| IgY References |
| Zheng L, Settle
M, Brubaker G, Schmitt D, Hazen SL, Smith JD, Kinter M. |
| Localization of nitration
and chlorination sites on apolipoprotein A-I catalyzed by myeloperoxidase
in human atheroma and associated oxidative impairment in ABCA1-dependent
cholesterol efflux from macrophages. |
J
Biol Chem. 2004 Oct 21; [Epub ahead of print]
Apolipoprotein A-I (apoAI), the major protein component of high
density lipoprotein (HDL), is a selective target for MPO-catalyzed
nitration and chlorination in both human atheroma and in serum
of subjects with cardiovascular disease. The extent of apoAI
nitration and chlorination are each strongly correlated with
functional impairment in reverse cholesterol transport activity
of the isolated lipoprotein. Tandem mass spectrometry was used
to map the sites of MPO-mediated apoAI nitration and chlorination
in vitro and within human atheroma, and to relate the degree
of site-specific modifications to loss of apoAI lipid binding
and cholesterol efflux functions. Of the seven tyrosine residues
in apoAI, Y192, Y166, Y236, and Y29 were nitrated and chlorinated
in MPO-mediated reactions under physiologically plausible conditions.
Site-specific LC-MS quantitative analyses demonstrate that the
favored modification site following exposure to MPO-generated
oxidants is Y192. MPO-dependent nitration and chlorination both
proceed with Y166 as a secondary site, and with Y236 and Y29
modified only minimally. Parallel functional studies demonstrate
dose-dependent losses of ABCA1-dependent cholesterol acceptor
and lipid-binding activities with apoAI modification by MPO.
Finally, LC-tandem MS analyses show that apoAI in human atherosclerotic
tissue is nitrated at the MPO-preferred sites, Y192 and Y166.
The present studies suggest that site-specific modifications
of apoAI by MPO are associated with impaired lipid binding and
ABCA1-dependent cholesterol acceptor functions, providing a
molecular mechanism that likely contributes to the clinical
link between MPO levels and cardiovascular disease risk. |
| Zheng L, Nukuna B, Brennan
ML, Sun M, Goormastic M, Settle M, Schmitt D, Fu X, Thomson
L, Fox PL, Ischiropoulos H, Smith JD, Kinter M, Hazen SL. |
| Apolipoprotein A-I is a selective
target for myeloperoxidase-catalyzed oxidation and functional
impairment in subjects with cardiovascular disease. |
J
Clin Invest. 2004 Aug;114(4):529-41.
Systemic levels of protein-bound nitrotyrosine (NO(2)Tyr) and
myeloperoxidase (MPO), a protein that catalyzes generation of
nitrating oxidants, serve as independent predictors of atherosclerotic
risk, burden, and incident cardiac events. Apolipoprotein A-I
(apoA-I), the primary protein constituent of HDL, is a selective
target for MPO-catalyzed nitration and chlorination in vivo
and that MPO-catalyzed oxidation of HDL and apoA-I results in
selective inhibition in ABCA1-dependent cholesterol efflux from
macrophages. Dramatic selective enrichment in NO(2)Tyr and chlorotyrosine
(ClTyr) content within apoA-I recovered from serum and human
atherosclerotic lesions is noted, and analysis of serum from
sequential subjects demonstrates that the NO(2)Tyr and ClTyr
contents of apoA-I are markedly higher in individuals with cardiovascular
disease (CVD). Analysis of circulating HDL further reveals that
higher NO(2)Tyr and ClTyr contents of the lipoprotein are each
significantly associated with diminished ABCA1-dependent cholesterol
efflux capacity of the lipoprotein. MPO as a likely mechanism
for oxidative modification of apoA-I in vivo is apparently facilitated
by MPO binding to apoA-I, as revealed by cross-immunoprecipitation
studies in plasma, recovery of MPO within HDL-like particles
isolated from human atheroma, and identification of a probable
contact site between the apoA-I moiety of HDL and MPO. The present
results provide the first direct evidence for apoA-I as a selective
target for MPO-catalyzed oxidative modification in human atheroma.
They also suggest a potential mechanism for MPO-dependent generation
of a proatherogenic dysfunctional form of HDL in vivo. |
| Douglas Hinerfeld, David
Innamorati, John Pirro and Sun W. Tam. |
| Serum/Plasma Depletion with
Chicken Immunoglobulin Y Antibodies for Proteomic Analysis from
Multiple Mammalian Species |
| Journal
of Biomolecular Techniques, 15:184-190
Plasma from different species is the most accessible and valuable
source for biomarker discovery in clinical and animal samples.
However, due to the high abundance of some proteins such as
albumin and immunoglobulins, low-abundant proteins are often
undetectable in proteomic analysis of plasma. A plasma depletion
scheme was established using chicken antibodies against various
abundant proteins. This immunoaffinity purification procedure
is able to deplete albumin across multiple species. The high
binding capacity and specificity of the chicken antibody enables
the efficient capture of its ligand from microliter volumes
of plasma sample. The resulting two-dimensional gel analyses
of the depleted and captured samples show significant enhancement
of the low-abundant proteins and specific capture of the abundant
ligand. By utilizing this sample preparation scheme, it is
now possible to analyze the plasma proteome from multiple
species in a potentially rapid and large-scale capacity for
biomarker discovery, drug target discovery, and toxicology
studies. sequence, and amino acid analysis. |
| Lee SC. Lee KN. Schwartzott
DG. Jackson KW. Tae WC. McKee PA. |
| Purification of human alpha
2-antiplasmin with chicken IgY specific to its carboxy-terminal
peptide. |
Preparative
Biochemistry & Biotechnology 27(4):227-37, 1997
Alpha 2-antiplasmin, a plasma glycoprotein of the serpin superfamily,
is the primary physiological inhibitor of plasmin, the key enzyme
in fibrin degradation. Previous purification methods utilize
lengthy multistep protocols with low yields or use monoclonal
antibodies that are expensive or difficult to make. With a relatively
small investment, a chicken was immunized with keyhole limpet
hemocyanin-conjugated to alpha 2-antiplasmin C-terminal 26 residue
synthetic peptide and the peptide-specific antibody ( IGY )
was isolated from the egg yolks of hens using the peptide affinity
column. Based on the interaction between this IgY and alpha
2-antiplasmin, pure alpha 2-antiplasmin was isolated from human
plasma in two steps: (a) citrated plasma was precipitated with
15% PEG-8000 to remove the bulk of plasma proteins while retaining
the majority of alpha 2-antiplasmin activity; and (b) the alpha
2-antiplasmin was affinity-purified from the supernatant using
the IgY column. Yields were typically 48% and the purity and
authenticity of the alpha 2-antiplasmin were verified by gel
electrophoresis, Western Blot analysis, N-terminal sequence,
and amino acid analysis. |
| Fortgens PH. Dennison
C. Elliott E. |
| Anti-cathepsin D chicken
IgY antibodies: characterisation, cross-species reactivity and
application in immunogold labelling of human splenic neutrophils
and fibroblasts. |
Immunopharmacology
36(2-3):305-11, 1997.
Hyperexpression, alteration of trafficking and secretion of
cathepsin D has been linked with tumour invasion and inflammation.
To study these phenomena in a variety of cells large quantities
of anti-cathepsin D antibodies and the appropriate immunogen
are required. As the human immunogen for studies on human tissue
is less easily accessed, antibodies to both human and porcine
cathepsin D were raised in chickens, as high levels of antibody
may be recovered from egg yolks, and the potential cross-reactivity
of the anti-porcine cathepsin D
IgY antibody was assessed. This preparation cross-reacted strongly
with human cathepsin D, comparing favourably with the reactivity
of the chicken antibody to the human immunogen. The necessity
for isolating human immunogen can thus be circumvented. The
cross-species-reacting chicken IgY was successfully used to
localise cathepsin D in immunogold labelling of human tissues.
To our knowledge, IgY antibodies have not previously been used
by other researchers for this purpose. Application of the cross-reacting
antibody to human splenic neutrophils (PMNs) has confirmed the
presence of cathepsin D in some granules. Double labelling has
shown these to be novel subpopulations of azurophil granules.
Cathepsin D may, therefore, be relevant in the invasive and
inflammatory activities of PMNs and a target for therapeutic
strategies. |
| Cain CC. Saslowsky DE.
Walker RA. Shirley BW. |
| Expression of chalcone synthase
and chalcone isomerase proteins in Arabidopsis seedlings. |
Plant
Molecular Biology. 35(3):377-81, 1997.
Antibodies have been developed against the first two enzymes
of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone
synthase (CHS) and chalcone isomerase (CHI) were overexpressed
and purified from Escherichia coli as fusion proteins with glutathione
S-transferase from Schistosoma japonicum. The recombinant proteins
were then used to immunize chickens and the resulting IgY fraction
was purified from egg yolks. Immunoblots of crude protein extracts
from Arabidopsis seedlings carrying wild-type and null alleles
for CHS and CHI showed that the resulting antibody preparations
provide useful tools for characterizing expression of the flavonoid
pathway at the protein level. An initial analysis of expression
patterns in seedlings shows that CHS and CHI proteins are present
at high levels during a brief period of early seedling germination
that just precedes the transient accumulation of flavonoid end-products. |
| Hatta H. Tsuda K. Ozeki
M. Kim M. Yamamoto T. Otake S. Hirasawa M. Katz J. Childers
NK. Michalek SM. |
| Passive immunization against
dental plaque formation in humans: effect of a mouth rinse containing
egg yolk antibodies (IgY) specific to Streptococcus mutans. |
| Caries
Research 31(4):268-74, 1997.
Passive immunization involving the delivery of antibodies
specific to pathogens of infectious diseases to the host has
been an attractive approach to establish protective immunity
against a variety of microbial pathogens, including Streptococcus
mutans, which is the principal etiologic agent of dental caries
in humans. The overall purpose of the present study was to
determine the effectiveness of a mouth rinse containing antibodies
to S. mutans in preventing the establishment of this bacterium
in dental plaque of humans. The antibodies were derived from
egg yolks obtained from hens immunized with whole cells of
S. mutans grown in sucrose-containing medium. The immunoglobulin
derived from the yolks (IgY) of immunized hens was characterized
in vitro and in vivo in human volunteers. Cross-reactivity
tests showed that immune IgY reacted with every serotype,
except serotype b, which had lost its GTase activity, when
the bacteria were cultured in sucrose-containing medium. Immune
IgY inhibited S. mutans adherence to saliva-coated hydroxyapatite
discs by 59.2%, while control IgY caused an inhibition of
only 8.2%. In the short-term (4-hour) test using a mouth rinse
containing 10% sucrose, immune IgY decreased the ratio of
the percentage of S. mutans per total streptococci in saliva.
In the long-term (7-day) test using a mouth rinse without
sucrose, the ratio in saliva was not significantly reduced
in the volunteers using the immune IgY due to the large standard
deviation. However, comparing the ratios of the percentage
of S. mutans per total streptococci in plaque of individual
subjects, there was a tendency for a reduction of the ratios
in the volunteers receiving the mouth rinse containing immune
IgY. These results support the effectiveness of IgY with specificity
to S. mutans grown in the presence of sucrose as an efficient
method to control the colonization of mutans streptococci
in the oral cavity of humans. |
| Chaudhuri J. Chakrabarti
A. Maitra U. |
| Biochemical characterization
of mammalian translation initiation factor 3 (eIF3). Molecular
cloning reveals that p110 subunit is the mammalian homologue
of Saccharomyces cerevisiae protein Prt1. |
Journal
of Biological Chemistry 272(49):30975-83, 1997.
Eukaryotic translation initiation factor 3 (eIF3), which plays
an essential role in initiation of protein synthesis, was purified
from rabbit reticulocyte lysates using an assay that specifically
measures its ability to stimulate the binding of Met-tRNAf (as
a Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits.
Purified eIF3 consisted of six major polypeptides of molecular
masses 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa
polypeptide reported to be a constituent of other eIF3 preparations.
Characterization of purified eIF3 lacking the 170-kDa polypeptide
showed that the eIF3-mediated 40 S initiation complex formed
in the presence of AUG codon efficiently joined 60 S ribosomal
subunits in an eIF5-dependent reaction to form a functional
80 S initiation complex. eIF3, which was originally bound to
the 40 S initiation complex, was released from the 40 S subunit
during the subunit joining reaction. Additionally, chicken antibodies
raised against rabbit reticulocyte eIF3 were used to immunochemically
characterize eIF3 subunits and to isolate a 3.1-kilobase pair
human cDNA that encodes the p110 subunit of mammalian eIF3.
The derived amino acid sequence (calculated Mr 95,214) shows
that the p110 subunit is the mammalian homologue of Saccharomyces
cerevisiae protein Prt1p, a subunit of yeast eIF3. |
| Troeberg L. Pike RN. Lonsdale-Eccles
JD. Coetzer TH. |
| Production of anti-peptide
antibodies against trypanopain-Tb from Trypanosoma brucei brucei:
effects of antibodies on enzyme activity against Z-Phe-Arg-AMC. |
Immunopharmacology
36(2-3):295-303, 1997.
Anti-peptide antibodies were produced against the cysteine proteinase
trypanopain-Tb from Trypanosoma brucei brucei and the effects
of these antibodies on enzyme activity against carboxybenzoyl
(Z)-Phe-Arg-aminomethylcoumarin (AMC) investigated. A peptide
was synthesised corresponding to a region of the trypanopain-Tb
active site around the active site histidine so that the resulting
anti-peptide antibodies specifically targeted the active site
of the enzyme. Such antibodies were considered more likely to
modulate enzyme activity compared with antibodies directed against
other regions of the enzyme. Trypanopain-Tb activity was modulated
by rabbit and chicken antibodies produced against both the free
and conjugated peptide. Rabbit anti-peptide antibodies enhanced
trypanopain-Tb activity by up to 64% at 500 micrograms/ml relative
to non-immune antibodies. Chicken antibodies on the other hand,
both enhanced (by up to 176% at 500 mg/ml) and inhibited (by
up to 85% at 250 mg/ml) trypanopain-Tb activity against Z-Phe-Arg-AMC.
The nature of the antibody effect depended on the stage during
the immunisation protocol at which the antibodies were produced.
Chicken antibodies also modulated trypanopain-Tb activity in
lysates of T.b. brucei, while rabbit antibodies were only effective
against the purified enzyme. Anti-trypanopain-Tb peptide antibodies
were thus shown to have the potential to affect trypanopain-Tb
activity. |
| Ebina T. |
| Prophylaxis of rotavirus
gastroenteritis using immunoglobulin. |
Archives of Virology
- Supplementum 12:217-23, 1996.
Oral inoculation of the human group A rotavirus MO strain (G
serotype 3) into 5-day-old BALB/c mice causes gastroenteritis
characterized by diarrhea. Using this small animal model, passive
protection of suckling mice against human rotavirus infection
was achieved with the use of immunoglobulin (IgY) from the yolks
of eggs of rotavirus-immunized hens. When IgY against the rotavirus
strain homotypic with the challenge virus (MO strain) was administered
to mice, complete protection was achieved. After immunizing
8-month old pregnant Holstein cows with human rotavirus MO strain,
colostrum containing neutralizing antibody to four different
G serotypes of human rotavirus, designated Rota colostrum, was
obtained. Rota colostrum completely protected suckling mice
against rotavirus infection, and purified IgG obtained from
Rota colostrum protected against infection with the homologous
virus. After randomly grouping 20 infants from a baby care center,
10 infants received 20 ml of Rota colostrum for 2 weeks and
10 control infants received none. Rotavirus-associated diarrhea
developed in 7 of the 10 infants in the control group. None
of the three infants in the group daily receiving the Rota colostrum
had such symptoms, and one of three infants in the group receiving
treatment, every other day developed rotavirus-induced diarrhea.
Oral administration of Rota colostrum seems to be an effective
and safe means of preventing diarrhea caused by human rotavirus
infection. Recently, the immunized cows were boosted by reinjection
of 4 serotypes of human rotavirus into a superficial cervical
lymph node two weeks after delivery, resulting in mass production
of cow's milk containing a high-titered antibody to human rotavirus.
Therefore, the hyperimmune cow's milk is a candidate for a "physiologically
functional food" in Japan. |
| Gouin-Thibault I. Dewar
L. Craven S. Kulczycky M. Wun TC. Ofosu FA. |
| Probable regulation of factor
VIIa-tissue factor and prothrombinase by factor Xa-TFPI and
TFPI in vivo. |
British
Journal of Haematology 95(4):738-46, 1996.
Given that factor VIIa-tissue factor (TF) probably initiates
coagulation in vivo, this study investigated the relationship
between plasma concentrations of factor VIIa and prothrombin
fragment 1 + 2 in plasma (the latter as an index of prothrombinase
activity in vivo). The relationships between these two parameters
and the concentrations of tissue factor pathway inhibitor (TFPI)
and factor Xa-TFPI in plasma were also investigated. TFPI inactivates
factor Xa in a reaction accelerated by heparin, whereas factor
Xa-TFPI inactivates factor VIIa-TF and prothrombinase. Established
enzyme-linked immunosorbent assays (ELISAs) were used to quantify
TFPI and prothrombin fragment 1 + 2, whereas we developed an
ELISA to quantify factor Xa-TFPI using affinity purified rabbit
(anti-human TFPI)-IgG and chicken anti-(human factor Xa-TFPI)-IgY
as the capture and detector antibodies, respectively. Plasma
factor VIIa was quantified using truncated tissue factor. The
concentrations of factor VIIa and prothrombin fragment 1 + 2
increased in parallel in the plasmas of up to 145 healthy adults
assayed (P = 0.007), as did the concentrations of factor VIIa
and TFPI (P = 0.0039), and prothrombin fragment 1 + 2 and TFPI
(P = 0.013). In contrast, there was an inverse relationship
between the concentrations of free factor Xa-TFPI and factor
VIIa (P < 0.0001) and free factor Xa-TFPI and prothrombin
fragment 1 + 2 (P = 0.0095). These results are consistent with
factor Xa-TFPI regulating factor VIIa-tissue factor and prothrombinase
in vivo. |
| Sugita-Konishi Y. Shibata
K. Yun SS. Hara-Kudo Y. Yamaguchi K. Kumagai S. |
| Immune functions of immunoglobulin
Y isolated from egg yolk of hens immunized with various infectious
bacteria. |
British
Journal of Haematology 95(4):738-46, 1996.
We studied the immune functions of IgY obtained from hens immunized
with a mixture of formalin-treated pathogenic bacteria. The
IgY inhibited the growth of Pseudomonas aeruginosa, the production
of Staphylococcus aureus enterotoxin-A, and adhesion of Salmonella
enteritidis to cultured human intestinal cells (Caco 2). The
results indicated that IgY specific for plural bacteria has
effects useful toward prevention of bacterial diseases. |
| Lundahl TH.
Lindahl TL. Fagerberg IH. Egberg N. Bunescu A. Larsson A. |
| Activated platelets
and impaired platelet function in intensive care patients analyzed
by flow cytometry. |
Blood
Coagulation & Fibrinolysis 7(2):218-20, 1996.
Intensive care patients often have disturbances in their coagulation
and fibrinolysis systems, which may result in haemorrhage or
disseminated intravascular coagulation (DIC). DIC is a dreaded
complication that may develop rapidly and has a high mortality
rate. Platelets play a central role in haemostasis and it is
thus important to have assays that rapidly can monitor platelet
activation and platelet function. We have used flow cytometry
to measure platelet activation and function in intensive care
patients. Fluorescein labelled chicken antibodies were used
to detect platelet bound fibrinogen as these antibodies have
advantages over mammalian antibodies in flow cytometry. We found
increased levels of circulating activated platelets and microparticles
in vivo and impaired platelet function after stimulation in
vitro. The two patients with the highest percentage of microparticles
died shortly after blood sampling. |
| Murata T.
Saito S. Shiozaki M. Lu RZ. Eto Y. Funaba M. Takahashi M. Torii
K. |
| Anti-activin
A antibody (IgY) specifically neutralizes various activin A
activities. |
Proceedings
of the Society for Experimental Biology & Medicine 211(1):100-7,
1996.
Activin A (beta A beta A), originally isolated from ovarian
follicular fluids as a follicule-stimulating hormone (FSH) secretion
stimulator, has also been identified as an erythroid differentiation
factor (EDF), a neuron survival factor and a mesoderm-inducing
factor. Thus, activin A is a multifunctional factor, and further
studies on its physiological function are important. However,
it is very difficult to produce a specific antibody to neutralize
the activity of activin A because of its highly conserved amino
acid sequence across mammalian species. In this study, we succeeded
in generating an antibody against activin A, which can neutralize
several activities of activin A, such as the stimulation of
FSH secretion from pituitary cells and the induction of the
differentiation of erythrocytes in vitro. This antibody did
not affect the activity of activin B (beta B beta B), which
induces the differentiation of erythrocytes in vitro, and the
activity of inhibin A (alpha beta A), which inhibits FSH secretion
from pituitary in vitro, but slightly neutralized that of activin
AB (beta A beta B). Western blotting analysis showed that this
antibody recognized both dimeric and monomeric forms of the
beta A subunit of activin and inhibin. These results suggest
that this antibody recognizes the beta A subunit of activin
and specifically neutralizes the activity of a dimer of the
beta A subunit, activin A. Furthermore, by the addition of this
antibody to the culture medium, the development of murine embryos
was suppressed, suggesting that endogenous activin A plays an
important role in murine development. These results indicate
the usefulness of this antibody for studies of endogenous activin
actions. |
| Warr GW. Magor
KE. Higgins DA. |
| IgY: clues to
the origins of modern antibodies. [Review] |
Immunology
Today 16(8):392-8, 1995
IgY is the functional equivalent of IgG in birds, reptiles and
amphibia, but many aspects of its biology are poorly understood.
Recent studies have increased awareness of the genetics and
functions of this molecule, and have revealed its position as
the ancestor of the uniquely mammalian antibodies IgG and IgE.
Here, Greg Warr, Kathy Magor and David Higgins review current
knowledge of IgY structure, function and expression in the context
of the evolutionary role of this primitive immunoglobulin. |
| Puffinbarger
NK. Hansen KR. Resta R. Laurent AB. Knudsen TB. Madara JL. Thompson
LF. |
| Production and
characterization of multiple antigenic peptide antibodies to
the adenosine A2b receptor. |
Molecular
Pharmacology 47:1126-32, 1995
A polyclonal antibody to the human adenosine A2b receptor (A2bR)
was produced by immunizing a chicken with a multiple antigenic
peptide consisting of eight copies of a 16-amino acid peptide,
corresponding to the presumed second extracellular loop of the
A2bR, linked to a branched lysine core. Western blotting with
affinity-purified antibody revealed the human A2bR to be a protein
of approximately 50-55 kDa, found in a variety of tissues including
thymus, colon, and small intestine. The antibody also recognized
mouse and rat A2bRs and revealed heterogeneity in size, with
a 35 kDa protein being detected in small intestine in addition
to the larger 50-52 dKa species in thymus, colon, and placenta.
The chicken anti-human A2bR peptide antibody recognized the
receptor in both frozen and formalin-fixed tissue sections.
In human colon, the A2bR was highly expressed in epithelial
cells of the crypts. A2bR immunoreactivity was also apparent
in syncytiotrophoblast cells of human placental villi and in
the basal zone of murine chorioallantoic placenta. These cell
type-specific patterns of expression are consistent with the
hypothesized roles of the A2bR in mediating electrogenic Cl
secretion and the resulting secretory diarrhea caused by colonic
crypt abscesses and in regulating morphogenesis of the placenta.
Insight into the multiple physiological consequences of A2bR
engagement will be forthcoming from an analysis of the cell
type-specific expression of this receptor in additional tissues. |
| Gutmann DH.
Geist RT. Rose K. Wright DE. |
| Expression of
two new protein isoforms of the neurofibromatosis type 1 gene
product, neurofibromin, in muscle tissues. |
Developmental
Dynamics 202(3):302-11, 1995
The neurofibromatosis type 1 (NF1) gene encodes
a tumor suppressor protein, termed neurofibromin, which is expressed
predominantly in neurons, Schwann cells, oligodendrocytes, and
leukocytes. There are at least three isoforms of neurofibromin
produced by the alternative use of exons 23a and 48a. Previously
we described the identification of an NF1 mRNA isoform containing
an additional 54 nucleotides from exon 48a (type 3 NF1) in human
skeletal, cardiac and smooth muscle tissues by reverse-transcribed
(RT)-PCR. To extend our initial observations, we have produced
high titer chicken IgY antibodies which specifically recognize
this muscle-specific neurofibromin isoform. An NF1 cDNA was
generated containing human exon 48a sequences and expressed
as a fusion protein in bacteria. The muscle-specific neurofibromin
antibodies detected this exon 48a fusion protein by Western
immunoblotting. Immunoprecipitation using these type 3 neurofibromin
antibodies also specifically detected a 250 kDa protein in human
and rat muscle tissues. Type 3 neurofibromin was found in rat
heart and muscle, but not in liver brain, kidney or spleen with
levels of expression declining after postnatal day 7. Expression
of total NF1 RNA during rat embryonic development was detected
at high levels in E15 heart, tongue, and limb bud. In addition,
using type 2 neurofibromin-specific antibodies, the existence
of a fourth isoform of neurofibromin (type 4 neurofibromin)
containing both exon 23a and 48a sequences was demonstrated
in rat heart muscle tissues. The identification of two muscle-specific
isoforms of neurofibromin expands our definition of this important
tumor suppressor protein and suggests additional roles for neurofibromin
in muscle development and differentiation. |
| Pchelintseva
O. Pak YK. Weiner H. |
| Identification
of protein-receptor components required for the import of prealdehyde
dehydrogenase into rat liver mitochondria. |
Archives
of Biochemistry & Biophysics 323(1):54-62, 1995.
Mitochondrial aldehyde dehydrogenase is synthesized as a high-molecular-weight
precursor in cytosol and transported into mitochondrial matrix
space where it is processed to the mature enzyme. To identify
components of the transport machinery on liver mitochondria,
anti-idiotypic antibodies against the rabbit anti-prealdehyde
dehydrogenase signal peptide antibodies were produced in chicken
eggs and rabbit. Both anti-idiotypic antibodies inhibited the
import of prealdehyde dehydrogenase (pALDH) into isolated rat
liver mitochondria. The rabbit anti-idiotypic antibody could
recognize by Western blotting five mitochondrial membrane proteins
with apparent molecular weights of 66, 60, 42, 34, and 29 kDa.
The anti-idiotypic antibodies were cross-linked to mitochondrial
membrane proteins using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate
which is an iodinatable, heterofunctional, and photoreactive
cross-linker. Mitochondrial proteins with apparent molecular
weights of 66, 60, and 42 kDa were identified using the chicken
antibody. The 66- and 34-kDa proteins were cross-linked to the
rabbit antibody as the major components and the 42-kDa protein
as a minor one. Antibodies against the 60- and 42-kDa proteins,
as well as Fab fragments, inhibited the import of pALDH, suggesting
that these proteins are receptor/translocator components for
pALDH import. |
| Danielpour
D. Roberts AB. |
| Specific and
sensitive quantitation of transforming growth factor beta 3
by sandwich enzyme-linked immunosorbent assay. |
Journal
of Immunological Methods 180(2):265-72, 1995.
Transforming growth factors beta (TGF-beta) consist of a highly
homologous family of 25 kDa dimers involved in a diverse array
of biological functions. Progress in understanding the biology
of the third isoform (TGF-beta 3) of this family has been limited
by the absence of a quantitative assay for TGF-beta 3. Here
we report the development of a sensitive and specific sandwich
enzyme-linked immunosorbent assay (SELISA), which is based on
an anti-TGF-beta mouse monoclonal IgG as the capture antibody
and chicken anti-recombinant-hTGF-beta 3 IgY as the secondary
antibody. This assay can quantitate TGF-beta 3 in complex biological
fluids, with a detection limit of 2 pg and no cross-reactivity
or interference with as high as 1000-fold molar excesses of
either TGF-beta s 1, 2 or 1.2. This TGF-beta 3 SELISA is the
first reported assay for the direct and sensitive quantitation
of TGF-beta 3 in complex biological fluids. |
| Phillips AO.
Steadman R. Donovan KD. Williams JD. |
| A new antibody
capture enzyme linked immunoassay specific for transforming
growth factor beta 1. |
International
Journal of Biochemistry & Cell Biology 27(2):207-13, 1995.
Previous studies which examined Transforming Growth Factor beta
1 (TGF-beta 1) generation have relied on the identification
of TGF-beta 1 mRNA or measurement of TGF-beta 1 by bioassay.
Quantitation of TGF-beta 1 message alone however is inadequate
since the regulation of TGF-beta 1 synthesis is often post-transcriptional.
TGF-beta 1 is poorly immunogenic, and sensitive and specific
immunoassays for this peptide have proved difficult to develop.
Bioassays depend on stimulation or inhibition of cell proliferation
in a TGF-beta 1 dependent manner, and are very rigid in their
requirements for optimal performance. The aims of this work
was therefore to develop a sensitive and reproducible immunoassay
for TGF-beta 1. Microtitre plates were coated with human recombinant
TGF-beta 1, unbound protein was discarded from the wells prior
to blocking with bovine serum albumin. Chicken anti-human TGF-beta
1 antibody was incubated with the test solution overnight at
4 degrees C and then added to the coated wells. Bound antibody
was detected with alkaline phosphatase conjugated anti-chicken
antibody. The assay is sensitive to 0.2 ng/ml with a range to
100 ng/ml. The assay detects the mature form of human recombinant
TGF-beta 1, natural platelet extracted TGF-beta 1, and TGF-beta
1 derived from human monocytes stimulated with Phorbol myristate
acetate (PMA). Active TGF-beta 1 is measured directly and latent
TGF-beta 1 can be measured indirectly following acid activation
of samples. Inter-assay precision ranged from 4.3 to 9.6%, (coefficient
of variation, %CV) and intraassay precision ranged from 2.8
to 8.6% (CV) |
| Grossmann
A. Eggers-Schumacher G. Sander E. |
| Stabilization
of erythrocytes by aldehydes and suitability of chicken IgY
for the detection of potato virus X (PVX) in avidin-biotin enhanced
reverse passive haemagglutination. |
Journal
of Immunological Methods 179(2):243-50, 1995.
Methods are described for the detection of potato virus X (PVX)
by reverse passive haemagglutination (RPH) by means of polyclonal
antiviral antibodies coupled to sheep red blood cells (sRBC)
and the chromic chloride method. The cells were stabilized with
pyruvic aldehyde, thus providing a stock suspension for numerous
coupling experiments lasting several months. Anti-PVX IgY, which
is readily isolated in large amounts from the egg yolk of immunized
chickens, was used in an avidin-biotin enhanced RPH assay with
stabilized sRBC. With this method the PVX detection rate achieved
was comparable to that of RPH assays using fresh non-fixed sRBC.
In addition, avidin-coated sRBC could be stored for weeks at
4 degrees C and subsequently used for coupling with biotinylated
IgY. |
| Concetti A.
Ripani E. Barboni L. Torregiani E. Bombardelli E. Gariboldi
P. Venanzi FM. |
| Immunorecognition
of ring skeleton of taxanes by chicken egg yolk antibodies. |
Biological
Chemistry Hoppe-Seyler. 375(6):419-23, 1994
Anti-10 deacetylbaccatin III (DAB) antibodies (IgY) were elicited
in hens immunized with a succinyl-DAB/BSA conjugate and extracted
from egg yolk. As shown by indirect competitive inhibition
enzyme immunoassay (CIEIA), the addition of free-DAB competitively
inhibited the binding of affinity purified anti-DAB IgY to
DAB/BSA solid phase conjugated antigen. The assay enabled
the detection of DAB in concentrations as low as 7.5ng/ml
(13.7 nM DAB), whereas anti-DAB IgY did not react with taxol
even at a concentration a thousand times higher. The structural
requirements of the diterpenoid nucleus for binding to IgY
were considered on the basis of the levels of cross-reaction
found with 10 authentic taxanes. The results indicate that
anti-DAB IgY represents the first high affinity antibody produced
capable of recognizing the ring skeleton of taxol precursors. |
| Meisel H. |
| Antibodies from
egg yolk of immunized hens against a bioactive caseinopeptide
(beta-casokinin-10). |
Biological
Chemistry Hoppe-Seyler. 375(6):401-5, 1994
Antibodies (IgY) directed against a synthetic, bioactive peptide
(beta-casokinin-10) were obtained from egg yolk of immunized
chickens. Using a beta-casokinin-10/BSA conjugate for immunization,
large quantities of high-titered anti-peptide antibodies were
obtained. ELISA standard curves for beta-casokinin-10 were linear
in the range 30-22,000 ng/ml. IgY-antibodies against beta-casokinin-10
recognized not only the immunogenic peptide structure but also
analogues epitopes in protein preparations containing bovine
beta- and alpha s-caseins, respectively, as well as in ovine
caseins. The anti-beta-casokinin-10 IgY-antibodies are intended
to be used as immunochemical reagents in future structure-activity
studies of bioactive casokinins that are inhibitors of the angiotensin-converting
enzyme. |
| Hatta H. Tsuda
K. Akachi S. Kim M. Yamamoto T. |
| Productivity
and some properties of egg yolk antibody (IgY) against human
rotavirus compared with rabbit IgG. |
Bioscience,
Biotechnology & Biochemistry. 57(3):450-4, 1993
Productivity and some properties of anti-Human Rotavirus (HRV)
hen egg yolk antibody (IgY) were compared with those of anti-HRV
rabbit serum antibody (IgG). The hens immunized with HRV (Wa
strain, serotype 1 and Mo strain, serotype 3) were found to
continuously to lay eggs without any change in the egg laying
rate and the yolk of the eggs laid over a year showed a high
level of neutralization titer against HRV. The production of
anti-HRV IgY by a hen (one year) was at least 15 times (anti-Wa)
and 120 times (anti-Mo) more effective than those by an immunized
rabbit in the neutralization titer of the antibodies. The stability
of anti-HRV IgY at temperature above 70 degrees C and low pH
2-3 was less than that of anti-HRV rabbit IgG. The temperature
corresponding to the maximum of denaturation endotherm (Tmax)
of IgY was 73.9 degrees C while that of rabbit IgG was 77.0
degrees C in the analysis by differential scanning calorimetry.
This discrepancy in heat and acidic pH stability found between
the two antibodies as discussed with regard to their protein
structures. |
| Kalvatchev
Z. Alexandrov E. Dontcheva R. Madjurova A. |
| Use of immunogold
assay for a rapid evaluation of antigens of spotted fever group
rickettsiae. |
Acta
Virologica. 37(2-3):184-6, 1993
Rabbit-antihuman IgG, yolk immunoglobulins (IgY) and monoclonal
antibodies against Rickettsia conorii were adsorbed to colloidal
gold. Thus obtained conjugates were found to be active and responded
specifically to the agent of the Mediterranean Spotted fever.
The suitability of the immunogold assay as a alternative to
other known immunoenzymatic or immunofluorescent methods for
assessing the presence and/or activity of rickettsial antigens
is discussed. |
| Akita EM.
Nakai S. |
| Production and
purification of Fab' fragments from chicken egg yolk immunoglobulin
Y (IgY). |
Journal
of Immunological Methods. 162(2):155-64, 1993
Methods were described for the production of Fab and Fab' fragments
from chicken egg yolk IgY also referred to as IgG by papain
and pepsin digestion respectively. Pepsin digestion was found
to be suitable for the large scale preparation and purification
of Fab'. Optimum yield of Fab' was obtained after peptic digestion
of IgY at pH 4.2 for 9 h at low NaCl concentration. This condition
led to the complete digestion of pFc' fragment leaving only
the Fab' fragment. By combination of ultrafiltration and anion
exchange, and conditions which allowed binding of the small
amount of contaminants in the digest to the anion exchange column,
pure Fab' fragments were easily obtained in the eluent. The
advantage of this approach is that a small column could be used
to purify large amount of protein, therefore, improving the
efficiency of purification. The Fab and Fab' fragments appeared
to be similar on the basis of their molecular weights as determined
by SDS-PAGE, reaction of identity in immunodiffusion assay and
similar antigen binding activities as shown by ELISA. |
| Larsson A.
Balow RM. Lindahl TL. Forsberg PO. |
| Chicken antibodies:
Taking advantage of evolution -- A review. |
Poultry
Science 72: 1807-1812, 1993.
Laying hens are highly cost-effective as producers of antibodies
compared with the mammals traditionally used for such production.
Also, chicken antibodies have biochemical advantages over mammalian
antibodies due to the phylogenetical differences between avian
and mammalian species, resulting in increased sensitivity as
well as decreased background in immunological assays. In contrast
to mammalian antibodies, chicken antibodies do not activate
the human complement system nor will they react with rheumatoid
factors, human anti-mouse IgG antibodies, or bacterial and human
Fc (fragment crystallizable)-receptors. Thus, chicken antibodies
offer many advantages over mammalian antibodies and may replace
such antibodies in the future. |
| Rosol TJ.
Steinmeyer CL. McCauley LK. Merryman JI. Werkmeister JR. Grone
A. Weckmann MT. Swayne DE. Capen CC. |
| Studies on chicken
polyclonal anti-peptide antibodies specific for parathyroid
hormone-related protein (1-36). |
Veterinary
Immunology & Immunopathology. 35(3-4):321-37, 1993
Chicken polyclonal antibodies were prepared against a synthetic
peptide corresponding to the first 36 N-terminal amino acids
of parathyroid hormone-related protein (PTHrP) by immunizing
laying hens. Significant increases of antibodies to PTHrP were
first detected after the second immunization. Production of
anti-PTHrP egg yolk antibodies peaked 1-2 weeks after the second
through sixth immunizations and declined over a period of 2-4
weeks. Polyclonal IgG (IgY) to PTHrP was purified from the egg
yolks with high levels of PTHrP specific binding. The anti-PTHrP
IgG was used to develop a radioimmunoassay for PTHrP that was
able to detect 100 pg PTHrP ml-1 (23 pM) in conditioned cell
culture medium. The anti-PTHrP IgG was bound to a solid phase
and utilized to immunopurify iodinated [Tyr36]-PTHrP (1-36).
Anti-PTHrP IgG inhibited the in vitro biologic activity of PTHrP
as demonstrated by the inhibition of adenylate cyclase stimulation
in a rat osteoblast-like cell line (ROS 17/2.8). The anti PTHrP
IgG was immunopurified and utilized for immunohistochemical
localization of PTHrP in canine skin. Chickens were advantageous
in producing large amounts of high affinity, neutralizing antibodies
to a highly conserved mammalian protein such as PTHrP. The antibodies
will be useful to investigate the function and metabolism of
PTHrP in vivo and in vitro. |
| Coetzer TH.
Pike RN. Dennison C. |
| Localization
of an immunoinhibitory epitope of the cysteine proteinase, cathepsin
L. |
Immunological
Investigations. 21(6):495-506, 1992
Antibodies, raised in chickens (IgY) and rabbits (IgG) against
the lysosomal proteinase cathepsin L, targeted the enzyme in
an ELISA and Western blot. In contrast to the rabbit IgG, the
chicken IgY was immunoinhibitory towards cathepsin L. An epitope
that elicits immunoinhibitory antibodies has been localized
to an active site-associated peptide sequence. The corresponding
free peptide, coated down in an ELISA, is recognised by the
chicken IgY, but not the rabbit IgG. This peptide was able to
inhibit the immunoinhibition of cathepsin L by chicken anti-cathepsin
L IgY, suggesting its complete or partial identity with an immunogenic
epitope for chickens in whole cathepsin L. |
| Suardet L.
Gaide AC. Calmes JM. Sordat B. Givel JC. Eliason JF. Odartchenko
N. |
| Responsiveness
of three newly established human colorectal cancer cell lines
to transforming growth factors beta 1 and beta 2. |
Cancer
Research. 52(13):3705-12, 1992
We have established 3 new human colorectal cancer cell lines
(LS411N, LS513, and LS1034) from clinical biopsy samples. These
lines are tumorigenic and grow s.c. as adenocarcinomas in nude
mouse xenografts. Specific marker chromosomes are observed in
each line. Carcinoembryonic antigen is expressed at the surface
of all 3 lines, but with marked quantitative differences. Indeed,
less than 10% of the cells from the HT-29 line used as a reference
express carcinoembryonic antigen while more than 90% of the
LS1034 cells do so. LS513 and LS1034 consistently express HLA
class I antigens and intercellular adhesion molecule 1 which
are not detected at the surface of the LS411N cells. No expression
of HLA class II antigens DR, DQ, and DP has been measured on
any of the lines. All three lines grow well in 5% fetal calf
serum medium without addition of exogenous growth factors. The
LS1034 line has been adapted to growth in serum-free conditions
and exhibits increased clonogenicity when cells are seeded in
serum-free methylcellulose medium, as compared with medium containing
5% fetal calf serum. The LS513 and LS1034 lines have proved
to be of particular interest since they respond to the growth-inhibitory
action of TGF-beta 1 and TGF-beta 2 in both liquid and semisolid
medium. Both factors were, at pM concentrations, equipotent
inhibitors of LS1034 cell proliferation. In contrast, higher
concentrations of TGF-beta 1 are inhibitory for proliferation
of LS513 cells, whereas TGF-beta 2 has no effect on the growth
of these cells in liquid assay. On this basis, using appropriate
anti-TGF-beta 1 and anti-TGF-beta 1 IgY, we developed a bioassay
for TGF-beta 1 and TGF-beta 2. Two of the three lines have indeed
been shown to produce latent-TGF-beta 1 activity. |
| Sturmer AM.
Driscoll DP. Jackson-Matthews DE. |
| A quantitative
immunoassay using chicken antibodies for detection of native
and recombinant alpha-amidating enzyme. |
Journal
of Immunological Methods. 146(1):105-10, 1992
A sensitive competitive ELISA has been developed for the detection
and quantitation of native and recombinant alpha-amidating enzyme.
Chickens immunized with purified enzyme (75 kDa) isolated from
a rat medullary thyroid carcinoma, produced IgY antibodies specific
for the native enzyme. The assay is defined by a standard curve
with a linear range of 0.78-12.5 ng/ml in phosphate-buffered
saline, and a limit of sensitivity for detection of the enzyme
of 0.20 ng/ml. The immunoassay is capable of detecting enzyme
from both tumor derived sources, and from cells genetically
engineered to secrete the enzyme into tissue culture medium
containing up to 10% fetal calf serum. |
| Gassmann M.
Thommes P. Weiser T. Hubscher U. |
| Efficient production
of chicken egg yolk antibodies against a conserved mammalian
protein. |
FASEB
Journal 4: 2528-2532, 1990.
The egg yolk of immunized chicken is a rich and inexpensive
source of specific polyclonal antibodies. In this paper we show
that 20-30 µg 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 extraced from 62
eggs of one immunized hen, yielding approximately 130 mg of
specific antibodies. |
| Carroll SB.
Stollar B.D. |
| Antibodies to
calf thymus RNA polymerase II from egg yolks of immunized hens. |
Journal
of Biological Chemistry 258(1): 24-26, 1983
Polyclonal antibodies to calf thymus RNA polymerase II were
raised in laying hens. Up to 75 mg of immunoglobulin per egg
yolk were extraced by the polyethylene glycol procedure of Roeder.
The concentration of specific antibody in egg yolks (IgY) was
comparable to that of serum as measured by enzyme-liked immunoassay.
Purified antibody was shown to be directed against enzyme by
removal of enzyme activity in immune complexes precipitated
by rabbit anti-chicken IgY. The antibodies recognized several
of the subunits of the enzyme as determined by their reactivity
with polypeptides transferred to nitrocellulose paper after
gradient SDS-PAGE. Production of antibodies in laying hens may
facilitate the study of other highly conserved antigens that
are poorly immunogenic in mammalian hosts. |
