Monna Crone

Eggs: Conveniently packaged antibodies. Methods for purification of yolk IgG

Eggs from immunized chickens may provide a convenient and inexpensive source of antibodies. We describe two simple and efficient methods for purification of IgG from yolk. The antibody is rendered useful for most currently employed immunological techniques. Amounts of antibody corresponding to almost half a litre of antiserum may be recovered from a chicken in one month.

The MHC haplotypes of the chicken.

The major histocompatibility complex (MHC) of Gallus gallus is the B complex of which three classes of cell-membrane antigens have been clearly defined by serological, histogenetic, and biochemical methods. Two of these classes are homologous to classes I and II of mammals (B-F and B-L, respectively), while the third (B-G) is a differentiation antigen of the erythroid cell-line; the mammalian homologue of this class is still undefined. The B haplotypes comprise at least one gene of each class that displays linkage disequilibrium of a remarkable strength. The present work is the first systematic comparison by serological and histogenetic methods of the allelic products (allomorphs) of 15 haplotypes, including all of the 11 that were accepted as “standard” B haplotypes at the recent international Workshop on the chicken MHC in Innsbruck, Austria. The analysis has revealed many similarities, but only four pairs of probable identities: G2 and G12, F4 and F13, L4 and L13, L12 and L19. It appears therefore that the B-G locus is comparable in its degree of polymorphism to the class I (B-F) locus. The “standard” haplotypes are almost all of White Leghorn derivation, and preliminary typings of other breeds of chickens, and of wild chickens, indicate the existence of a much wider spectrum of allomorphs.

The Elusive T Cell Receptor

Less than five years ago, at the Cold Spring Harbor Symposium on Antibodies, there was no one to question the assumption that recognition of antigen by potentially reactive lymphocytes is effectuated by specific immunoglobulin receptors. Nothing but nodding approval from the audience could, for example, meet either Mitchison or Jeme when, each in their fashion, they made axiomatic this generally shared assumption. The thought of multipotential lymphocytes referred to by Jeme (1967) as heresy seems but mild revisionism compared to the suggestion that recognition structures may not be immunoglobulins after all. It is nevertheless towards the latter suggestion we are becoming inclined with regard to cell mediated immunity. The present paper wiU describe our own investigations of the receptor mechanism involved in the GVH reaction in chicken embryos and discuss the findings in relation to conflicting findings in the literature. The principal approach has been to try neutralizing the GVH reaction with specific heteroand isoimmune sera. As a concurrently pursued interest we have investigated the same antibodies for their ability to inhibit antigenbinding cells in their rosette formation with sheep red blood cells (SRBC). Many of the best experiments have combined these two reactions by employing for the GVH reaction donor animals which were pre-immunized with SRBC. Three principal groups of antisera have been used, namely a) rabbit anti chicken L-chain (anti-L), b) rabbit anti chicken thymus (ATS), or bursa (ABS), and c) isoimmune chicken sera raised by cross-immunizing homozygous B 1/1 and B 2/2 birds (anti-Bl and anti-B2). The latter group, of course, are antisera directed against alloantigens determined by the major histocompatibility and blood group locus of the chicken, the B locus.

The chicken erythrocyte-specific MHC antigen. Characterization and purification of the B-G antigen by monoclonal antibodies

Mouse monoclonal antibodies with B-G antigen (major histocompatibility complex class IV) specificity were obtained after immunization with erythrocytes or partially purified B-G antigen. The specificities of the hybridoma antibodies were determined by precipitation of B-G antigens from 125I-labeled chicken erythrocyte membranes (CEM) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The B-G antigen had an approximate molecular mass of 46–48 kd in reduced samples, depending on the haplotype, and in unreduced samples contained either dimers (85 kd), when labeled erythrocytes were the antigen source, or trimers (130 kd), when B-G was purified and precipitated from CEM. The B-G antigen was unglycosylated as studied by (1) in vitro synthesis in the presence or absence of tunicamycin, (2) binding experiments with lectin from Phaseolus limensis, and (3) treatment of purified B-G antigen with Endoglycosidase-F or trifluoromethanesulfonic acid. Two-way sequential immunoprecipitation studies of erythrocyte membrane extracts with anti-B-G alloantisera and monoclonal antibodies revealed only one population of B-G molecules. Pulse-chase experiments have shown B-G to be synthesized as a monomer, with dimerization taking place after 20–30 min. No change in the monomers molecular mass due to posttranslational modifications was revealed. The antigen was purified from detergent extract of CEM by affinity chromatography with a monoclonal antibody, and then reduced and alkylated and affinity-purified once more. Finally, reverse-phase chromatography resulted in a pure product. The B-G antigen was identified in the various fractions by rocket immunoelectrophoresis. The final product was more than 99% pure, as estimated by SDSPAGE analysis followed by silver stain of proteins. The yield from the affinity chromatography step was 3–4 μg B-G/ml blood, calculated from Coomassie-stained SDS-PAGE of B-G using ovalbumin standards. The monoclonal antibodies were also used to identify the B-G (class IV) precipitation arc in crossed immunoelectrophoresis. No common precipitate with the B-F (class I) antigen was observed.

B‐L Antigens (Ia‐like Antigens) of the Chicken Major Histocompatibility Complex

This paper reviews the present knowledge of B‐L antigens encoded by the chicken B complex as regards to the following aspects: (1) identification and cellular expression, (2) structural studies, (3) evidence for two distinct populations of B‐L antigens, (4) mapping of B‐L loci of the B complex, (5) B‐L and immune response, and (6) the role of the B‐L antigens for the control of mixed lymphocyte reactions (MLR) and graft‐versus‐host (GVH) reactions. It is concluded that B‐L antigens of the chicken exhibit extensive homology with mammalian la antigens. A genetic map of the B complex is presented.

Evidence for two populations of B-L (Ia-Like) molecules encoded by the chicken MHC

Two specific alloantisera detecting B-L (Ia-like) antigens on chicken lymphocytes of the B6 and B15 haplotypes were found to cross-react strongly. Anti-B-L6 and anti-B-L15 alloantisera both reacted with B-L molecules on B6 and B15 lymphocytes as demonstrated by immunofluorescence and SDS-PAGE analysis of 125I-labeled B-L antigens isolated by incubation with anti-B-L alloantisera. Absorption studies showed that the anti-B-L alloantisera reacted with at least two kinds of antigenic determinant, one set shared by B-L6 and B-L15 molecules and another set specific for each haplotype. In spite of the absence of genetic evidence for more than one B-L locus in the chicken B complex, it was shown by sequential antibody incubations that these two different B-L antigenic determinants are associated with at least two separate species of B-L molecules, indicating the presence of at least two B-L loci within the MHC of the chicken.

Binding of corticosterone by thymus cells, bursa cells and blood lymphocytes from the chicken

Abstract The binding of tritiated corticosterone in lymphocytes from thymus, bursa of Fabricius and peripheral blood from chickens were studied in vitro . A high-affinity binding of corticosterone was found in all populations of lymphocytes that were investigated. The affinity of the binding was nearly identical to that previously found in rat thymocytes. No high-affinity binding of corticosterone was found in chicken thrombocytes, in chicken erythrocytes or in sheep erythrocytes.

Isolation and characterization of chicken and turkey beta2-microglobulin

Abstract Chicken and turkey beta2-m were isolated from citrated plasma in sequential use of three Chromatographie steps: affinity chromatography, gel filtration chromatography and anion-exchange chromatography. The purified protein was identified as beta2-m by reaction with a beta2-m specific monoclonal antibody and by the ability to recombine with the chicken MHC class I heavy chain. The purity was estimated by SDS-PAGE and IEF. The pI was between 5.1 and 5.3 for chicken beta2-m and 4.7 and 4.8 for turkey beta2-m, which fact is reflected in their different electrophoretic mobilities in agarose gel (turkey migrates in the alpha and chicken migrates in the beta region). The mol. wt of both chicken and turkey beta2-m was 14,500 estimated by SDS-PAGE whereas calculations based on the amino acid compositions gave mol. wts of 11,000. E M 280 was 15.9 for chicken beta2-m and 16.4 for turkey beta2-m. The amino acid compositions and sequences of the two avian beta2-m molecules have been compared with earlier data from the literature. The sequence of the 23 N -terminal amino acids was found to be identical in our preparations from both chicken and turkey, namely DLTPKVQVYSRFPASAGTKNVLN, and is incompatible with a previously published sequence also thought to be from turkey beta2-m. Reasons for our opinion that the molecules isolated and sequenced in this paper are the correct ones are given.

The still elusive T cell receptor: on the possibility of a common V-gene pool for B- and T-cell-antigen receptor molecules.

The contention that VH constitutes a part of T‐cell receptors for antigens was probed by purifying rabbit T cells and analysing these cells for non‐immunoglobulin VH, i.e. VH not associated with L chain, A number of anti‐VH antisera were employed for this purpose, the most important being goat antiserum, reacting with common al allotype determinants (allotype determinants expressed on free VH and H chain as well as on intact immunoglobulins), rat antibody against common non‐allotype VH determinants (VH framework determinants expressed on VH and H chain as well as on intact immunoglobulins) and chicken antibody against unmasked non‐allotype determinants (VH determinants accessible only in the absence of L chain). VH and L chain was quantified by radioimmunoassays on extracts and supernatants from unstimulated T cells as well as from T cells stimulated by concanavalin A and by allogeneic cells. Absolute depletion of Ig‐containing and ‐producing cells was not achieved but in no case was an excess of VH over L chain observed. This indicates that all detected VH originated from cells of the B lineage. The cells were also cultured in the presence of labelled amino acids followed by analysis of detergent extracts and supernatants by immunoadsorption and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) fluorography. Again, no evidence for T‐cell VH could be found. Affinity purified anti‐VH antibody was used to label viable rabbit T cells through the use of secondary fluorescence‐labelled anti‐immunoglobulin antibody. No VH‐specific labelling of T cells could be observed. Mixed lymphocyte cultures were carried out in the presence of affinity‐purified anti‐VH antibodies. No inhibition of the reaction could be discerned. The failure to detect T‐cell VH is in agreement with the recent finding that the VH‐genome in T cells is not rearranged in a functional manner similar to that in B cells.

Antigen Binding by Non-Bursa-Derived Chicken Leukocytes

Antigen‐binding peripheral blood leukocytes (PBL) from normal and bursectomized agammaglobulinemic chickens were labeled by incubation in vitro with radioiodinated antigen at 4°C in the presence of sodium azide. [125I] TGAL‐binding cells could be detected by autoradiography of PBL from normal, unimmunized chickens at a frequency of 1 to 4 labeled cells per 104 leukocytes. No [125I] TGAL‐binding cells were found in PBL from bursectomized chickens, even after incubation with 25 μg/ml of labeled antigen followed by prolonged autoradiographic exposure. The binding to normal PBL was specific as judged by inhibition with unlabeled TGAL but not with unlabeled TIGAL. The binding was, furthermore, inhibited by preincubation with rabbit anti‐chicken L chain antibody but unaffected by normal rabbit IgG [125I] TIGAL was, in contrast, found to bind to PBL from both normal and bursectomized chickens at a frequency of 6 to 80 labeled cells per 104 leukocytes. The labeling was specific, since it was inhibited by cold TIGAL but not by cold TGAL. The binding of [125I] TIGAL to PBL from bursectomized chickens showed from none to slight inhibition on preincubation of the cells with anti‐L chain antibody, whereas preincubation with normal rabbit IgG resulted in almost complete inhibition. To our knowledge this is the first demonstration of antigen binding to PBL from agammaglobulinemic chickens.