Chicken antichromatin antibodies: specificity to different chromatin fractions.

Specific complement-fixing and precipitin antibodies were induced in chicken to chromatin from WI-38 human diploid fibroblast. Chicken antibodies were chosen because they present two major advantages with respect to rabbit antibodies: (a) the optimal NaCl concentration for chicken precipitin is 1.5 M, which is also an optimal solvent for chromatin proteins that are insoluble at the lower salt concentration required by rabbit precipitin; (b) chicken antichromatin antibodies require an antigen concentration much lower with respect to rabbit antibodies for saturation at the complement-fixation reaction. These antibodies are species specific and they react at the complement-fixation and precipitin assay not only with the whole chromatin but also with dissociated proteins, albeit at a higher concentration of both antigen and antibodies. The specificity of these antibodies to different fractions of chromosomal proteins and of the chromatin after washing with a solution at a different sodium chloride concentration has been investigated.     

Chicken developmental antigens: analysis of erythroid populations with monoclonal antibodies

Fusions were performed between the mouse PAI myeloma cell line and spleen cells from Balb/c mice immunized with intact erythrocytes from 1-day Cornell K-strain White Leghorn chickens. Following single cell cloning, four hybridoma clones were found to secrete erythroid specific monoclonal antibodies. Based on its pattern of reactivity, the antibody (IgG2a, kappa) secreted by clone 10C6 detects a specific avian oncodevelopmental antigen associated with the hematopoietic system: chicken fetal antigen (CFA). Two other clones, designated as 3F12 and 4C2, produced antibodies (IgM, kappa) that recognize another avian developmental antigen: chicken adult antigen (CAA). A fourth clone, 9F9, produced an antibody (IgM, kappa) that reacts with all peripheral erythrocytes from both Japanese quail and chicken regardless of age. Clone 10C6 antibody apparently detects an erythrocyte specific (ES) determinant of CFA associated with determinant #8 while antibodies of clones 3F12 and 4C2 recognize a chicken specific determinant of CAA. Analysis by complement mediated microcytotoxicity indicated that the epitopes detected by 10C6 vs 3F12 and 4C2 antibodies were expressed on erythrocytes in a reciprocal fashion during development. Furthermore, strain variations in the incidence of erythrocytes carrying these epitopes were observed. The usefulness of these monoclonal antibodies for the study of erythroid populations is discussed.     

Reaction of phosphofructokinase 2/fructose 2,6-bisphosphatase with monoclonal antibodies. A proof of the bifunctionality of the enzyme

Monoclonal antibodies were derived from mice immunized against homogeneous chicken liver phosphofructokinase 2/fructose 2,6-bisphosphatase. Of 112 clones, 30 were found to secrete antibodies that specifically reacted with the antigen in enzyme-linked immunoabsorbant assay (ELISA) while 17, which were ELISA-negative, produced antibodies that affected the enzymic activity of the antigen. Four clones were subcloned and used for an extensive investigation of the reaction of the corresponding antibodies with the supposedly bifunctional enzyme. A definite proof of the bifunctionality of the enzyme was obtained from the two following observations. First, the two activities were similarly retained by the four antibodies that had been coupled to Sepharose. Second, one of the antibodies inhibited both activities with the same efficiency. Furthermore, the antigen-antibody reaction led to the formation of aggregates with an apparent molecular mass of several megadaltons, showing that the two subunits of the antigen reacted with the same antibody and were therefore identical. The four monoclonal antibodies affected the activity of phosphofructokinase 2. This effect was seen as an up to 17-fold activation as well as an up to 85% inhibition. Only one of the four antibodies (antibody 10) had inhibitory effects on fructose 2,6-bisphosphatase, an effect which was in part explained by a decrease in the rate of formation of the intermediary phosphoenzyme. All the effects described above were obtained on both the chicken liver and the pigeon muscle enzymes but with lower doses of antibody in the case of the former enzyme. Antibody 10 was also shown to react with mouse liver phosphofructokinase 2/fructose 2,6-bisphosphatase, and with phosphofructokinase 2 from chicken brain, heart and testis and from frog skeletal muscle and liver. None of the four antibodies cross-reacted with phosphofructokinase 2 from Saccharomyces cerevisiae or from spinach leaves.     

Antibodies against a nonapeptide of polyomavirus middle T antigen: cross-reaction with a cellular protein(s).

Antibodies were raised against the sequence Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met -Glu, which represents a part of the middle T antigen of polyomavirus that is considered to be important in inducing the phenotype of transformed cells. The antibodies reacted with native as well as denatured middle T antigens. In addition, the antibodies immunoprecipitated a cellular protein with an apparent molecular weight of 130,000 (130K) from mouse and rat cells. In some cases, a 33K protein was also immunoprecipitated. Immunoprecipitation of middle T antigen as well as 130K and 33K proteins was blocked by the peptide. The antibodies labeled microfilaments of untransformed mouse, rat, human, and chicken cells by immunofluorescence. This labeling was also blocked by the peptide. The labeling pattern and distribution under a variety of conditions were indistinguishable from those of anti-actin antibodies, although no evidence has been obtained to indicate that the anti-peptide antibodies react with actin. The 130K protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than chicken gizzard vinculin (130K) and slightly faster than myosin light-chain kinase of chicken smooth muscle (130K). Neither of these proteins absorbed the anti-peptide antibodies. The 33K protein does not seem to be tropomyosin (32K to 40K).     

Induction of antibody response to bromelain-treated mouse erythrocytes by low-density lipoproteins from chicken egg yolk

The administration of low-density lipoproteins from chicken egg yolk (EyLDL) to mice induced T-dependent antibodies against bromelain-treated mouse erythrocytes (BrMRBC). The anti-BrMRBC antibodies produced antigen specifically by EyLDL appear to be identical to the antibodies produced polyclonally by lipopolysaccharide (LPS); both antibodies reacted with the liposomes of phosphatidylcholine and showed identical idiotypic patterns. It is suggested that the anti-BrMRBC antibodies may be produced antigen specifically by exogenous antigens to be useful to the mouse. The activation of anti-BrMRBC B cells specifically by EyLDL or polyclonally by LPS resulted in neither expansion nor regression of anti-BrMRBC LPS-reactive B cells. This finding indicates that the population size of anti-BrMRBC LPS-reactive B cells is controlled independently by exogenous specific or polyclonal stimuli.     

Ovalbumin-like immunoreactivity detected in chicken sensory neurons by antibodies to aldehyde-treated ovalbumin

Summary A method has been developed to raise an antiserum against ovalbumin that can detect this antigen immunohistochemically in chicken sensory ganglia. Ovalbumin-like immunoreactivity has been identified in a subpopulation of chicken dorsal root ganglion neurons by the generation of antibodies to aldehyde-conjugated ovalbumin but not by the antibodies to native ovalbumin, although both antibodies recognize the much higher concentrations of ovalbumin in sections of the oviduct. Biochemical analysis demonstrated that the antigen is more readily detectable in fixed tissue extracts than in fresh tissue extracts. Sensitive immunoblot analysis combined with affinity purification of the antigen, has confirmed that the antigen is of the same molecular weight as ovalbumin. Furthermore, the immunoreactive material elutes at a position identical to native ovalbumin on a molecular sieve column. These findings argue that molecules sensitive to aldehyde fixation may be more readily detected by the use of antisera prepared against aldehyde-modified antigens. The function of the ovalbumin-like antigen in these neurons is unknown.     

Avian developmental antigens: Expression on erythrocytes of chickens,Japanese quail,and quail-chicken hybrids

Monoclonal and polyclonal antibodies were used to examine the expression of three erythroid developmental antigen systems in the chicken, Japanese quail, and quail-chicken hybrid. Chicken fetal antigen (CFA), quail fetal antigen (QFA), and chicken adult antigen (CAA) each represent a series of cell-surface glycorproteins associated with the development of avian hematopoietic cells. Monoclonal anti-CFA antibodies from clones 190-4 and 288-1.1.1.2 supernatants were shown to react against epitopes associated with CFA determinants 8 and 2, respectively. Using complement-mediated microcytotoxicity, these reagents permitted the identification of different erythroid subpopulations in the neonatal chicken and hybrid; therefore, heterogeneity in cell surface CFA determinants among mature peripheral erythrocytes should serve as a useful tool for analyzing erythroid development. In the case of CAA, erythrocytes from adult hybrids were found to express the same complement of CAA determinants identified in the chicken, and CAA appeared much earlier in the hybrid than in either of the parental species. Similarly, two species-restricted fetal antigens associated with similar glycoproteins, CFA8 and QFA, had similar developmental profiles in their respective species, the chicken and quail. In contrast, these antigens were dominantly expressed but exhibited different developmental profiles on erythrocytes from the hybrids. While quail-chicken hybrids exhibited apparent genomic interactions in the expression of these developmental antigens, no evidence for the existence of hybrid-specific fetal antigens was obtained.     

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.     

Role of the B-G-region antigen in the humoral immune response to the B-F-region antigen of chicken MHC

In the chicken MHC there exist two regions, designated F and G, which were separated by crossing-over. The F region contains genes controlling all functions characteristic of the MHC. So far only one gene has been assigned to the G region and it is responsible for the presence of an RBC antigen. When cross immunizing animals of the congenic lines CB and CC with erythrocytes, we have found that both F- and G-specific antibodies were produced. By using the recombinant haplotypes B ^R1 and B ^R2 we were able to dissociate the F from the G antigen and immunize with them separately. It was found that production of F antibodies required the copresence of the G antigen, whereas G antibodies were formed regardless of the presence or absence of the F-region antigen. It could be demonstrated that a prerequisite of the role of the G antigen with respect to the F antigen was the localization of both antigens on the same erythrocyte. Possible mechanisms underlying this phenomenon are discussed.Abbreviations used in this paper MHC major histocompatibility complex - RBC red blood cells - PBL peripheral blood lymphocytes - GVH graft-versus-host - MLR mixed lymphocyte reaction - i.v. intravenous - PBS phosphate-buffered saline - ME mercaptoethanol     

Immunochemical analysis of histone H1 and H5 from pigeon erythroid cells]

An antiserum with the antibody titer of 1 : 4096 was obtained by immunization of rabbits with the tRNA-histone H5 complex from pigeon erythrocytes. The specificity of the antiserum was studied quantitatively from the reaction of the complement binding to a homologous antigen (histone H5) and its modifications (I, II, III), differing in the degree of phosphorylation. It was shown that phosphorylation of histone H5 increases the ability of the antigen to bind to antibodies, which is especially well-pronounced at the antiserum dilutions as high as 20480. The comparison of the antigenic properties of histones H5 from pigeon and chicken erythrocytes revealed beside structural differences of the proteins the presence of common antigenic determinants. A similar observation was made when histones H5 and H1 from pigeon erythrocytes were compared. Histone H1 from chicken erythrocytes and histone H1 from calf thymus did not produce criss-cross reactions with antiserum H5.     

Monoclonal antibodies to tissue-specific chromatin proteins

Antisera raised in mice to chromatins from different tissues of the chicken reacted preferentially with the chromatin type that was used for immunization. This tissue specificity was also evident in the spectrum of monoclonal antibodies generated when mice were immunized with erythrocyte chromatin. Three erythroid-specific antigens and one antigen that was present in a number of chicken tissues were characterized in further detail. These antigens, which comprised less than 0.1% of the erythrocyte chromatin proteins, were nuclear localized although three were also detected in the cytoplasm. Two of the erythroid-specific antigens existed as multiple polypeptides in isolated chromatin. The multiple chromatin forms of one antigen were derived from a precursor protein that was selectively cleaved within 1 min after erythrocyte lysis. Analysis of this antigen in extracts from erythrocytes and reticulocytes indicated that the cleavage of the precursor protein was developmentally regulated in vivo.     

Structural characterization of developmentally expressed antigenic markers on chicken erythrocytes using monoclonal antibodies

Monoclonal antibodies selected for embryonic and adult erythrocyte specificity have been used to characterize developmentally expressed markers on the surfaces of mature circulating erythrocytes of young and adult chickens. The data presented demonstrate that the antigenic changes which occur on the avian erythrocyte membrane with organismic maturation can be accounted for, at least in part, by changes in the expression of structurally different, but possibly related polypeptides. Monoclonal antibodies selected for specific reactivity with the erythrocytes of newly hatched chicks recognize a glycoprotein of 48,000 daltons apparent molecular weight. On two-dimensional isoelectric focusing gels, this antigen, which appears identical in all strains studied, displays microheterogeneity; consisting of eight to nine closely spaced spots with an isoelectric midpoint of approximately 5.5. This antigen is not expressed on the circulating erythrocytes of mature birds; however, an antigen with similar, but perhaps not completely identical structure, can be detected within the adult bone marrow. The monoclonal antibodies which show preferential binding to the circulating erythrocytes of adult birds also immune precipitate an antigen of 48,000 daltons apparent molecular weight, but this antigen has a more basic isoelectric point. The adult antigen is polymorphic. Slightly different patterns were obtained on two-dimensional gels with erythrocytes from inbred birds having different major histocompatibility genotypes. It has a major component near pH 7.0 and additional focusing spots usually occurring at a slightly lower molecular weight near pH 6.8 or 6.6 depending upon strain. Competitive radiobinding assays with B-system-specific alloantisers suggest that these antigens may in fact be antigens of the polymorphic BG locus of the chicken major histocompatibility complex. One-dimensional peptide mapping of the immune precipitated embryonic and adult erythrocyte polypeptides demonstrate that the antigens are borne on distinct but possibly related polypeptides. Both common and unique peptide fragments are found in the digestion products. Selective solubilization of the chicken erythrocyte membrane suggests that the antigens are integral membrane proteins extractable with nonionic detergent but not with reagents which remove peripheral proteins.     

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.     

Reconciling the Structural Attributes of Avian Antibodies

Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation.     

Monoclonal antibodies distinguish titins from heart and skeletal muscle

Murine monoclonal antibodies specific for titin have been elicited using a chicken heart muscle residue as antigen. The three antibodies T1, T3, and T4 recognize both bands of the titin doublet in immunoblot analysis on polypeptides from chicken breast muscle. In contrast, on chicken cardiac myofibrils two of the antibodies (T1, T4) react only with the upper band of the doublet indicating immunological differences between heart and skeletal muscle titin. This difference is even more pronounced for rat and mouse. Although all three antibodies react with skeletal muscle titin, T1 and T4 did not detect heart titin, whereas T3 reacts with this titin both in immunofluorescence microscopy and in immunoblots. Immunofluorescence microscopy of myofibrils and frozen tissues from a variety of vertebrates extends these results and shows that the three antibodies recognize different epitopes. All three titin antibodies decorate at the A-I junction of the myofibrils freshly prepared from chicken skeletal muscle and immunoelectron microscopy using native myosin filaments demonstrates that titin is present at the ends of the thick filaments. In chicken heart, however, antibodies T1 and T4 stain within the I-band rather than at the A-I junction. The three antibodies did not react with any of the nonmuscle tissues or permanent cell lines tested and do not decorate smooth muscle. In primary cultures of embryonic chicken skeletal muscle cells titin first appears as longitudinal striations in mononucleated myoblasts and later at the myofibrillar A-I junction of the myotubes.     

Specific phosphorylation of yeast hexokinase induced by xylose and ATPMg. Properties of the phosphorylated form of the enzyme.

Rabbit antibodies to partially purified nicotinamide mononucleotide adenylyltransferase precipitated the enzyme, which remained fully active in the insoluble complexes. Precipitation of antigen-excess soluble complexes with sheep anti-rabbit γ globulin increased the sensitivity of the immunoassay. With this double-antibody assay, the enzymes from chicken erythrocytes, liver, kidney, and thymus showed nearly identical reactivity. Goose, pheasant, and turkey enzymes were highly cross-reactive with the chicken form; pigeon liver enzyme was markedly less reactive. There was no cross-reactivity with fish, amphibian, or mammalian enzymes. The specificity of the antiserum was increased by absorption of antibodies to nonenzyme proteins. The absorbed serum still precipitated the enzyme; in complement fixation assays, it reacted with an antigen that behaved like the enzyme. This antigen was detectable in whole chromatin and in the proteins extracted from chromatin by high salt or urea concentrations. Its immunological reactivity survived exposure to 0.5 m urea, but was reduced by exposure to 6.0 m urea plus 0.4 m guanidine. The enzyme was present as an inactive, partially denatured protein in nonhistone chromatin proteins prepared with these reagents.     

B-L antigens (Class II) of the chicken major histocompatibility complex control T-B cell interaction

The detailed study of the genetic control of T-B cell interactions in the chicken has been hampered by the lack of defined major histocompatibility complex (MHC) recombinant chicken lines. In the present study we have used some recently described MHC recombinant chicken lines separating regions encoding antigens that are homologous to class I and class II antigens of mammals in adoptive bursa cell transfer experiments, in which bursa cells from newly hatched chicks were transplanted into cyclophosphamide (Cy)-treated chicks. Subsequent immunizations of the recipients with a thymus-dependent antigen (SRBC) and a thymus-independent antigen (Brucella abortus) showed that the generation of germinal centers in the spleen and the production of antibodies to SRBC required identity between donor and recipient class II antigens (B-L antigens), whereas response to Brucella antigen did not require identity at any of the known MHC loci of the chicken. The results thus reveal that also in the chicken class II (B-L) region genes encode cell-surface glycoproteins that serve as restriction elements in T-B cell cooperation.This work has been presented in part at the Ninth International Congress of the Transplantation Society (Vainio et al. 1983a).     

Efficient production of chicken egg yolk antibodies against a conserved mammalian protein

The egg yolk of immunized chicken is a rich and inexpensive source of specific polyclonal antibodies. In this paper we show that 20-30 micrograms of a highly conserved mammalian protein, as exemplified by proliferating cell nuclear antigen, are sufficient to induce an immune response. Immunoblot analysis revealed that specific antibodies appeared 20 days after immunization, reached a plateau after 30 days, and remained high until at least day 81. A total amount of 4 g immunoglobulin was extracted from 62 eggs of one immunized hen, yielding approximately 130 mg of specific antibodies.     

Isolation of monoclonal antibodies reactive with Marek's disease tumor-associated surface antigen (MATSA)

Two hybridoma clones producing monoclonal antibodies were obtained from mice immunized with the Marek's disease (MD)-lymphoblastoid cell line MSB1. These monoclonal antibodies reacted with the surface of MD-lymphoblastoid cell lines at higher titers than with avian lymphoid leukosis cell lines or with normal chicken thymus, bursa or peripheral blood lymphocytes. The serological specificity of these monoclonal antibodies seemed to correspond with that of rabbit antiserum reactive with MD tumor-associated surface antigen (MATSA).     

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR)

Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody–antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (<1.25 μg/ml) in a physiologically relevant range (1–40 μg/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.