Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-11-02
2002-07-23
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S007100, C435S975000, C436S547000, C436S548000, C436S808000, C530S388100, C530S866000, C530S864000
Reexamination Certificate
active
06423546
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
The present invention relates to novel monoclonal antibodies reactive with lipid transfer proteins typically found in foaming beverages. More specifically, the present invention relates to novel monoclonal antibodies raised against the native and denatured forms of barley lipid transfer protein 1, and an assay for determining the content of said proteins in foaming beverages at various stages of their production.
Foaming beverages, e.g. beer and some soft drinks, are popular items in today's marketplace. In addition to taste, the appearance of the beverage and its ability to form a stable head of foam when poured are important characteristics. In beer, the foam head is one of the first characteristics that a consumer generally uses to judge beer quality.
Foam formation and retention are two factors considered when defining foam quality. The rate at which the foam head forms and collapses depends upon, among other things, the composition of the beer.
Several different beer and foam proteins have been suggested to be important in foam formation and head retention. One such protein is the lipid transfer protein found in cereal grains. In particular, several different studies have analyzed the lipid transfer protein from barley and have shown that its presence in beer exhibits a positive effect on foam formation and stability. Evans and Hejgaard, “The Impact of Malt Derived Proteins on Beer Foam Quality. Part I: The Effect of Germination and Kilning on the Level of Protein Z4, Protein Z7, and LTP1
”, J. Inst. Brewing
, 105:3:159-169 (1999); Evans et al., “The Impact of Malt Derived Proteins on Beer Foam Quality. Part I: The Influence of Malt-positive Proteins and Non-starch Polysaccharides on Beer Foam Quality”,
J. Inst. Brewing
, 105:2:171-177 (1999); Lusk et al., “Foam tower fractionation of beer proteins and bittering acids,” European Brewery Convention Beer Foam Quality Symposium (Amsterdam, Oct. 25-27, 1998); Bock et al., “New Analytical Techniques with Relevance for the Brewing Industry”,
Brygmesteren
, 54(5):47-55 (1997); Lusk et al., “Independent role of beer proteins, melanoidins and polysaccharides in foam formation,”
J. Am. Soc. Brew. Chem
., 53(3):93-103 (1995); Sorenson et al., “Barley Lipid Tranfer Protein 1 is Involved in Beer Foam Formation”,
MBAA Tech. Quarterly
, 30:136-145 (1993).
Two members of the lipid transfer protein gene family are expressed in barley grain, LTP1 and LTP2. Of the two proteins, only LTP1 is found in beer (see, Evans and Hejgaard, supra). LTP1 is an albumin protein primarily expressed in the aleurone layer of the barley seed. It has a molecular weight of 9,694 Daltons and contains 91 amino acid residues, including 8 cysteines. The amino acid sequence of LTP1 is set forth in SEQ ID NO:1. Studies by Bock et al., supra, have shown that LTP1 is modified during the malting and brewing process to a denatured form (fLTP). It is this denatured form that is believed to effect foam formation and stability.
Other studies have suggested that other proteins and polypeptides are important in foam formation and stability. In particular, it has been suggested that beer and foam proteins of a molecular weight greater than 5,000 Dalton tend to be foam-positive, while polypeptides of molecular weights below 5,000 Dalton tend to be foam-negative. For example, studies by Sharpe et al. have suggested that head retention was related to the ratio of high and low molecular weight polypeptides. (Sharpe et al., “Rapid methods of measuring the foam-active nitrogenous components of worts and beers”,
Proc. Eur. Brewing Conv
.: 18
th Cong
., 607-614 (1981)). Meanwhile, Yokoi, et al, has suggested that protein Z, a 40,000 Dalton barley albumin, plays the most significant role in foaming and head retention (Yokoi et al., “Characterization of beer proteins responsible for the foam of beer”,
Proc. Eur. Brewing Conv
.: 22
nd Cong
., 503-512 (1989)). On the other hand, Kauffman et al. has suggested that the prolamin storage proteins of barley, called hordeins, are also important in foam formation and stability (Kauffman et al., “Immunological Characterisation of Barley Polypeptides in Lager Foam”,
J. Sci. Food Agric
., 66:345-355 (1994)).
Most of the above conclusions have resulted from investigations generally involving the fractionation of beer proteins and a determination of their foaming effect. More recently, there has been considerable interest in tracing the origin of foam proteins using immunological methods. Polyclonal antibodies against barley, malt, beer and yeast proteins have been developed and used in these studies. For example, Hollemans and Tonies used polyclonal antibodies to remove polypeptides from beer to establish their effect on foaming (Hollemans and Tonies, “The role of specific proteins in beer foam”,
Proc. Eur. Brew. Conv
.: 22
nd Cong
., 561-568 (1989)); Ishibashi et al., used polyclonal antibodies to analyze both foam and haze proteins in beer (Ishibashi et al., “Development of a new method for determining beer foam and haze proteins by using the immunochemical method ELISA”,
J. Am. Soc. Brew. Chem
., 54(3):177-18)); and Bech et al. used polyclonal antibodies to determine the concentration of LTP1 in wort, beer, and barley and malt extracts from several different barley varieties (EP 0728188). The information obtained using polyclonal antibodies, however, is partly limited due to problems of polyspecificity resulting from the presence of immunodominant repetitive hordein sequences (Mills et al., “Immunological Study of Hydrophobic Polypeptides in Beer”,
J. Agric. Food Chem
., 46:4475-4483 (1998)). Accordingly, more exact methods for performing immunological studies on beer and foam proteins are needed.
Monoclonal antibodies have been employed in some cases to avoid the problems associated with the use of polyclonal antibodies. For example, Kaufman et al., supra, has reported the use of monoclonal antibodies against wheat prolamins to study hordein-type material found in beer and foam fractions. Sheehan and Skerritt have also used monoclonal antibodies to examine modifications of hordeins during beer production (Sheehan and Skerritt, “Identification and Characterisation of Beer Polypeptides Derived from Barley Hordeins”,
J. Inst. Brew
., 103:297-306 (1997)). Mills et al. have reported the creation of a monoclonal library to beer proteins and polypeptides believed to be derived from the hordeins in malts (Mills et al., “Immunological Study of Hydrophobic Polypeptides in Beer”,
J. Agric. Food Chem
., 46:4475-4483 (1998)). Meanwhile, European Patent 0863153 by Ishibashi et al., and Kukai et al., “Development of Monoclonal Antibody Sandwich-ELISA for Determination of Beer Foam-Active Proteins”,
J.Am. Soc. Brew. Chem
., 56(2):43-46 (1998), both report the production and use of monoclonal antibodies in ELISA experiments directed against foam-active proteins having molecular weights between 40 and 50 kDa.
With respect to the lipid transfer proteins, only a single monoclonal antibody disclosed by Dickie has been reported (Dickie, “Immunological Determination of Foam-Positive Hydrophobic Polypeptides in Barley and the Effects of Malting”,
BRI Quarterly
, 15-18 (October, 1997)). This antibody, identified as IFRN 1625, recognizes a ca. 8 kDa polypeptide found in Group 5 foam fractions, and is believed to be one of the proteins involved in lipid transfer. Dickie postulates that this 8 kDA polypeptide originates from LTP1 but presents no evidence to support this hypothesis.
What is needed is a set of monoclonal antibodies exhibiting specificity against lipid transfer proteins in either their native or modified forms. What is also needed is an assay capable of measuring and characterizing the content of native and denatured lipid transfer proteins in foaming beverages during various stages of their production process.
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized
Goldstein Henry
Lusk Lance T.
Navarro Alfonso L.
Ryder David S.
Wagner Randall J.
Miller Brewing Company
Nguyen Bao-Thuy L.
Quarles & Brady LLP
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