Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
2000-06-09
2003-08-19
Chan, Christina (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C530S389100, C530S861000
Reexamination Certificate
active
06608172
ABSTRACT:
BACKGROUND OF THE INVENTION
1) Field of the Invention
The present invention relates to a method for the preparation and purification of IgY(&Dgr;Fc) antibody from avian yolk, and the IgY(&Dgr;Fc) antibody produced thereby. The present invention also relates to uses of the novel IgY(&Dgr;Fc) antibody for quantitatively or qualitatively analyzing an etiological agent of interest.
2) Description of the Related Art
Antibodies are used widely in many biological investigations and clinical applications. Sera obtained from hyperimmunized mammalians are the most common source of polyclonal antibodies. Antibodies derived from such immune sera belong to a group of proteins called ‘immunoglobulins,’ among which the immunoglobulin G (IgG) is the most abundant. The IgG molecule consists of three domains, namely two Fab regions and one Fc region. The Fab portion involves mainly in antigen binding. The Fc portion, though having no ability to bind with an antigen, directs several biological activity of an antibody, such as complement fixing and Fc receptor binding.
In the art of immunodiagnostics, an intact IgG molecule is not suitable for use in detection systems and immunological assays involving mammalian sera since the Fc region on an IgG molecule is capable of binding to Fc receptors, activating the complement system, and reacting with rheumatoid factor in mammalian sera. Removal of the Fc portion of an IgG molecule frequently lead to a reduction in the interference (E. Lamoyi,
Methods in Enzymology
121:652-663. (1986)).
Some of the suggested uses of antibody in immunotherapy include treating patients with intoxicated bacterial toxins or snake venoms (see, for example, U.S. Pat. Nos. 5,340,923 and 5,601,823), and protection of neonatal piglets against fatal enteric colibacillosis (see, for example, H. Brussow et al.,
J. Clin. Microbiol.
25:982 (1987); and C. O. Tacket et al.,
New Eng. J. Med.
318:1240 (1988)). Since the Fc fragment of an antibody molecule is known to be the most antigenic portion of the immunoglobulin (E. M. Akita et al.,
J. Immunol. Methods.
162:155-164 (1993)), cleavage of the same which results in the formation of an F(ab′)
2
fragment will reduce significantly a number of potential allergenic sites on the immunoglobulin molecule and is thus beneficial to human or an animal administered with the immunoglobulin.
Recently, the divalent F(ab′)
2
antibody fragment has been shown to be more useful in the immunodiagnostic tests (M. Muratsugu et al.,
J. Colloid Interface Sci
147:378 (1991); and J. L. Ortega-Vinuesa et al.,
J. Immunol Methods
90:29 (1996)) and more suitable for development of the immunoassays involving mammalian sera than the parent IgG.
The F(ab′)
2
antibody fragment, however, has not found widespread use in clinical immunodiagnostic kits as one might expect. This may be attributed to the difficulties and cost-ineffectiveness of large scale production of the F(ab′)
2
fragments, which is conventionally made by pepsin digestion of IgG and subsequent purification via chromatography.
Ducks and their phylogenetically close relatives and some reptiles, such as turtles, have three kinds of serum immunoglobulins: a macromolecular immunoglobulin IgM (800 kDa in duck), and two isoforms of low molecular weight IgG with sedimentation coefficients of 7.8S (in duck, 180 kDa) and 5.7S (in duck, 130 kDa), respectively. (E. R. Unanue etal.,
J. Exp. Med.
121:697-714 (1965); H. M. Grey,
J. Immunol
98:811-819 (1967); and B. Zimmerman et al.,
Biochemistry
10:482-448 (1971)). Avian IgG is oftentimes called IgY due to their existence in egg yolk. The 5.7S IgY, constituted with shorter heavy chains, is structurally and antigenically similar to the F(ab′)
2
fragment of the 7.8S IgY (FIG.
1
), and this fact leads to the nomenclature of IgY (equivalent to 7.8S IgY) and IgY(&Dgr;Fc) (equivalent to 5.7S IgY) to represent both isoforms of IgY (K. E. Magor et al.,
J. Immunol.
149:2627-2633 (1992)).
Studies conducted in the infected or experimentally immunized birds showed that duck antibodies are deficient in a number of biological effector functions, including complement fixation and Fc receptors binding, without sacrificing their binding activity to corresponding antigens (G. W. Litman et al.,
Immunochemistry
10:323 (1973); and T. E. Toth et al.,
Avian Dis.
25:17-28 (1981)). This may reasonably result from the apparent lack of Fc-equivalent region of the IgY(&Dgr;Fc) antibody that constitutes the quantitatively major component of duck antibody response. It is thus believed that the IgY(&Dgr;Fc) antibody, which appears to be a structural and functional analog of the F(ab′)
2
fragment, would provide magnificent advantages in immunological uses, if a promising process for manufacturing the antibody could be found, and the appropriate physical requirements for its activity could be identified.
Avian yolk antibodies have been reported to exhibit useful properties for both research and clinical applications as mammalian antibodies do (see, for example, U.S. Pat. Nos. 5,340,923; 5,585,098; 5,601,823; and 5,976,519). Egg yolks derived from a laying hen is inexpensive and more convenient and safer to handle as compared to the hyperimmunized mammalian sera. More importantly, yolk antibodies are able to stand up to the scrutiny under modern animal protection regulations (A. Polson et al.,
Immunol. Commun.
9:475 (1980); and B. Gottstein et al.). These facts suggest a potential use of egg yolk as a commercial source of antibodies.
Efforts have been made to isolate and purify IgY from egg yolks. For instance, materials, including agar, pectin (Japanese Kokai No. 64-38098 published in Feb. 8, 1989), dextran sulfate (J. C. Jensenius et al.,
J. Immunol. Methods
46:63 (1981)), natural gums (H. Hatta et al.,
J. Food Science
53:425(1988)) and polyethyl glycol (PEG) (A. Polson et al.,
Immunol. Invest.
14:323 (1985); see also U.S. Pat. No. 4,550,019 issued to A. Polson) were used to precipitate non-aqueous bio-molecules, mainly lipids and yolk granules, to thereby harvest a water soluble phase containing abundant yolk antibodies. A. Hassl et al. developed a two-step chromatographic process, comprised of hydrophobic interaction chromatography and size exclusive chromatography, for further isolation of yolk antibodies from a PEG-purified fraction (A. Hassl and H Aspock,
J. Immunol. Methods
110:225 (1988)). Akita et al. described an improved method for isolating IgY, in which yolk antibodies were extracted from chick eggs by diluting the egg yolks with a large volume of water and subjecting the resultant supernatant to size exclusive chromatography and/or ion exchange chromatography (E. M. Akita et al.,
J. Immunol. Methods.
160:207 (1993); and E. M. Akita and S. Nakai,
J. Food Sci.
57:629 (1993)).
However, all these studies and patents only focus on the isolation of the entire population of yolk antibodies (which includes at least IgY and IgY(&Dgr;Fc)) from avian eggs, rather than on the purification of IgY(&Dgr;Fc) antibody alone. Moreover, since the IgY(&Dgr;Fc) antibody is only present in the birds belonging to Order Anseriformes, including duck and goose, the isolation methods implemented on the galliform birds, such as chicken and turkey, provide no suggestion of a successful purification of IgY(&Dgr;Fc) antibody.
In 1989, Higgins tried to prepare the antibodies from hyperimmunized duck sera, but the antibodies affinity-purified at pH 8.0 and 0.5 M NaCl generally failed to exhibit effective precipitation or agglutination reactions (D. A. Higgins,
Comp. Biochem. Physiol.
93B:135-144 (1989)). The optimal pH value for forming duck antibody precipitins, as Higgins alleged in the literature, ranges from pH 8.5 to pH 9.05. Since then, no critical study regarding the isolation of the IgY(&Dgr;Fc) antibody and its potential uses has been reported.
Therefore, there is a need in the art for a rapid, cost-effective, high-throughput process that provides easy isolation of the desired IgY(&Dgr;Fc) antibody from the antibody pool while ma
Chan Christina
Fish & Richardson P.C.
Good Biotech Corporation
Huynh Phuong N
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