Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-11-10
2001-01-09
Minnifield, Nita (Department: 1665)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007100, C436S546000, C436S544000, C436S543000, C530S350000
Reexamination Certificate
active
06171807
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is in the field of fluorescence polarization assays for endotoxin.
2. Related Art
Fluorescence polarization was first described in 1926 (Perrin,
J. Phys. Rad.
1:390-401 (1926)) and has been a powerful tool in the study of molecular interactions. When fluorescent molecules are excited with plane polarized light, they emit light in the same polarized plane, provided that the molecule remains stationary throughout the excited state (4 nanoseconds in the case of fluorescein). However, if the excited molecule rotates or tumbles out of the plane of polarized light during the excited state, then light is emitted in a different plane from that of the initial excitation. If vertically polarized light is used to excite the fluorophore, the emission light intensity can be monitored in both the original vertical plane and also the horizontal plane. The degree to which the emission intensity moves from the vertical to horizontal plane is related to the mobility of the fluorescently labeled molecule. If fluorescently labeled molecules are very large, they move very little during the excited state interval, and the emitted light remains highly polarized with respect to the excitation plane. If fluorescently labeled molecules are small, they rotate or tumble faster, and the resulting emitted light is depolarized relative to the excitation plane. “Introduction to Fluorescence Polarization,” Pan Vera Corp., Madison, Wis., Jun. 17, 1996.
Fluorescence polarization (FP) is defined as:
FP
=
Int
&LeftDoubleBracketingBar;
-
Int
⊥
Int
&LeftDoubleBracketingBar;
+
Int
⊥
Where Int(parallel) is the intensity of the emission light parallel to the excitation light plane and Int(perpendicular) is the intensity of the emission light perpendicular to the excitation light plane. FP, being a ratio of light intensities, is a dimensionless number and has a maximum value of 0.5 for fluorescein. The Beacon System expresses polarization in millipolarization units (1 Polarization Unit=1000 mP Units). “Introduction to Fluorescence Polarization,” Pan Vera Corp., Madison, Wis., Jun. 17, 1996.
Fluorescence anisotropy (A) is another term commonly used to describe this phenomenon. Polarization and anisotropy are related in the following way.
A
=
Int
&LeftDoubleBracketingBar;
-
Int
⊥
Int
&LeftDoubleBracketingBar;
+
2
Int
⊥
and
A
=
2
P
3
-
P
As discussed above, polarization is related to the speed at which a fluorescently labeled molecule rotates. The Perrin equation can be used to show that polarization is directly proportional to the correlation time, the time that it takes a molecule to rotate through an angle of approximately 68.5°. The correlation time is sometimes referred to as the rotational relaxation time or the rotational correlation time. Correlation time is related to viscosity (n), absolute temperature (T), molecular volume (V), and the gas constant (R) by the following equation.
Correlation
Time
=
3
η
V
RT
It follows then, that if viscosity and temperature are held constant, correlation time, and therefore polarization, are directly related to the molecular volume. Changes in molecular volume may be due to molecular binding, dissociation, synthesis, degradation, or conformational changes of the fluorescently labeled molecule.
Light from a monochromatic source passes through a vertical polarizing filter to excite fluorescent molecules in the sample tube. Only those molecules that are orientated in the vertically polarized plane absorb light, become excited, and subsequently emit light. The emission light intensity is measured both parallel and perpendicular to the exciting light. The fraction of the original incident, vertical light intensity that is emitted in the horizontal plane is a measure of the amount of rotation the fluorescently labeled molecule has undergone during the excited state, and therefore is a measure of its relative size. See, “Introduction to Fluorescence Polarization,” Pan Vera Corp., Madison, Wis., Jun. 17, 1996. Other publications describing the FP technique include G. Weber,
Adv. Protein Chem.
8:415-59 (1953); W. B. Dandliker and G. A. Feigen,
Biochem. Biophys. Res. Commun.
5:299-304 (1961); W. B. Dandliker et al.,
Immunochemistry
10:219-27 (1973); and M. E. Jolley,
J. Anal. Toxicol.
5:236-240 (1981). “Chapter 4—Introduction to Fluorescence Polarization,” the FPM-1™ Operators Manual, pp. 9-10, Jolley Consulting and Research, Inc. Grayslake, Ill.
One of the most widely used fluorescence polarization applications is the competitive immunoassay used for the detection of therapeutic and illicit drugs. A small, fluorescently-labeled drug, when excited with plane polarized light, emits light that is depolarized because the fluorophore is not constrained from rotating during the excitation state. When the labeled drug is added to a serum-antibody mixture, it competes with the unlabeled drug in the sample for binding to the antibody. The lower the concentration of unlabeled drug in the sample, the greater amount of labeled drug that will bind to the antibody. Once bound to antibody, the labeled drug rotates and tumbles more slowly. Light emitted by the fluorescently labeled drug/antibody complex will be more polarized, and the fluorescence polarization value of the sample will be higher. By constructing a standard curve of serum samples with known drug concentrations versus polarization value, the concentration of drug in a patient sample can be easily determined. “Introduction to Fluorescence Polarization,” Pan Vera Corp., Madison, Wis., Jun. 17, 1996; Perrin,
J. Phys. Rad.
1:390-401 (1926).
FP measurements can be made using Pan Vera's Beacon® Fluorescence Polarization system, which can detect as little as 10 femtomoles/ml of fluorescently labeled sample. Measurements are made on samples directly in solution and, as a result, the need for any separation or filtration of bound from unbound elements is eliminated.
Most buffers and salts are contaminated with fluorescence and give background readings that are too high for measurements in the pM or even nM range. Pan Vera offers a line of low fluorescence buffers and reagents that allows one to measure fluorescence polarization values to low pM concentrations.
The abridged version of the Beacon® Fluorescence Polarization Applications Guide provides examples of a wide range of molecular binding (and enzymatic degradation) experiments performed on the Beacon FP system.
The Limulus Amoebocyte Assay (LAL) is an assay for endotoxin or bacteria which have endotoxin on their cell surfaces.
Another assay for endotoxin employs the endotoxin binding and neutralizing protein (ENP). ENP may be isolated from the horseshoe crab (Seq ID NO: 1) or produced recombinantly. When produced recombinantly in a yeast host, a modified ENP is obtained which contains GluAlaGluAla at the N-terminus (SEQ ID NO:3). ENP can be used to detect endotoxin in samples in a qualitative and quantitative fashion, as well as for treating endotoxemia in vivo. See International Application Publication No. WO92/20715 and U.S. application Ser. No. 08/264,244, filed Jun. 22, 1994.
SUMMARY OF THE INVENTION
The invention relates to fluorescently labeled ENP.
The invention also relates to a method of preparing fluorescently labeled ENP, comprising combining in a solution ENP and a fluorescence labeling reagent, and isolating the fluorescently labeled ENP from the solution.
In particular, the invention relates to a method of preparing fluorescently labeled ENP, comprising combining in an aqueous citrate buffer solution ENP having SEQ ID NO: 3, urea and FS, and isolating the fluorescently labeled ENP from the solution.
The invention also relates to a method of detecting endotoxin in a liquid sample suspected of containing endotoxin, comprising admixing said liquid sample with fluorescently labeled ENP for a time sufficie
Novitsky Thomas J.
Ridge Richard J.
Sloyer Jack L.
Associates of Cape Cod, Inc.
Devi S.
Minnifield Nita
Sterne Kessler Goldstein & Fox P.L.L.C.
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