IgG2a monoclonal antibody, KS1/4, and its deacetylvinblastine (DAVLB) conjugate. The UV assay is rapid, but it requires an extensive calibration of the response factor, and impurities may cause a high bias. Amino acid analysis (AAA) is an absolute method that has few interferences, but it requires evaluation of hydrolysis recovery factors. Kjeldahl nitrogen is very sensitive to minor impurities, and it requires a conversion factor to calculate percent protein. The Kjeldahl assay also is less precise (observed RSD of 3-4%) than the UV and AAA assays (observed RSD of 2-3% for both). The bicinchoninic acid assay, a representative of colorimetric assays, inherently requires comparison to a calibrated standard of the same material and tends to be less precise than the other assays. Thus, the UV and AAA assays are the techniques of choice for measurement of the total protein content of KS1/4 and its DAVLB conjugate.     

Functional heterogeneity of monoclonal antibodies obtained using different screening assays

Two alternate screening methods have enabled the detection of monoclonal antibodies with different specificities toward the lysosomal enzyme alpha-mannosidase of Dictyostelium discoideum. Spleen/myeloma hybrid cell cultures were screened for antibody production by separate assays: an indirect enzyme-linked immunoadsorbent assay (ELISA) based on the antibody binding to enzyme adsorbed on plastic, and a direct assay of the antibodies' ability to precipitate enzyme activity with fixed Staphylococcus aureus cells (Pansorbin). Fourteen stable antibody-producing cell lines resulted from a single fusion; these fell into three distinct classes based on their screening characteristics. A group of eight were positive in both assays, and these immunoprecipitated a 140,000 Mr precursor form of alpha-mannosidase in addition to the 58,000 and 60,000 Mr mature enzyme subunits from [35S]methionine-labeled total secreted protein preparations. Two of the antibodies were positive only in the immunoprecipitation assay; these failed to precipitate the 140,000 Mr precursor. The third class consisted of four antibodies that were positive only in the ELISA method. These exclusively recognized an altered conformation of the enzyme (precursor and mature forms) that was immobilized either on plastic or on nitrocellulose paper. In addition, only members of this class were able to bind to immobilized fragments of protease-treated enzyme. The implications of these findings for the general design of monoclonal antibody screenings and for the alternative structures of this enzyme are discussed.     

Novel recombinant monoclonal antibodies for vitellogenin assays in cyprinid fish species

Various polyclonal and monoclonal antibodies have been developed for vitellogenin (Vtg) bioassays in different aquatic species. Preparation of these reagents is time-consuming and expensive. In the present study, a phage-displayed, recombinant, single-chain variable fragment (scFv) format antibody library was constructed using splenic mRNA from non-immunized mice. After 3 rounds of panning, 3 scFv antibodies with specificity for the highly conserved N-terminal region of cyprinid fish Vtg were isolated. One of these, antibody H4, bound purified Vtg from common carp Cyprinus carpio, zebrafish Danio rerio and Chinese rare minnow Gobiocypris rarus with similar affinities and detected Vtg in zebrafish plasma samples. This study provides a simple, low cost Vtg bioassay for plasma samples from a variety of cyprinid fish.     

Characterization of the interaction between murine tumour necrosis factor and monoclonal antibodies

We characterized the interaction between murine TNF (mTNF) and the neutralizing monoclonal antibodies TN3 and 1F3F3. The epitopes were localized by comparing the detection efficiency for a panel of TNF chimaeric proteins and site-specific muteins in ELISA. Mutation of mTNF amino acid Q131 inhibited the interaction with 1F3F3, whereas mutation of D71/Y72 inhibited the binding to TN3. As D71/Y72 are located in an exposed loop near the TNF intersubunit groove, binding of TN3 promoted the dissociation and/or interfered with the reassociation of subunits into mTNF trimers. 1F3F3, on the other hand, prevented the spontaneous dissociation of bound (hetero)trimeric mTNF.     

Surface markers in acute non-lymphoid leukemia: analysis with a panel of 36 monoclonal antibodies

The reactivity of a panel of monoclonal antibodies was studied in fifty-four cases of acute myeloid (AML) or undifferentiated (AUL) leukemias. Thirty-six antibodies from the Myeloid section of the Second Workshop on Human Leukocyte Differentiation Antigens were used in an indirect immunofluorescence assay. The antibodies could be classified into three groups recognizing respectively granulocytic, monocytic or granulomonocytic leukemias. Most antibodies stained erythroblastic and megakaryoblastic leukemias. In each group, it was possible to define antibodies staining either the less differentiated forms (FAB M 1 and M 5 a) or the more differentiated forms (M 2, M 3, M 4 and M 5 b). Six out of eight AUL were stained by some of the antibodies (mainly from the monocytic group). However, a heterogeneity of stainings in a same blast population was observed.     

Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones.

Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique.     

Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.     

Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies.

Monoclonal antibodies to varicella-zoster virus were used to study viral glycoproteins by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on the viral glycoproteins immunoprecipitated, the five monoclonal antibodies fell into three groups. Two antibodies, 4B7 and 8G9 (group 1), immunoprecipitated a single glycoprotein of molecular weight (MW) 118,000 (118K glycoprotein) and had high neutralizing activity in the absence of complement. One antibody, 3C7 (group 2), which lacked neutralizing activity, immunoprecipitated two glycoproteins of MWs 120,000 and 118,000 and a glycoprotein giving a diffuse band in the region of 64,000 to 65,000. Pulse-chase experiments and experiments with monensin as an inhibitor of glycosylation suggested that the 120K polypeptide was derived by glycosylation of the 118K polypeptide and that a 43K antigen was processed into the 64 to 65K glycoprotein. Two antibodies, 3G8 and 4E6 (group 3), both had neutralizing activity only in the presence of complement, and both immunoprecipitated at least five polypeptides, with MWs ranging from 50,000 to 90,000. Antibody 3G8 was isotype immunoglobulin G2b (IgG2b), and its immunoprecipitating activity was stronger than that of 4E6, which was isotype IgG1. Pulse-chase experiments with antibody 3G8 showed that lower-MW glycopeptides chased into three polypeptides of MWs 90,000, 80,000, and 60,000 by 24 h. Immunoprecipitation experiments with antibody 3G8 on infected cells treated with glycosylation inhibitors 2-deoxyglucose, monensin, and tunicamycin, suggested that a prominent, early-appearing 70K polypeptide may have been processed into the glycoproteins of higher MWs and that the 60K polypeptide may have been derived by glycosylation of polypeptides of lower MWs.     

Leukocyte surface markers in Rana catesbeiana,identified using mouse monoclonal antibodies

Monoclonal antibodies (mAbs), each reactive with thymocytes, neutrophils, and thrombocytes of the bullfrog, Rana catesbeiana, were raised and used for studies of hematopoiesis of metamorphic larvae. The mAbRc-T1, which immunoreacted exclusively with thymocytes in adults, was also strongly reactive to larval thymocytes. Thus the determinant Rc-T1 may provide an appropriate marker for thymocytes, although antigenic sharing is also seen in larval ovarian tissue. Neutrophils detectable by both mAbRc-N1 and Rc-N2 originated primarily in the mesonephros and showed up increasingly in peripheral blood of larvae. Thrombocytes detectable by mAbRc-P may originate in the larval spleen and/or liver but not in the mesonephros.     

Induction of arthritis with monoclonal antibodies to collagen.

mAb were developed from DBA/1 mice immunized with chick type II collagen. A total of 69 IgG antibodies was isolated and characterized. The majority (36%) reacted with a CNBr-derived peptide CB11 previously identified as containing a major immunogenic and arthritogenic epitope(s). Seven of the antibodies reactive with CB11 crossreacted strongly with mouse type II collagen. These were administered to DBA/1 mice in an attempt to induce arthritis. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of antibodies induced severe arthritis with marked destruction of articular cartilage. The minimal effective combination consisted of three antibodies. Arthritis developed within 48 to 72 h after injection of the antibodies and persisted for the duration of the observation period of 3 wk. Antibody levels were measured at intervals and persisted for the 3 wk observation period although at diminishing levels. Competitive binding assays demonstrated that each of the effective antibodies bound independently suggesting that some spatial or quantitative relationship was important possibly related to their ability to activate complement.     

Mechanism of action of therapeutic monoclonal antibodies: Promises and pitfalls of in vitro and in vivo assays

Therapeutic monoclonal antibodies (mAbs) are mostly used in cancer, as anti-infectious agents and as immunomodulatory drugs, and are amongst the most active area of research and development in the pharmaceutical industry. This class of drugs comprises unconjugated antibodies or antibody fragments, antibody-drug conjugates, radio-immunoconjugates and bispecific/trispecific molecules. A better understanding of the mechanism of action of successful mAbs is fundamental for the selection of more active and less toxic mAbs of new generation. Furthermore reliable screening of new compounds at an early stage of preclinical development, for both efficacy and toxicity, should allow the selection of the best molecules at an early stage, and improve the rate of success of this class of drugs. Here we review the major methods that are employed for testing the activity of therapeutic mAbs in vitro and in vivo in small animal models and point out to some of the pitfalls in these assays.     

Immunoelectron microscopy of Chlamydia psittaci with monoclonal antibodies.

An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydia psittaci and infected cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.     

Surface antigenic analysis of rosette-forming cells in rat thymus--using monoclonal antibodies against T cell surface markers.

We have assumed that rat thymocytes capable of rosette-formation with guinea pig erythrocytes were double positive (CD4+8+) cells. Therefore surface-binding erythrocytes on rosette-forming lymphocytes (RFLs), separated in highly pure state, were eliminated by a hypotonic shock. The surface markers of RFLs, without surface erythrocytes, were analyzed by flow cytometry using two kinds of monoclonal antibodies (anti-CD4, anti-CD8) labelled with different fluorescence materials. About 96.2% of cells in the RFL population were sorted in two areas, into which double positive cells were separated. This result substantiates the assumption mentioned above. It was, furthermore, discussed that the RFLs without surface erythrocytes would be one of the valuable materials for disclosing the mechanisms of differentiation and maturation from double positive thymocytes to single positive ones.