Chicken Immunoglobulin Research Articles

Mapping of the chicken immunoglobulin heavy‐chain constant region gene locus reveals an inverted α gene upstream of a condensed υ gene

Chicken antibodies are increasingly being used as diagnostic and therapeutic tools. As only the genomic organization of the micro encoding gene was previously known, we analysed the chicken immunoglobulin Y (IgY) and IgA (upsilon and alpha chain) immunoglobulin heavy-chain constant region (IGHC) genes and the organization of the chicken IGHC locus. The alpha gene is encoded by four separate exons, whereas, surprisingly, there is no intervening DNA sequence between the CH1 and CH2 domains of the IgY heavy chain, which is thus encoded by three exons separated by two introns. DNA sequence analysis shows that the exon boundaries of the chicken IGHC genes are not consistent with published domain borders. Furthermore, differences in DNA sequence confirm the existence of IgY, IgA and IgM allotypes in chickens. Finally, our results show that the IGHC genes of chicken (IgY, IgA and IgM) are all colocated on chromosome E18C15W15, where the alpha gene is located upstream of the upsilon gene in an inverted transcriptional orientation. The distances between the mu and alpha genes and the alpha and upsilon genes are about 18 and 15 kilobases, respectively, and thus, the size of the whole chicken IGHC locus is approximately 67 kilobases.

Adjuvant effects of various lipopeptides and interferon-γ on the humoral immune response of chickens

The adjuvant effects of various lipopeptides and recombinant chicken interferon γ (IFN-γ) on the humoral immune response of laying hens was investigated in four immunization studies. We used the lipopeptide Pam3Cys-Ser-(Lys)4 (PCSL), the conjugate P-Th1 consisting of the lipopeptide P3CS and the T-helper epitope Th1 (FISEAIIHVLHSRHPG), and the conjugate P-Th2 of the lipopeptide P3CSS and the T-helper epitope Th2, which corresponds to the peptide EWEFVNTPPLV, as adjuvants. Human serum albumin (HSA), recombinant bovine somatotropin (RBST), and human immunoglobulin G (IgG) served as antigens in the different experiments. All tested adjuvants enhanced the humoral immune response with various intensities. Chickens showed high antibody titers after the immunization with HSA even without adjuvant, but the adjuvant effects of PCSL and the combination of PCSL and recombinant chicken interferon-γ (IFN-γ) were much more pronounced using the antigens RBST and IgG. Especially after the third immunization, higher titers of antibodies were induced by the coadministration of P-Th1 and, to a greater extent, by the combination of PCSL and P-Th1 compared with the use of PCSL. Also, chickens that had received PCSL and P-Th2 showed the highest immune response, even after the second booster. The average concentrations of chicken immunoglobulin Y were significantly higher in 5-mo-old chickens (9.4 mg/mL serum and 10.1 mg/mL egg yolk) compared with 9-mo-old chickens (5.9 mg/mL serum and 5.1 mg/mL egg yolk). The specific serum antibody response was higher in the older chickens than in the younger chickens. Because chicken antibodies are likely to be used increasingly for diagnostic and therapy in the future, lipopeptides and recombinant chicken IFN-γ may find many applications as adjuvants, thus contributing to the welfare of experimental animals.

Methods for the generation of chicken monoclonal antibody fragments by phage display

Phage display has become an important approach for the preparation of monoclonal antibodies from both immune and nonimmune sources. This approach allows for the rapid selection of monoclonal antibodies without the restraints of the conventional hybridoma approach. Although antibodies to a wide variety of antigens have been selected using phage display, some highly conserved mammalian antigens have proven to be less immunogenic in mammalian animals commonly used for immunization. In order to optimize methods for constructing chicken immunoglobulin phage display libraries in the pComb3 system, we have immunized chickens with the hapten fluorescein, and generated combinatorial antibody libraries from spleen and bone marrow RNA. Herein we present methods for the isolation of scFv, diabody and Fab fragment libraries from chickens. Chicken Fab fragment libraries are constructed using human constant regions, facilitating detection with readily available reagents as well as humanization. Analysis of the selected V-genes revealed that gene conversion events were more extensive in light-chain variable region genes as compared to heavy-chain variable region genes. In addition, we present a new variant of the pComb3 phage display vector system.

Quantitation of Rat Lacrimal Secretion: a Novel Sandwich ELISA with High Sensitivity

Modulation of lacrimal acinar cell tear secretion may involve multiple factors acting both in subtle and pronounced ways. Functional screens of recombinant protein products arising from gene array technologies, or protein fractions derived from lacrimal conditioned media or extracellular matrix, will require a highly sensitive assay capable of monitoring tear protein secretion by small replicate cultures. To improve significantly on current methods, a rat- and mouse-specific sandwich ELISA was developed. For this purpose, chickens and rabbits were immunized with serum-free secretion media from carbachol and VIP-stimulated rat lacrimal acinar cell cultures. Immune sera were characterized by ELISA, Western blotting and immunohistochemistry, and subsequently optimized for use in a sandwich ELISA. Both antisera detected a wide range of different rat tear proteins, and immunostained only the secretory granule-rich juxtalumenal region in sections of rat lacrimal gland. Chicken, but not rabbit, antiserum cross-reacted with rabbit and human tears. In sandwich ELISA, capture with purified chicken immunoglobulin fraction and detection with rabbit antiserum detected as little as 1ngml−1tear protein in 10000-fold diluted rat secretion medium—a level of sensitivity 8000 times greater than the rat tear peroxidase assay. Such specificity and sensitivity greatly reduces the quantity of media needed for assay, and makes feasible functional screens for scarce factors that may influence lacrimal secretory processes, and in turn possibly play a role in human lacrimal insufficiency syndromes.

Detection of endogenous avian leukosis virus envelope in chicken plasma using R2 antiserum

Immunologic tolerance to oncogenic avian leukosis virus (ALV) is mediated, in part, by the interaction of endogenous ALV (EV) envelope and immune competent cells. A flow cytometry method is described for detecting the EV envelope in chicken plasma or serum. The method employs two types of target red blood cells (RBC) obtained from chickens lacking EV; RBC susceptible to EV infection (containing EV receptors), and those resistant to EV infection (lacking EV receptors). RBC from susceptible chickens will bind EV envelope glycoprotein (gp85) when present in plasma. The gp85-bound RBC are subsequently incubated with a highly specific chicken alloantibody, termed R2. Using flow cytometry, gp85 is detected indirectly with a fluoresceine-tagged antibody to chicken immunoglobulin; plasmas lacking gp85 are nonreactive and fluorescence remains at a background level. Because RBC from resistant chickens are nonreactive regardless of the presence or absence of EV gp85, a specific binding index was calculated to compare relative binding of EV gp85 on susceptible and resistant RBC, and thus identify chickens that express EV gp85. The specificity of the assay was demonstrated using plasma from chickens of 14 standard laboratory lines previously defined for EV envelope expression including two sets of highly congenic lines that differ in EV expression. This assay detects differences attributable to EV gp85 in chickens of commercial breeding lines of White Leghorns and broilers. Moreover, if chickens lack EV, the R2 plasma assay can differentiate between EV-susceptible and EVresistant siblings.

Cloning and expression of the chicken immunoglobulin joining (J)-chain cDNA.

The J chain is a component of polymeric immunoglobulin (Ig) molecules and may play an important role in their polymerization and the transport of polymeric Ig across epithelial cells. In this study, the primary structure of the chicken J chain was determined by sequencing cDNA clones. The cDNA had an open reading frame of 476 nucleotides encoding a putative protein of 158 amino acid residues including the signal sequence. The 3' untranslated region consisted of 1216 nucleotides and a poly(A) tail. The deduced amino acid sequence of the chicken J chain had a high degree of homology to that of human, cow, rabbit, mouse, frog, and earthworm, with eight conserved Cys residues identical to the mammalian J chains. Northern blot hybridization performed with total RNA from various chicken tissues revealed high levels of J-chain mRNA expression in spleen, intestine, Harderian gland, and bursa of Fabricius, and low levels in the thymus. The J chain was expressed in the bursa as early as day 15 of embryogenesis. These data indicated that the chicken J-chain gene displays a high degree of homology with that of other species, and is expressed at an early stage of development of the chicken immune system.

Detection of multiple toxic agents using a planar array immunosensor

A planar array immunosensor, equipped with a charge-coupled device (CCD) as a detector, was used to simultaneously detect 3 toxic analytes. Wells approximately 2 mm in diameter were formed on glass slides using a photoactivated optical adhesive. Antibodies against staphylococcal enterotoxin B (SEB), ricin, and Yersinia pestis were covalently attached to the bottoms of the circular wells to form the sensing surface. Rectangular wells containing chicken immunoglobulin were used as alignment markers and to generate control signals. After removing the optical adhesive, the slides were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction lens array was used to focus the sensing surface onto the CCD. Solutions of toxins were then placed on the slide. After rinsing, Cy5-labeled antibodies were introduced. The identity and amount of toxin bound at each location on the slide were determined by quantitative image analysis. Concentrations as low as 25 ng/mL of ricin, 15 ng/mL of pestis F1 antigen, and 5 ng/mL of SEB could be routinely measured.

Octamer function in the chicken lambda immunoglobulin light chain promoter.

The chicken immunoglobulin (Ig) lambda light chain promoter contains an octamer motif (-106) and a TATA box (-70). Two lymphoid cell lines, DT40 (B-cell line) and MSB1 (T-cell line), were used to test the activity of the germline promoter and the function of the octamer motif. The promoter was functional only in B cells in the presence of SV40 enhancer. Mutation of the octamer motif eliminated the transcriptional activity of the Ig promoter. These results suggest that the chicken Ig promoter functions similarly to mammalian Ig promoters in that it plays a role in tissue specificity and requires both an enhancer and an intact octamer motif for activity.

Zur systemischen verfügbarkeit von bovinemimmunglobulin g und hühner‐immunglobulin y aus gefüttertem kolostrum bzw. volleipulver bei neugeborenen kälbern

Bei 26 neugeborenen männlichen Kälbern aus 23 verschiedenen Betrieben wurden im Rahmen eines Versuchs zur Prophylaxe von infektiösen Durchfallerkrankungen mit spezifischen Dotterantikörpern auch Untersuchungen zur systemischen Verfügbarkeit von kolostralem bovinen Immunglobulin G (blgG) und von Hühner‐Immunglobulin Y (IgY) aus zugefüttertem Volleipulver durchgeführt. Den Kälbern wurde dazu in den ersten 14 Lebenstagen täglich zum gleichen Zeitpunkt eine Blutprobe entnommen. In Verbindung mit der Kolostrumgabe war die blgG‐Konzentration am Tag 1 post natum am höchsten und lag im Mittel bei einem Wert von 9,3 mg/ml Serum. Der gemittelte blgG‐Spiegel nahm anschließend kontinuierlich ab und erreichte am Tag 12 post natum einen signifikant niedrigeren Wert von 4,9 mg/ml Serum. Bis zum Ende der Beobachtungsperiode (Tag 14 post natum) blieb die mittlere blgG‐Konzentration weitgehend stabil (5,2 mg/ml). Die Gesamtproteinkonzentrationen im Serum blieben hingegen im Beobachtungszeitraum relativ konstant (im Mittel 56,8 mg/ml; SD 11,2). Die Anzahl der Kolostrumgaben je Tier hatte keinen signifikanten Einfluß auf die Höhe der bIgG‐Konzentrationen. Die Streubreite der individuellen Konzentrationen war an den einzelnen Tagen der ersten beiden Lebenswochen enorm. Der mittlere Variationskoeffizient lag bei 52,1 % (SD 5,7). Bei der Berechnung der individuellen bIgG‐Kurvenverläufe mittels einer Regressionsgleichung ergaben sich zwei Gruppen mit signifikant verschiedenem Eliminationsverhalten. So zeigte eine Gruppe (n= 10) mit Eliminationskonstanten von k < —0,02 eine mittlere Halbwertszeit für blgG von 24,3 Tagen (SD 4,6). Bei den übrigen Kälbern (n = 16) mit einer Eliminationskonstante für blgG von k >—0,02 lag die berechnete Halbwertszeit möglicherweise aufgrund der frühzeitiger erfolgten blgG‐Eigensynthese bei einem theoretischen Wert von 68,5 Tagen (SD 36,7). An 18 Kälbern wurde im selben Versuchszeitraum täglich 20 g Volleipulver mit einem IgY‐Gehalt von 15 mg/g verabreicht. Wurde das Eipulver bereits erstmals in den ersten 12 Lebensstunden zugefüttert, so erreichten die Kälber maximale IgY‐Konzentrationen von im Mittel 1,9 ug/ml Serum. Ein späterer Beginn der Eipulverzulage hatte signifikant niedrigere IgY‐Spiegel zur Folge. Wurde sie beispielsweise zwischen der 25igsten und 48igsten Lebensstunde begonnen, so zeigte sich eine mittlere IgY‐Konzentra‐tion von nur 0,035 μg/ml Serum. Aus der Berechnung der individuellen Eliminationskonstanten mittels einer Regressionsgleichung (r2 = 0,84) ergab sich eine mittlere Halbwerfszeit für IgY von 5,0 Tagen (SD 2,5). Um die Resorption und damit auch die mögliche systemische Wirksamkeit von speziesfremden Antikörpern weitgehend zu verhindern, sollte Eipulver zur Prophylaxe von infektiösen Durchfallerkrankungen beim neugeborenen Kalb demzufolge erst 24 bzw. besser 48 Stunden post natum zugefüttert werden. Entscheidend für eine wirksame Prophylaxe gegen infektiöse Durchfallerkrankungen ist allein die hohe intestinale und nicht die systemische Verfügbarkeit von spezifischen Antikörpern.

Monoclonal and polyclonal antibodies to chicken immunoglobulin isotypes specifically detect turkey immunoglobulin isotypes

Turkey immunoglobulin (Ig) isotypes IgG and IgM were isolated from blood and IgA was isolated from bile. Isolation was accomplished by gel filtration of the ammonium sulphate cut on Sephacry S-200. Using immunoelectrophoresis and indirect ELISA, the cross-reactivity between antibodies, of monoclonal and polyclonal origin, specific for the Ig isotypes of chicken, and the purified turkey Ig isotypes was evaluated. Commercially available polyclonal antibodies, anti-chicken/IgA (α-chain specific, affinity purified), anti-chicken/IgG (Fc-fragment specific) and anti-chicken/IgM (μ-chain specific) showed an interspecies cross-reactivity with the corresponding turkey Ig isotypes. The monoclonal antibody (MAb) AV-G3 specifically detected turkey IgG, whereas MAb M1 reacted exclusively with turkey IgM. This panel of anti-immunoglobulins represents a useful tool for examining the humoral immune responses of turkeys.

Use of a Heterologous Solid-Phase Antigen in an Indirect Competitive Antibody-Capture Enzyme-Linked Immunosorbent Assay for T-2 Mycotoxin

Anti- T-2 toxin (T-2) antibodies raised in chicken hosts were isolated as total IgY (chicken immunoglobulins of IgY isotype) from serum and the egg yolk. They were used for quantitation of T-2 toxin in an indirect competitive enzyme-linked immunosorbent assay (ELISA) which employed molecularly distinct toxin-carrier conjugates as solid-phase assay antigens. The sensitivities of the assays utilizing any of the purified IgY or unfractionated serum were similar but varied with respect to the type of conjugate used and could be improved by lowering the concentration of assay antigens or antibodies. Low concentrations of both immunoreactants resulted in assays of superb sensitivity, although the standard curves generated with some assay antigens decreased markedly in slopes and impractically long incubation times were required to develop sufficiently high read-out signals. However, high optical signals were obtained in a very short period of time when an amplified substrate system instead of para-nitrophenyl phosphate (pNPP) was used for the detection of a reporter enzyme, alkaline phosphatase. The use of an amplified substrate and of a solid-phase antigen that was prepared with Iso T-2 toxin attached directly to a carrier protein yielded an ELISA that had an improved ability to detect T-2.

Chicken immunoglobulin μ-chain gene: germline organization and tandem repeats characteristic of class switch recombination

We have isolated the phage clones covering the region spanning from the heavy (H)-chain joining (J) region to the end of the μ-chain gene of the chicken immunoglobulin (Ig). The distance from J H to the first exon of the μ-chain constant (C) region is approximately 13 kb, and introns between the C region exons measure more than 3 kb. These distances are significantly larger than those of known μ-chain genes. We found a region cross-hybridizing to the switch regions of the mouse Cμ and Cα genes just in front of the first exon of Cμ. Partial nucleotide sequencing of this region revealed that this region consists of tandem repeats of pentamers ( C T )( C A ) CAG complementary to the mammalian switch repetitive region. These findings suggest that this region is a good candidate for a class switch region of the μ-chain gene of chicken Ig.

Chicken egg yolk antibodies for cytokinin analysis

The production, isolation, and purification of specific chicken immunoglobulins (Igs) against three main groups of naturally occurring cytokinins are reported. The specific Igs directed against, respectively, zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine are extracted from the egg yolk and used in radioimmunoassays that allow the quantification in parallel of pmol of the cytokinins in plant extracts. As little as 50 fmol of zeatin riboside, 20 fmol of isopentenyladenosine, and 40 fmol of dihydrozeatin riboside can be detected. The levels of cytokinins measured in the radio-immunoassay correlate well with physicochemical analysis methods such as high performance liquid chromatography (HPLC) with UV spectrum detection and HPLC-coupled mass spectrometric detection. Cross-reactivity studies indicate that the assay is not affected by most of the structurally related compounds. The respective antibody preparations recognized zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine and the corresponding free bases. The results obtained when analyzing crude plant extracts are expressed as zeatin riboside equivalents, dihydrozeatin riboside equivalents, and isopentenyladenosine equivalents.

Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes

In chickens, single functional immunoglobulin variable and joining gene segments at each of the heavy and light chain loci undergo V(D)J rearrangement. Diversity is subsequently introduced by conversions templated by upstream pseudo V region genes in such a way that practically all V regions in mature B cells have identical ends. This greatly simplifies the representative amplification of V region genes. Furthermore, the entire naive repertoire of the adult chicken is produced in the bursa of Fabricius of the young bird. These special properties of the generation of immunoglobulin diversity in chickens have been exploited in the development of procedures to produce large libraries of diverse antibody combining sites derived from chicken Ig genes and expressed on filamentous bacteriophage. The utility of this library was assessed by selection of specifically binding phage using three solid phase-bound protein antigens, hen egg white lysozyme, bovine thyroglobulin and bovine serum albumin. The sequences of the V region genes thus isolated demonstrated that selection was specific and that the library contained useful diversity of binding sites. This library provides access to a repertoire whose diversity is based on a mechanism different from that underlying previously available libraries. The demonstrated feasibility of generating chicken phage antibodies may lead to the production of monoclonal reagents from immunised chickens, and the derivation of reagents for studying immunoglobulin mediated selection in avian B cell development.

The chicken immunoglobulin lambda light chain gene is transcriptionally controlled by a modularly organized enhancer and an octamer-dependent silencer.

Characterization of the regulatory elements involved in V(D)J recombination is crucial for understanding development of the B and T cell immune repertoire. Previously we have shown that the chicken immunoglobulin lambda light chain gene (CLLCG) undergoes lymphoid-specific rearrangement in transgenic mice. The whole gene is only 10 kb in length and contains all phylogenetically conserved target sites for recombinational and transcriptional regulation. In this study we have localized an enhancer element in a region 4 kb downstream of the constant (C) region. The 467 bp element can be subdivided into three subfragments. The previously detected silencer element on the V-J intervening sequence is shown to be localized on a 500 bp fragment. Partial silencer activity is retained on a 250 bp fragment, which includes an octamer motif. By mutational analysis this octamer is shown to be essential for B cell- but not for T cell-specific silencer function. The silencer represses transcription directed by heterologous elements like the SV 40 promoter or the Ig kappa 3' enhancer. We propose that transcription of the unrearranged and rearranged Ig genes is regulated by complex interactions between different modules from the promoter, enhancer and silencer, which is eliminated by recombination during B cell development.

Isolation and Characterization of Serum Immunoglobulin Classes of the Ostrich (Struthio camelus)

Immunoglobulins were separated from ostrich sera by ammonium sulfate precipitation and ion-exchange chromatography. Two classes of immunoglobulin could be identified, corresponding to IgG and IgM of other species, based on elution profiles from ion-exchange columns and molecular mass estimation on gel-filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On SDS-PAGE, the heavy chains of IgG and IgM were shown to have molecular masses of 67.5 kDa and 65 kDa, respectively, and the light chains common to both were shown to have a molecular mass of 27 kDa. The ostrich immunoglobulins were not recognized by monoclonal and polyclonal antibodies against immunoglobulins of many species of animals tested, nor by antibodies against chicken immunoglobulins.

Rearrangement of the chicken lambda light chain locus: a silencer/antisilencer regulation

Chicken immunoglobulin λ light chain rearrangement is regulated by different controlling elements. One negative control element, acting as a strong transcriptional silencer, is located in the V-J intervening sequence which is excised during the rearrangement process. Positive control elements include the Vλ promoter, the enhancer of transcription (3′ of Cλ), and one (or two) putative antisilencer element(s) located on each side of the silencer. It is proposed that these antisilencer elements counteract transiently the silencer thus allowing rearrangement of one allele in chicken B cell progenitors. The implications of this regulation for mouse B cell development are discussed.

Polymorphism of the functional immunoglobulin variable region genes in the chicken by exchange of sequence with donor pseudogenes.

We have isolated a number of new allelic variants of the unique functional genes encoding chicken immunoglobulin heavy and light chain variable regions (VH1 and VL1, respectively). The distribution and nature of nucleotide variation among these and previously identified VH1 and VL1 alleles demonstrates that random point mutations are likely not the predominant cause of allelic variation at these loci. Comparison of the variant nucleotides with sequences from the pseudo-VH and pseudo-VL gene families, which lie 5' to VH1 and VL1, respectively, suggests that the great majority of allelic variants can be accounted for by segmental transfer of sequence from donor pseudogenes into the germ-line VH1 and VL1 genes. These results demonstrate that the chicken VH1 and VL1 genes are susceptible to sequence replacement at the germ-line level as well as somatically during antibody diversification. The limited repertoire of B cell specificities produced by gene rearrangement in the chicken has led to speculation that these specificities may play a critical role in the progression of chicken B cell development. The results presented here do not support this hypothesis since many of the allelic variant nucleotides described here encode non-conservative amino acid substitutions within the antigen-binding sites of the Ig molecule.

Application of the immune complex for immune protection against viral disease

An immune complex (IC), composed of antigenic subunits of the Newcastle disease virus (NDV) and specific polyclonal allogeneic antibodies, was used to protect chickens against NDV. Antibodies in the IC were chicken immunoglobulin G. The antibody; antigen ratio in IC was 2.03. The IC was prepared at equivalence by direct mixing of NDV-infected allantoic fluid, treated with Triton X-100, and chicken anti-NDV serum. In order to bind NDV antigenic subunits to specific antibodies, previous isolation and purification of antigen is not required. Chickens were immunized with 1 mg IC, containing 0.3788 mg of viral antigens. The IC, prepared in the form of an oil-emulsion, was administered intramuscularly. The IC generated high levels of anti-NDV antibodies and successfully protected chickens against live virus challenge. Therefore, the IC could be recommended as a safe and environmentally convenient vaccine.

RAG-2 expression is not essential for chicken immunoglobulin gene conversion.

Chicken B cells diversify their immunoglobulin genes by gene conversion in the bursa of Fabricius. The avian leukosis virus-induced B-cell line DT40 continues to diversify its immunoglobulin light chain locus by gene conversion during in vitro passage. Since a variable(diversity)joining recombination-activating gene, RAG-2, is specifically expressed in chicken B cells undergoing immunoglobulin gene conversion, it has been suggested that RAG-2 may be involved in the immunoglobulin gene conversion process. We previously reported high ratios of targeted to random integration after transfection of genomic DNA constructs into DT40. This allows us to easily investigate the function of a gene product by gene disruption. We show here that subclones of DT40 maintain the ability to diversify their immunoglobulin light chain locus by gene conversion even after both copies of the RAG-2 coding regions are deleted. These results demonstrate that the RAG-2 product is not required for gene conversion activity in the immunoglobulin light chain locus.