Chemistry: analytical and immunological testing – Involving immune complex formed in liquid phase – Separation of immune complex from unbound antigen or antibody
Reexamination Certificate
1998-12-10
2001-05-01
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving immune complex formed in liquid phase
Separation of immune complex from unbound antigen or antibody
C436S538000, C436S545000, C436S161000, C436S162000, C436S804000, C436S501000, C436S544000, C436S546000, C436S050000, C436S056000, C436S172000, C435S007100, C435S007800, C435S007930, C435S007940, C435S007950, C435S008000, C435S807000, C073S019020, C073S023220, C073S061520
Reexamination Certificate
active
06225132
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention provides chromatographic methods and reagents suitable for the detection and quantification of analytes of interest at femtomolar (10
−15
M) concentrations. More specifically, the invention relates to binding a radioactive derivatizing agent to an analyte of interest present in a mixture at low concentration, separating the analyte of interest, and detecting it using multiphoton detection.
2. Background Information
The separation and detection of minute concentrations of chemicals in gas, liquid and solid environments is important in many applications, such as environmental diagnostics, biomedical applications and materials processing, particularly in the chemical and petroleum industries. Sensitive albeit inconvenient methods of measuring separation products have been developed, including mass spectroscopy (MS) and neutron activation for chemical applications, and fluorescence, luminescence, color spectroscopy and radiographic detectors for biomedical applications. Scientists continue to push detection sensitivity to lower and lower chemical concentrations. A few decades ago the operative term was microtraces. Today, femtotraces, i.e. materials present in sub-femtogram (10
−15
g) quantities, are of interest.
Chromatography is the method of choice when the detection and quantitation of specific compound(s) within mixture are needed, especially if the mixture contains several compounds with relatively similar structure. High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), Gas Chromatography (GC) and Capillary Electrophoresis (CE) have emerged as the preferred separation methods for a wide variety of applications due to their excellent sensitivity, selectivity, reproducibility and convenience of use.
Recent reviews on chromatography and detectors in use are provided in R.P.W. Scott, “Chemical Society Reviews,”
Modern Liquid Chromatography,
(June 1992); P. R. Fielden et al., “Developments in LC Detector Technology,”
J. Chrom. Science,
30, 1992; C. A. Bruckner et al., “Column Liquid Chromatography: Equipment and Instrumentation,”
Anal. Chem.,
66, 1R, 1994; and J. G. Dorsey et al., “Liquid Chromatography: Theory and Methodology,”
Anal. Chem.,
66, 500R, 1994. With recent broadening of chromatography applications, in many important cases chromatographic techniques are limited by the sensitivity of existing methods of detection. R. Stevenson, “A critical review of the development of HPCE instrumentation,”
American Laboratory,
Dec. 1994, pp. 29-33; M. E. McNally, A. C. Barefoot III; “Chromatography: Future directions in the industrial systems,”
American Laboratory
, Dec. 1994 pp. 28N-28W; W. Willis, “Analytical chemistry instrumentation and systems: A view to the future”,
American Laboratory
, Dec. 1994 pp. 13-17.
There is an acute need for more sensitive detection, identification and quantification of analytes separated by chromatography. Better understanding of different mechanisms calls for the study of constituent materials which are present in increasingly lower concentrations. Recently, applications appeared wherein the materials are available in picogram quantities, and sub-femtogram fractionation outputs are of interest.
Fractions leaving an HPLC column are typically quantitated by electrochemical or UV absorption detectors. This applies particularly to mixtures containing very small amounts of material. When using HPLC there is a tradeoff between sensitivity and specificity. Often, the column separation process is preceded by some pre-purification step which employs alumina, solid-phase extraction or liquid-liquid extraction to remove undesired components of the mixture, which enables better specificity of the chromatography. Pre-purification, however, often causes relatively low recoveries of some reactants. In recent years there have been attempts to improve the sensitivity of detection by employing small-bore columns (i.d. <2.0 mm). One important advantage of the microbore columns is that very small samples may be analyzed. C. P. Martucci et al.,
Pharmacol. Ther.
57 (2-3), 237, 1993; F. F. Hsu et al.,
Analyt. Biochem.,
216, 401, 1994.
To introduce properties compatible with a particular detector and mixture components, derivatizing agents are sometimes used. Derivatized mixtures may be separated better than non-reacted components. Pre-column derivatization is more often used than the post-column process. The chemistry of these derivatizing agents can be very diverse (see review in R. Yost et al.,
Biol. Mass. Spectrom.,
23, 131, 1994).
Currently, the most sensitive detection methods used in chromatography are mass-spectroscopy (MS), UV detection (UVD), electrochemical detection (ECD) and laser induced fluorescence (LIF). MS is typically used with gas chromatography whereas UVD and ECD are most useful for liquid chromatography. Furthermore, radiolabeling methods based on tritium and C
14
are often used, especially in thin layer chromatography.
Detectors currently used with chromatography respond to solute concentrations over the range of about 10
−7
to 10
−12
g/ml. The following Table shows the present practical limits of the most popular chromatographic detection methods.
Refractive
Electro-
UV-VIS
Fluorescence
Index
chemical
Conductivity
2 × 10
−10
g/ml
10
−11
g/ml
10
−7
g/ml
10
−12
g/ml
10
−8
g/ml
Current HPLC detector sensitivity is typically around a picomole/ml (10
−12
mole/ml) and is rarely better than 10
−14
mole/ml. This corresponds to nanomolar to picomolar concentrations. It would be extremely desirable to obtain a sensitivity of three orders of magnitude higher, that is, to femtomolar concentrations.
Absorbance Detectors
UV-VIS absorbance detectors are very popular in HPLC and CE since they are relatively inexpensive and can be focused down to the dimensions of the column or capillary. Also, most compounds of interest have UV-absorbing chromophores. The few that do not, such as common anions and small molecules, are candidates for indirect detection. The principal problem with absorbance detection in HPLC is that the path length of the cell is only as wide as the column, which is only a few millimeters. This limits the retention time within the detector. Recently, a special UVD for HPLC was designed where one looks down a long vertical segment. This improves detection by about a factor of 50. In some detectors, a bubble is induced in the flow cell to increase the path length and decrease the apparent peak width. With all of these improvements, however, detection limits are still in the range of 10
−7
mole/ml. The noise levels of these detectors are already very low and probably will continue to improve by not more than a factor of a few per decade. In TLC, the UVD performance is further limited by light absorption and scatter in the granulated medium.
Fluorescence Detection
Fluorescence detection offers the potential of improving detection limits by about a hundred times over UV absorbance. The detected intensity is function of the incident light and the quantum yield for the fluorophore group. It depends upon the performance of the light collection optics. The major requirement is that the excitation beam be isolated as much as possible from the emission optics in order to have the best possible signal-to-noise ratio. A variety of fluorescence detectors have been developed. One novel design uses a microscope to focus the excitation beam on the capillary and then collect the fluorescence signal through the same optics.
Fluorescence detectors with non-laser excitation sources are capable of detection limits in the 10
−9
mole range. Lasers can increase the flux of excitation light, which extends detection limits to about 10
−13
mole/ml in favorable cases. Stronger lasers might increase the LOD still further, but one quickly runs into problems of photo-bleaching and heating of the cell. Scattering from the cell wall can also be an important limitati
Bielski Roman
Drukier Andrzej K.
BioTraces, Inc.
Cook Lisa V.
Gollin Michael A.
Le Long V.
Venable
LandOfFree
Enhanced chromatography using multiphoton detection does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Enhanced chromatography using multiphoton detection, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Enhanced chromatography using multiphoton detection will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2549798