Isolation of rabbit IgA antihapten antibody and demonstration of skin-sensitizing activity in homologous skin.

Multiple antibody components of rabbit antisera against p-azobenzenearsonate (R(p)) were studied with respect to their globulin nature and skin-sensitizing activity. IgA antibody was characterized by isolating two IgA-rich fractions from a specifically purified antibody preparation. Examination of these fractions showed that IgA antibodies existed in two molecular forms, one with a sedimentation constant of 7S and the other 9S. Skin-sensitizing activity was examined by a P-K type test and a PCA test with R(p)-rabbit serum albumin in homologous (rabbit) species. Only the 7S but not 9S IgA antibody sensitized rabbit skin. IgM antibody showed no activity and IgG antibody showed very low activity. In contrast, only IgG antibody was active in the P-K type test to sensitize a heterologous species (guinea pig). None of the antibodies of other classes showed sensitizing activity in heterologous skin. The 7S IgA antibody lost its sensitizing activity upon reduction and alkylation, although no change in its molecular size could be observed. The loss of sensitizing activity was not due to the destruction of antigen-binding activity since the treated 7S IgA antibody retained this activity as shown by radioimmunoelectrophoresis and by binding to the specific immunoadsorbent. The 9S IgA antibody was more resistant to these treatments than the IgM antibody and showed no indication of dissociation. The treated 9S IgA also retained antigen-binding activity. Both the P-K type and PCA reactions were considerably stronger when the interval between injections of antibody and antigen was 24 hr rather than 4 to 5 hr.

Recovery of antibody activity from inactive hybrids of H and L chains.

Hybrid IgG molecules were prepared from the heavy and light chains of specifically purified antibody against two different haptens. One hybrid consisted of the H chains from the anti-p-azobenzoate antibody from one rabbit and L chains from the anti-p-azobenzenearsonate antibody from the second rabbit, and the second hybrid consisted of the opposite combination, light chains from the first rabbit and heavy chains from the second. The two hybrids were mixed at pH 8 and were found to be so stable in the mixture that even after 2 wk at 5 degrees C there was still only low hapten-binding activity toward p-iodobenzoate and p-iodobenzenearsonate. However, exposure of the mixture of hybrids to 1 M propionic acid followed by buffer at pH 8 resulted in recovery of binding activity for p-iodobenzoate and p-iodobenzenearsonate. Thus no exchange occurred between the light and heavy chains of the hybrids in the buffer, but exchange did occur on exposure to propionic acid, and this exchange favored a preferential combination among the chains in such a manner that effective antibody sites resulted.

Specificity in the combination of Fd fragments with L chains to form hapten-binding sites.

In the work reported here we have shown that light chains and Fd fragments can be separated completely in propionic acid and then recombined to form Fab fragments with antibody activity. This experiment indicates that in the recombination a correct alignment of the Fd fragments and the L chains occurs to give a competent antibody site, just as occurs with the recombination of separated heavy and light chains of the antibody; thus the Fc fragment is not required for correct alignment. Fd fragments of antibody alone show very low binding activity toward the specific hapten. As is the case for the combination of heavy and light chains, the combination of Fd fragments and light chains also requires that both components come from antibody from the same rabbit in order to give binding sites. When they are derived from different rabbits producing antibody against the same antigen, they still give Fab fragments as shown by immunoelectrophoresis but do not have competent binding sites. An important observation is that the subunits of the papain digest fractions, Fab(I) and Fab(II), have the capacity to cross-combine to form active Fab fragments with competent binding sites. Fd(I) from Fab(I) combines with L(II) chains from Fab(II) to give the composite (Fd(I)-L(II)) with good binding activity. Likewise, the composite (Fd(II)-L(I)) has good binding activity. The composites from the two types of antibody molecules yielding different Fab fragments have antibody activity although heretofore these molecules have appeared to be different on the bases of chromatography and amino acid analysis. There is also a preferential combination of the Fd fragments to combine with the correct L fragments to give binding sites since this combination takes preference over the combination of Fd fragments of antibody with light chains of normal globulin (or of light chains of antibody with Fd fragments of normal globulin).

Immunological dissection of human Ia molecules.

The immunochemical analysis of Daudi Ia molecules by a variety of alloantisera led to the recognition of at least three molecular species carrying different antigenic determinants: DRw6, DC-1, and DC-2. Genetic as well as structural evidence indicates that DRw6 and DC-1 molecules are controlled by separate, HLA-linked loci, rather than by alleles at the same locus. The alloantigenic determinants appear to be expressed on the small Ia subunit. DC-1 and DC-2 determinants discussed had not been defined by serological analysis at the population level, but were demonstrated to be present by immunochemical analysis at the molecular level.

Antibody molecules: discontinuous heterogeneity of heavy chains.

The heavy polypeptide chains of antibody ( and of gammaG-immnunoglobulin) molecules show discrete bands on disc electrophoresis. The same bands are present for chains from antibodies of the same or diverse specificities. Individual bands are of different intensities for chains from the different rabbits tested even if the antibodies are directed against the same hapten. The bands appear to represent classes of heterogeneous H-chains of the same size having discrete differences in mizobilities with respect to a single charge difference.

Specific binding activity of isolated light chains of antibodies.

Free light chains isolated from specifically purified antibody have been shown to bind specific hapten. This proves that part of the binding site does exist on the light chain. The light chains were obtained from antibody directed against the 4-azonaphthalene-1-sulfonate group, and the binding of the simple hapten 4-anilinonaphthalene-1-sulfonate was determined by the fluorescence-enhancement technique. Since this hapten undergoes a striking increase in fluorescence on binding to light chains (and also on binding to specific antibody), the presence of small amounts of bound hapten could be determined, even in the presence of the high concentrations of unbound hapten required because of the low binding constant.

Cells isolated from the embryonic neural retina differ in behavior in vitro and membrane structure.

Several subpopulations of cells were isolated from trypsin-dissociated embryonic (14 days) chick retinas. The cells of each subpopulation differed in associative behavior measured by cell aggregation and stationary culture assays and in glycoproteins that contain glucosamine. Freeze-fracture analysis showed that these populations also differed in intramembrane particle content.

Immunoglobulin M antibodies with ten combining sites.

Immunoglobulin M rabbit antibodies to a hapten are shown to have ten binding sites per molecule. The affinity for the specific hapten is approximately 100 times greater for one-half of the sites than for the other half. All sites are retained in the five 7S subunits produced by reduction and alkylation of the immunoglobulin M. Each of the 7S subunits of the IgM molecule apparently has one strong and one weak site.

Rabbit antiserum against a non-T, non-B leukemia cell line that carries the Ph1 chromosome (NALM-1): antibody specific to a non-T, non-B acute lymphoblastic leukemia antigen.

Rabbit immune sera against human cells of a non-T, non-B leukemia cell line bearing the Ph1 chromosome (NALM-1) were characterized. After proper and exhaustive absorption, the sera were tested by indirect membrane immunofluorescence on a panel of 19 human hematopoietic cell lines with T-, B-, or non-T, non-B cell surface characteristics. The absorbed sera reacted specifically with NALM-1 cells but not with cells of another Ph1-positive cell line, K-562. Four of the 6 leukemia T-cell lines, 2 of the 4 leukemia-lymphoma B-cell lines, all of 4 acute lymphoblastic leukemia (ALL) lines with non-T, non-B cell characteristics, and uncultured cells of all patients with non-T, non-B cell ALL were reactive with the antisera. A cross-absorption study of the antisera suggested that a single antigenic complex is involved in this antigen specificity. The antigen involved appears to be a common non-T, non-B ALL one that has been described previously.

Electron microscope study on human lymphocyte-sheep erythrocyte rosettes.

Nonimmune rosette formation of human peripheral blood lymphocytes and cultered MOLT 3 and MOLT 4 cells with sheep red blood cells was studied by transmission electron microscopy. At the cell contact area of the rosette, lymphoid and red cell membranes were parallel and 80-100 A apart. The inner leaflet of the lymphoid cell membrane seemed denser, and amorphous substance attached to its cytoplasmic face. The cell contact area was 100-1000 nm wide and frequently on the lymphoiid cell body rather than on microvilli, though some cells extended microvilli near red cells.

Carcinoembryonic antigen-binding immunoglobulin isolated from normal human serum by affinity chromatography.

A human immunoglobulin that binds carcinoembryonic antigen (CEA) was isolated from four individual normal human sera by affinity chromatography with the use of a CEA-Sepharose solid adsorbent. The yield of isolated protein, termed human CEA-binding protein (HCBP), ranged from 1.8 to 10 microgram/ml serum. HCBP is a gamma-globulin of restricted electrophoretic heterogeneity as shown by immunoelectrophoresis. HCBP was shown to bind radioiodine-labeled CEA both by a radioimmune precipitation assay and by a radioimmunoelectrophoresis assay. This protein was of practical interest because of its potential usefulness as a carrier of radioactivity or therapeutic agents to a CEA-producing tumor for therapeutic or diagnostic purposes.

A tumor-associated organ-specific antigen characteristic of spontaneously metastatic rat mammary carcinomas.

An organ-specific tumor-associated antigen (TAA) was present in several metastatic and nonmetastatic mammary carcinomas induced in WF female rats by 3-methylcholanthrene. The level of TAA was high in 2 metastatic carcinomas tested (TMT-081 and SMT-2A) and much lower--by a factor of 50--200--in 2 nonmetastatic mammary carcinomas (MT-100 and MT-W9B). The TAA in the 2 metastatic tumors was identical, as demonstrated by immunodiffusion and supported by cross-reactivity with antibody against TAA from TMT-081 in a binding inhibition radioimmunoassay. The TAA was shed in relatively large amounts by the metastatic tumors maintained in short-term organ culture. The high level and shedding of TAA thus appeared to be characteristics of the metastatic tumors but not of the nonmetastatic ones. This suggests that TAA on the cell membrane or in the circulation may be involved in the metastatic process as a factor blocking potentially cytotoxic cells or in other ways leading to suppression of the immune response against the tumor.

Radioimmunoassay for myeloma idiotype.

Rabbit anti-idiotype antisera were prepared against four human myeloma proteins. These antisera demonstrated a capacity to bind the 125I-labeled autologous purified monoclonal IgG, but failed to demonstrate any binding to 125I-labeled normal IgG or to labeled myeloma IgG obtained from other myeloma patients. The anti-idiotypic antisera were used with 125I-labeled autologous myeloma IgG preparations and goat antirabbit IgG for specific radioimmunoassay with a sensitivity limit of 20 ng/ml. Little or no cross-reaction occurred between these anti-idiotypic antisera and normal IgG preparations or other myeloma IgG proteins.

The development and use of radiolabeled antitumor antibodies.

The use of radioactive tumor-localizing antibodies is developed historically, starting with the demonstration that antibodies can be radioiodinated without destroying antibody activity. Then, antibodies against normal organs were shown to contain antibodies which localize in the normal organ. Subsequently, antibodies capable of localizing in tumors were demonstrated, and these localized well enough to permit their use diagnostically by scanning for radioactivity and their use therapeutically by localizing sufficient radioiodine. Various relevant problems are discussed.

Detection of liver-bound metabolites of azocarcinogens by the use of anti-hapten antibodies.

The presence of the azocompounds, p-dimethylaminoazobenzene and 3'-methyl-p-dimethylaminoazobenzene, and p-amino-N-acetyl-N-methylaniline (or their metabolites) bound to components of the liver cells of rats fed a single large dose of each compound has been detected using rabbit antibodies raised against the p-azo-N-acetyl-N-methylaniline hapten in the indirect fluorescent antibody technique. Binding of these antibodies was seen on liver sections from rats fed any one of these compounds. When the anti-p-azo-N-acetyl-N-methylaniline antiserum was absorbed with either liver sediments or cytosol fractions from rats fed p-amino-N-acetyl-N-methylaniline, the antibodies reacting with the liver-bound compounds were removed from the antiserum. Also, absorption of the antiserum with liver sediments or cytosol fractions of rats fed either one of the azocompounds selectively removed all of the antibodies reacting with the livers of rats fed that compound but did not remove other antibodies that were still capable of reacting with liver cells of rats fed the other azocompound or p-amino-N-acetyl-N-methylaniline. Thus this antiserum appears to contain several different anti-p-azo-N-acetyl-N-methylaniline antibodies with different structural requirements for reaction. Some can react with the azocompounds or certain of their metabolites, while others require more of the p-azo-N-acetyl-N-methylaniline structure for reaction. Some of the antibodies appear to react with liver-bound p-dimethylaminoazobenzene but not with liver-bound 3'-methyl-p-dimethylaminoazobenzene, while still others react with 3'-methyl-p-dimethylaminoazobenzene but not with p-dimethylaminoazobenzene.

Effect of antimyeloma cell antiserum on immunological enhancement.

Growth of Sarvoma 180 in AKR mice was enhanced by immunization with frozen-thawed homogenates of this tumor. Treatment of host mice with rabbit antimyeloma cell antiserum, either furing immunization or shortly after tumor implantation, resulted in a decreased incidence of tumor rejection or an increased rate of tumor growth. A slow-growing subline from a spontaneous, DBA/2Ha-DD mammary tumor is rejected after initial growth in DBA/2J mice. The incidence of rejection was reduced by treatment with the antiserum studied.

An experimental model for evaluation of factors in tumor escape from immunological attack.

A model system is presented for studying the factors involved in tumor immunity. The initial observations with this system concern the importance of dose and route of administration of tumor cells on tumor growth. The data show that myeloma tumor cells, when inoculated i.v.in relatively large numbers, are eradicated by the immune response of an allogeneic host; tumor cells administered i.v. in smaller number escape from immune attack even though the host has the potential to mount an immune response. BALB/c mouse myeloma cells (MOPC-21) were transplanted s.c., i.p., or i.v. into H-2-compatible allogeneic DBA/ 2 mice. There was a marked difference in the response of the host to tumor given s.c. or i.p. as compared to tumor given i.v. Thus s.c. or i.p. inoculation resulted in lethal tumor growth when 5 x 10-3 or more tumor cells were given. In contrast, the outcome of i.v. inoculation depended on tumor cell dose. Although small cell doses ( 5x 10-4 down to 10-2) resulted in lethal tumor gosulted in lethal tumor growth with only 10% survival, large cell doses (10-5 to 5 x 10-7) resulted in tumor rejection and 70% survival. DBA/2 mice possess the immunological ability to react agaist the tumor when large doses of tumor cells (10-7) are given i.v. or i.p., since spleen cells obtained from such mice were found to be able to suppress the growth of MOPC-21 when a mixture of spleen cells and tumor cells was inoculated. On the basis of these initial observations, our model appears to relate especially to the idea that, in autochithonous tumor development or in metastasis of tumor, a small number of antigenic tumor cells, perhaps even a single cell, usually grows into a frank tumor in spite of the immunological competence od the host to respond to the tumor cells.

Binding of the azocarcinogen 3'-methyl-p-dimethylaminoazobenzene to cellular components of normal rat liver and azocarcinogen-induced hepatomas.

The location of binding sites for 3'-methyl-p-dimethylaminoazobenzene (3'-Me-DAB) or metabolites on components of rat liver cells and hepatoma cells in tumors induced by this carcinogen was determined at 2 stages during the induction of tumors in rats: (a) in normal liver immediately following the application of a massive dose of the azocarcinogen by intragastric feeding, and (b) in liver and tumor after hepatomas had developed following repeated exposures to the carcinogen by s.c. injections. Bound 3'-Me-DAB or metabolites were detected by the use of rabbit antisera directed against either p-azoazobenzene or p'-azo-p-dimethylaminoazobenzene in an indirect fluorescent antibody technique. Soon after massive intragastric doses of 3'-Me-DAB, the staining observed when the anti-p-azoazobenzene antiserum was used was principally on cytoplasmic components of liver cells with some staining of the intranuclear components. When the second antiserum, anti-p'-azo-p-dimethylaminoazobenzene antiserum, was used, the most intense fluorescent staining was on the nuclear membranes, although there was some cytoplasmic and intranuclear staining as well.

Lactoperoxidase-catalyzed iodination of human IgM. Differences between 7 S IgM and 19 S IgM and between cell surface 7 S IgM and serum 7 S IgM.

We have studied the lactoperoxidase-catalyzed iodination of human IgM and have measured the ratio of radioactivity incorporated into the mu chain to that incorporated into the L chain (i.e. the mu/L ratio). Both 7 S and 19 S IgM were examined. The ratio of radioactivity was found to be larger for 7 S IgM than for 19 S IgM for all four of the monoclonal IgM proteins examined. The data suggest that some tyrosines of the mu chain which are buried and not available for iodination in 19 S IgM become exposed on conversion of 19 S IgM to 7 S IgM. The mu/L ratio for the IgM found on the cell surface of RPMI 8392 cells was significantly smaller than the ratios for all of the five 7 S IgM proteins studied in solution. It appears, therefore, that a portion of the mu chain of the cell surface IgM of the RPMI 8392 cells is buried in the membrane.

The effect of adjacent residues on the reactivity of tyrosyl with iodine.

The effect of the adjacent amino acid side chain groups on the iodination rate of the tyrosine was studied. The model peptides used were Gly-Tyr-Gly, Leu-Tyr-Leu, Glu-Tyr-Glu, and Lys-Tyr-Lys, in which the tyrosine is sandwiched between two hydrophobic, two negatively charged, or two positively charged residues. The results show only minor differences in the iodination rate of tyrosine in these four peptides. These differences are very small in comparison with those previously observed between the tyrosines of kappa Bence-Jones proteins.