Particulate solid phase immobilized protein quantitation

Details Particulate solid phase immobilized protein quantitation Particulate solid phase immobilized protein quantitation

2000-02-24

2004-02-24

Chin, Christopher L. (Department: 1641)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

C436S008000, C436S172000, C436S523000, C436S546000, C436S015000, C436S164000, C436S165000, C436S166000, C436S169000, C436S800000, C435S004000, C435S007100, C435S007920, C435S007930, C435S007940, C435S007950

Reexamination Certificate

active

06696304

ABSTRACT:

SUMMARY OF THE INVENTION
The embodiment of the present invention entails a novel combination of separately existing chemistries and instrumentations to offer an ultrasensitive method for the quantitation of an analyte of interest such as a protein immobilized on a particulate solid phase, i.e., the surface of a microparticle, which serves as a carrier, support, or matrix. Due to amine-reactive nature of the signal label, any analyte of interest other than protein and having at least one amine group can be used in this process. As used hereinafter the term protein refers not only to a protein, but also to any substance of interest having at least one amine group. An amine can be selected from the group consisting of aliphatic amines, aromatic amines, diamines, polyamines, and substituted amines, e.g., acetamidyl, amidyl, or aminyl.
It is a further object of this invention to provide means of detecting and measuring such analytes. Analytes readily comprise most diverse classes of chemicals including proteins, peptides, prosthetic proteins, biogenic amines, various drugs with amine residues, transaminated nucleic acids, etc. Analytes also comprise fluorescent dyes having an amine group(s), e.g., phycobiliproteins, bound to a microparticle and is either labeled with another amine-reactive dye such as fluorescein isothiocyanate (FITC) or not labeled. One skilled in the art easily recognizes other similar substances like peptide nucleic acids having nucleobases attached to a polyamide backbone and containing alkylamine side chains as disclosed for example in the U.S. Pat. No. 5,786,461 and incorporated herein by way of reference.
The preferred method of making a standard microparticle preparation with a known average amount of a reference substance bound thereto comprises the steps of binding or associating a known average amount of a reference substance to a microparticle; and measuring the amount of microparticle-associated or bound reference substance by a standard calorimetric protein assay.
The preferred steps involved in this process are preferably, but not necessarily, carried out in the following sequence: immobilizing an analyte of interest on a particulate solid phase (e.g., microparticle) either by adsorption or by covalent conjugation; covalently binding to the analyte of interest a light-emitting signal molecule or signal label; comparing the intensity of the light emitted from the label to standard microparticle preparations with known amount of reference material or substance, which is labeled with the same light-emitting label under essentially same conditions, and determining the presence and/or amount of the analyte of interest. Typical standards will contain varying amounts of an immobilized reference protein extending across attogram to femtogram concentrations. For example, a series of standards ranging from 0.1 to 1000 femtograms per unit solid phase are sufficient for most common practical applications
More specifically a process is provided for determining the relative average amount of at least one protein of interest, which is immobilized on each of a plurality of particulate solid phases, comprising: subjecting under substantially the same protein labeling conditions a plurality of particulate solid phases to each of which is immobilized an unknown.average amount of at least one protein of interest and a plurality of standard particulate solid phases to each of which is immobilized a known average amount of at least one reference protein, to provide protein of interest and reference protein both labeled with at least one light-emitting label; and comparing the average amount of light emitted by labels found on the standard particulate solid phases harboring the reference protein with that emitted by labels found on particulate solid phases harboring the protein of interest, to provide a relative average amount of the protein of interest immobilized on each of the particulate solid phases.
An alternative preferred method of measuring the concentration of an analyte bound to a particulate solid phase is also provided. This method consists of associating the analyte of interest with the particulate solid phase, e.g., microparticle, which has a light-emitting label embedded within or immobilized thereon. The label is chosen in such a way that it is capable of changing its light absorption and/or emission pattern as a function of the concentration of the analyte. In addition at least one and preferably more reference samples are prepared with the known amount of the reference substance bound to the solid phase. This solid phase has the same light-emitting label and the reference substance is bound to the microparticle by essentially identical procedure as the analyte. The measurement of the amount of bound analyte is then achieved by comparing the light emission characteristics or the optical signal with the light emission spectra (optical signal) of the reference sample(s).
The preferred method comprises quantifying the amount of at least one protein of interest immobilized directly or indirectly on a particulate solid phase comprising, subjecting one or more particulate solid phases, to each of which is immobilized directly or indirectly an unknown amount of at least one protein of interest, to labeling conditions effective to affix to each particulate solid phase an effective amount of a light-emitting label to provide a labeled amount of the at least one protein of interest which is proportional [sufficient to label] to the amount of the at least one protein of interest immobilized to the particulate solid phase; and elating the amount of light emitted from each particulate solid phase to the amount of the at least one protein of interest immobilized to the particulate solid phase using a standard curve. The average amount of the reference substance bound to the microparticle is in a range between about 0.01 and 1,000 femtograms per microparticle.
The preferred method of measuring the quantity of an analyte bound to a particulate solid phase, said method comprising the steps of binding an analyte with the particulate solid phase, said particulate solid phase having a fluorescent label capable of emitting a fluorescent signal embedded within said particulate solid or immobilized thereon, said label being capable of changing its fluorescence signal as a function of the concentration of the analyte; providing at least one standard microparticle preparation with a known average amount of a reference substance bound thereto, said standard microparticle preparation having the same fluorescent label as said particulate solid phase and measuring the quantity of the analyte [associated with] bound to the particulate solid phase by comparing the fluorescence signal emitted from said particulate solid phase with the fluorescence signal emitted from the standard microparticle preparation.
To achieve higher precision it is preferable that the reference substance is labeled with the light-emitting label under the same or essentially the same reaction condition as the analyte. Typically, this means that both analyte (protein) and reference substance (protein) are labeled at the same time and in the same reaction mixture. To achieve even better precision it is preferable that both analyte and reference substance are same or closely related classes of proteins. It is preferable that reference protein is the same as, similar to, analogous to, homologous to, or functionally equivalent to the protein of interest. For example if the analyte is an immunoglobulin it is preferable that the reference substance is also an immunoglobulin of the same isotype. If a single standard is available only at one known concentration then one can only make inferences about the relative amount of the analyte (protein) of interest. Better precision and absolute quantitation is possible when a plurality of standards with known amounts of reference proteins are available which permits constructing a standard curve and based on that one skilled in the art can interpolate the qua

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