Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-26
2001-05-08
Nolan, Patrick J. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S810000, C530S387100, C530S387300, C530S387500
Reexamination Certificate
active
06228599
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an antibody. In particular, the present invention relates to an antibody that is capable of recognizing a certain motif on a pectin structure.
BACKGROUND OF THE INVENTION
Primary plant cell walls are fibrous composite structures consisting of cellulose microfibrils embedded in a heterogeneous polymer matrix of which a major constituent is pectin. In addition to a directly structural role, the pectic network provides a dynamic operating environment within the primary cell wall matrix and is involved in cell adhesion, regulation of cell wall porosity, cell wall extensibility and ionic status.
Pectins are immensely complex not only in their composition but also in their linkages and in their intermolecular bonds. In most cell types they principally comprise the following three types of polysaccharides: homogalacturonan (HG), rhamnogalacturonan I (RGI) and rhamnogalacturonan II (RGI).
The organization, integration and precise structures and functions of these pectic polysaccharides within primary cell walls is still not fully understood.
However, it is known that HG can contain long interrupted sequences of contiguous (&agr;-1,4-linked-GalA residues (known as HG blocks) that may be interspersed with rhamnose residues either occasionally or as repeating GalA-Rha structures. In vivo, HG is thought to be synthesized in a form with extensive methyl-esterification at the C-6 carboxyl position.
It is also thought that the controlled de-esterification of pectin by pectin methyl esterases within the cell wall influences pectic gel formation, principally by means of the formation of calcium bridges between stretches of de-esterified HG blocks at ‘junction zones’. Furthermore, de-esterification also influences the susceptibility of HG to hydrolytic and degradative enzymes such as polygalacturonases.
The mechanical properties of pectin gels are important both structurally, in that they contribute to the resisting of turgor pressure, and physiologically in that they determine the porosity of the cell wall. It is thought that cell wall porosity may regulate, by physical exclusion, the access of cell wall modifying enzymes to polymer substrates and so influence changes in cell wall architecture. HG is also an important source of biologically active oligogalacturonides (OGAs) that appear to have roles as signalling molecules in both plant development and defence.
Thus, HG is a multi-functional pectic polysaccharide of primary cell walls involved in calcium cross-linking and gel formation, the regulation of the ionic status and porosity of the primary cell wall matrix and is a source of oligosaccharins functioning in plant development and defence.
In addition to the above commentary, it is known that pectin comprises highly branched regions with an almost alternating rhamno-galacturonan chain. These highly branched regions may also contain other sugar units (such as D-galactose, L-arabinose and xylose) attached by glycosidic linkages to the C3 or C4 atoms of the rhamnose units or the C2 or C3 atoms of the galacturonic acid units. The long chains of (&agr;-1-4 linked galacturonic acid residues are commonly referred to as “smooth” regions, whereas the highly branched regions are commonly referred to as the “hairy regions”.
As indicated above, some of the carboxyl groups of the galacturonic residues are esterified (e.g. the carboxyl groups are methylated). Typically esterification of the carboxyl groups occurs after polymerization of the galacturonic acid residues. However, it is extremely rare for all of the carboxyl groups to be esterified (e.g. methylated).
Usually, the degree of esterification (“DE”) will vary from 0-90%. If 50% or more of the carboxyl groups are esterified then the resultant pectin is referred to as a “high ester pectin” (“HE pectin” for short) or a “high methoxyl pectin”. If less than 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a “low ester pectin” (“LE pectin” for short) or a “low methoxyl pectin”. If 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a “medium ester pectin” (“ME pectin” for short) or a “medium methoxyl pectin”. If the pectin does not contain any—or only a few—esterified groups it is usually referred to as pectic acid.
A Protocol for determining the degree of esterification of the PME substrate may be found on page 58 of WO-A-97/03574. For ease of reference, this Protocol is recited below.
DEGREE OF ESTERIFICATION (%DE)
To 50 ml of a 60% isopropanol and a 5% HC1 solution is added 2.5 g pectin sample and stirred for 10 min. The pectin solution is filtered through a glass filter and washed with 15 ml 60 % isopropanol/5% HC1 solution 6 times followed by further washes with 60% isopropanol until the filtrate is free of chlorides. The filtrate is dried overnight at 80° C.
20.0 ml 0.5 N NaOH and 20.0 ml 0.5 N HC1 is combined in a conical flask and 2 drops of phenolphthalein is added. This is titrated with 0.1 N NaOH until a permanent color change is obtained. The 0.5 N HC1 should be slightly stronger than the 0.5N NaOH. The added volume of 0.1 N NaOH is noted as V0.
0.5 g of the dried pectin sample (the filtrate) is measured into a conical flask and the sample is moistened with 96% ethanol. 100 ml of recently boiled and cooled destined water is added and the resulting solution stirred until the pectin is completely dissolved. Then 5 drops of phenolphthalein are added and the solution titrated with 0.1 N NaOH (until a change in color and pH is 8.5). The amount of 0.1 N NaOH used here is noted as V1. 20.0 ml of 0.5 N NaOH is added and the flask shaken vigously, and then allowed to stand for 15 min. 20.0 ml of 0.5 N HC1 is added and the flask is shaken until the pink color disappears. 3 drops of phenolphthalein are then added and then the resultant solution is titrated with 0.1 N NaOH. The volume 0.1 N NaOH used is noted as V2.
The degree of esterification (% DE: % of total carboxy groups) is calculated as follows:
%
DE
=
V2
-
V0
V1
+
(
V2
-
V0
)
As indicated above, the structure of the pectin, in particular the degree of esterification (e.g. methylation), dictates many of the resultant physical and/or chemical properties of the pectin. For example, pectin gelation depends on the chemical nature of the pectin, especially the degree of esterification. In addition, however, pectin gelation also depends on the soluble-solids content, the pH and calcium ion concentration. With respect to the latter, it is believed that the calcium ions form complexes with free carboxyl groups, particularly those on a LE pectin.
A Protocol for determining calcium sensitivity may be found on page 57 of WO-A-97/03574. For ease of reference, this Protocol is recited below.
CALCIUM SENSITIVITY INDEX (CF)
Calcium sensitivity is measured as the viscosity of a pectin dissolved in a solution with 57.6 mg calcium/g pectin divided by the viscosity of exactly the same amount of pectin in solution, but without added calcium. A calcium insensitive pectin has a CF value of 1.
4.2 g pectin sample is dissolved in 550 ml hot water with efficient stirring. The solution is cooled to about 20ø C. and the pH adjusted to 1.5 with IN HC1. The pectin solution is adjusted to 700 ml with water and stirred. 145 g of this solution is measured individually into 4 viscosity glasses. 10 ml water is added to two of the glasses (double determinations) and 10 ml of a 250 mM CaC12 solution is added to the other two glasses under stirring.
50 ml of an acetate buffer (0.5 M, pH about 4.6) is added to all four viscosity glasses under efficient magnetic stirring, thereby bringing the pH of the pectin solution up over pH 4.0. The magnets are removed and the glasses left overnight at 20°C. The viscosities are measured the next day with a Brookfield viscometer. The calcium sensitivity index is calculated as follows:
CF
=
Viscosity
of
a
solution
with
57.6
mg
Ca
2
+
Knox John Paul
Mikkelsen Jorn Dalgaard
Willats William George Tycho
Danisco A/S
Ewoldt Gerald R.
Knobbe Martens Olson & Bear LLP
Nolan Patrick J.
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