John A. Sogn

Microdetermination of rabbit immunoglobulin allotypes by ELISA using specific antibodies conjugated with peroxidase or with biotin

Enzyme-linked immunoadsorbent assays (ELISA) specific for the allotypes of rabbit heavy chain variable region (group a) and light chain constant region (group b) have been developed. These assays utilize affinity-purified allogeneic anti-allotype antibodies to coat polyvinyl microtiter plates. After test samples are added to the plates, the same affinity-purified antibody, coupled to the enzyme peroxidase, is used to detect binding of allotype-positive IgG samples to the coated plate. These assays can detect as little as 40 ng/ml of IgG of the appropriate allotype; the assays are highly specific and allow quantitative determination of allotype-defined IgG samples. In addition, the procedure can be modified by coupling of the antibody with biotin followed by development with avidin-peroxidase; this adaptation avoids the difficulties encountered in antibody-peroxidase conjugations. The ELISA assay is not influenced by the presence of anti-allotype antibody in the test sample, giving a distinct advantage over solid-phase radioimmunoassay procedures conventionally used for the quantitative determination of rabbit allotypes.

Genetic Characterization of a new allele of the rabbit group b Ck allotypes

An inherited variant (b4v) of the rabbit kappa-chain allotype b4 is characterized by the presence of serine in place of alanine at position 121 and leucine in place of glutamine at position 124. The variant was traced through eight generations of a pedigreed rabbit family. Genetic analysis of this trait demonstrated that it is an allele of the other group b allotypes, and recent breedings have produced rabbits homozygous for this light-chain type. Two findings, other than the amino acid sequence differences, distinguish b4v from b4 in our colony. First, the level of expression of b4v in heterozygous rabbits is less than that of b4. For example, in b4b4v rabbits, approximately 30% of the preimmune IgG carries b4v. In b4vb5 animals, 46% of the IgG carries the allotype b5, although in animals of allotype b4b5, 38% of the IgG is b5. Second, retrospective analysis of some litters revealed an abnormally low frequency of b4v in male heterozygotes. However, male b4vb4v homozygotes were found at the expected frequency in prospective crosses between b4vb5 rabbits.

Cell surface glycoproteins of rabbit lymphocytes: Characterization with monoclonal antibodies

The structural characteristics of antigens recognized by a panel of monoclonal antibodies prepared against a rabbit T-lymphocyte cell line have been investigated. Those antigens which could be isolated using immunoadsorbents prepared from the monoclonal antibodies had mol. wts of 42,000, 90,000 and 120,000. The 42,000 mol. wt molecule is similar or identical to a rabbit class I major histocompatibility complex antigen and its characterization has been reported elsewhere. Three different 90,000 mol. wt proteins can be distinguished by their reactivity with lectins and by sequential immunoprecipitation. The 120,000 mol. wt protein is a very abundant surface glycoprotein that appears to be a specific marker for T-cells in the rabbit. It is the immunodominant antigen in a lentil lectin bound glycoprotein pool. Over half of the antibodies were directed against this antigen. All antigens detected by the panel of monoclonal antibodies have been detected on normal lymphoid cells.

Primary structural sequence polymorphism in the human class II MHC antigen 33.1.

Monoclonal antibody 33.1 defines a non-DR, class 11, human major histocompatibility complex antigen, 33.1, which appears to be distinct from other class II antigens in its cellular distribution and primary structure. To characterize the structure more fully and to determine the degree of polymorphism within 33.1, a comparative N-terminal sequence study has been undertaken using a series of ten B lymphoblastoid cell lines with different DR and MB types. The results confirm that both the α and β chains of 33.1 are homologues of the corresponding chains of the murine I-A antigen and indicate that while 33.1 does not appear to be identical with MB, it is closely related. Sequence analyses revealed two major variants of 33.1, corresponding to cells with specificities MB1 and MB 3, respectively. Within each MB type, other polymorphisms have been detected. Cells that are MB2 do not react with monoclonal antibody 33.1. Suggestive evidence is presented that monoclonal antibody 33.1 reacts predominantly with the β chain of the antigen. The preferential expression of 33.1 on activated B cells suggests that expression of at least the 33.1 β chain gene is greatly enhanced in the course of B-cell activation, but the specific function of 33.1 remains to be determined.

Cancer Vaccines: The Perspective of the Cancer Immunology Branch, NCI

The Cancer Immunology Branch, NCI, is supporting a great deal of exciting research relevant to cancer vaccine development. The few areas highlighted here are representative of ongoing research opportunities, but further progress depends largely on a continued infusion of investigator-initiated ideas to realize the potential of current research areas and open new ones.

Cancer Immunology: Highlights of the NCI Extramural Immunology Program

The long-term goal of research supported by the Immunology Program is to better understand immune mechanisms and their regulation in order to develop more effective strategies to strengthen the immune response against cancer. While there has been much progress in the field of immunology in recent years, many major questions remain unanswered. The role of MHC antigens in regulating the immune response to tumors is still unclear, as is the nature of putative tumor-associated antigens which are the targets of this response. The efficacy of various immune cell subsets in tumor cell killing is differentially affected by changes in tumor cell surface MHC antigen expression. Furthermore, although we now know much more about the cellular interactions in the immune response, little is actually known about the particular cell subsets which participate in an immune response is regressing versus progressing tumors. Interleukins have been shown to stimulate a variety of immunes response, and some of these immune modulators are now being tested in clinical trials, in various stages, to determine their antitumor effects. However, systemic administration of large quantities of interleukins can result in very different effects than those created by the local release of effector molecules from specific T-cell populations. Effector T cells can deliver lymphokines to precise target structures, whereas systemically administered lymphokines would affect preferentially those cells expressing the largest numbers of high affinity receptors for the lymphokines. The specificity of lymphokines as mediators of immunologic response rests largely or exclusively in the local release of such materials by T cells upon activation by antigen: MHC complexes on a stimulating cell. Because lymphokines show specificity only for nonantigen-specific, non-MHC-restricted receptor molecules on target cells, the effect of lymphokine injections is likely to be determined solely by the expression of these receptors. Thus, lymphokines function well as effector molecules in a number of specific immune reactions, but it remains to be determined whether they will be useful in regulating immune responses in specific disease situations. It may be critical to recruit specific immune cells to the area of tumor growth where they, in turn, can release lymphokines to activate appropriate antitumor effector cells. Adoptively transferred T cells of the helper phenotype can induce an effective antitumor immune response in recipient mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Phagocytic rabbit cell lines expressing class II MHC products: establishment of cell lines by viral transformation.

Continuous rabbit macrophage‐like cell lines were established after in vitro infection of spleen cells with either Simian virus 40, lymphotropic papovavirus or herpesvirus sylvilagus. These cell lines are characterized as morphologically similar to mature macrophages, esterase positive, and have unrearranged immunoglobulin genes. They possess macrophage functionality because they are highly phagocytic and are able to mediate an antibody dependent cell mediated cytotoxicity assay on chicken red blood cells. Northern blot analysis of total cellular RNA with a DQα probe indicates that the cell lines constitutively express message for class II gene products. In addition, cell sorter analysis indicates that two of these lines display class II antigen at the cell surface.

A plate-binding elisa for use with hybridoma supernatant fluids

An enzyme-linked immunosorbent assay (ELISA) is described for initial screening of hybridoma supernatants. The protocol is flexible enough to be used with a variety of different antigen types with only a few minor modifications. The method is uncomplicated and does not require any special instnumentation or expertise.

The detection of HLA-DR, MB and MT determinants on purified class II molecules by inhibition of microcytotoxicity

An assay has been developed which makes it possible to determine the HLA allospecificities carried by molecules in purified fractions of detergent lysates from EBV-transformed human lymphocytes. It is based on inhibition of the standard microlymphocytotoxic test used for identifying HLA class I and II antigens with alloantisera. Soluble cell membrane products from EBV-transformed cell lines homozygous for the HLA region gave specific inhibition of standard typing antisera. The test requires preincubation of microliter volumes of soluble antigen preparations maintained in 0.05% NP-40 with selected antisera prior to adding EBV-transformed cells as target cells. It was possible using this assay to follow isolation of the structurally related human class II molecules bearing the MB and DR specificities. Detergent lysates of cells were fractionated on affinity columns prepared from monoclonal antibodies directed against distinct class II antigens. Eluates from these columns contained the expected DR and MB specificities. The assay is easy to perform, highly reproducible and allows multiple determinations.

The Role of Rabbit Immunoglobulin Allotypes in an Immune Network

The expression of latent allotypes is a well documented phenomenon in rabbits. Speculation about their molecular genetic basis and the mechanisms that control expression of these unexpected markers lead inevitably to questions about the Ig gene complement of the rabbit. This central question is under current study using probes derived originally from the mRNA of rabbit-mouse hybridomas secreting rabbit Ig chains. Some of the basic features of rabbit Ig genes are already clear from these studies, and DNA fragments that might encode latent allotypes have been identified with the first set of probes. Further gene closing sequencing should shortly provide a definitive answer to the question of latent allotypy and will also provide a detailed understanding of nominal Ig expression in rabbits.