Stock material or miscellaneous articles – Composite – Of polyamide
Reexamination Certificate
2001-11-30
2004-09-28
Seidleck, James J. (Department: 1711)
Stock material or miscellaneous articles
Composite
Of polyamide
C427S002130
Reexamination Certificate
active
06797393
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to fabricating protein microarrays in general and in particular to a method that utilizes a gelatin-based substrate wherein the gelatin surface is modified to improve specific attachment of biological molecules.
BACKGROUND OF THE INVENTION
The completion of Human Genome project spurred the rapid growth of a new interdisciplinary field of proteomics which includes: identification and characterization of complete sets of proteins encoded by the genome, the synthesis of proteins, post-translational modifications, as well as detailed mapping of protein interaction at the cellular regulation level.
While 2-dimensional gel electrophoresis in combination with mass spectrometry still remains the dominant technology in proteomics study, the successful implantation and application of DNA microarray technology to gene profiling and gene discovery have prompted scientists to develop protein microarray technology and apply microchip based protein assays to the field of proteomics. For example, in WO 00/04382 and WO 00/04389, a method of fabricating protein microarrays is disclosed. A key element in the disclosure is a substrate consisting of a solid support coated with a monolayer of thin organic film on which protein or a protein capture agent can be immobilized.
Nitrocellulose membrane was widely used as a protein blotting substrate in Western blotting and enzyme linked immunosorbent assay (ELISA). In WO 01/40312 and WO 01/40803, antibodies are spotted onto a nitrocellulose membrane using a gridding robot device. Such spotted antibody microarrays on a nitrocellulose membrane substrate have been shown to be useful in analyzing protein mixture in a large parallel manner.
In WO 98/29736, L. G. Mendoza et al. describe an antibody microarray with antibody immobilized onto a N-hydroxysuccinimidyl ester modified glass substrate. In U.S. Pat. No. 5,981,734 and WO 95/04594, a polyacrylamide based hydrogel substrate technology is described for the fabrication of DNA microarrays. More recently, in
Anal. Biochem
. (2000) 278, 123-131, the same hydrogel technology was further demonstrated as useful as a substrate for the immobilization of proteins in making protein microarrays.
In the above cited examples, the common feature among these different approaches is the requirement of a solid support that allows covalent or non-covalent attachment of a protein or a protein capture agent on the surface of said support. In DNA microarray technology, a variety of surfaces have been prepared for the deposition of pre-synthesized oligos and PCR prepared cDNA probes. However, unlike DNA, proteins tend to bind to surfaces in a non-specific manner and, in doing so, lose their biological activity. Thus, the attributes for a protein microarray substrate are different from those for a DNA microarray substrate in that the protein microarray substrate must not only provide surface functionality that are capable of interacting with protein capture agents, but must also resist non-specific protein binding to areas where no protein capture agents have been deposited.
Bovine serum albumin (BSA) has been demonstrated to be a useful reagent in blocking proteins from non-specific surface binding. Polyethylene glycol and phospholipids have also been used to passivate surfaces and provide a surface resistant to non-specific binding. However, all of these methods suffer disadvantages either because surface preparation takes a long time or because the method of surface modification is complex and difficult, making the method less than an ideal choice for large scale industrial manufacture.
Hence, there is still need to discover other low cost and readily manufacturable materials that serve as a matrix on a solid support for the attachment of protein capture agents. The art needs a substrate with chemical functionality for the immobilization of protein capture agents, but such substrate must not bind proteins to areas on the gelatin surface that are without immobilized protein capture agents.
SUMMARY OF THE INVENTION
The present invention seeks to solve some of the problems discussed above by providing:
A gelatin-based substrate for fabricating protein arrays, the substrate comprising: gelatin and a trifunctional compound A—L—B; wherein A is a functional group capable of interacting with the gelatin; L is a linking group capable of interacting with A and with B; and B is a functional group capable of interacting with a protein capture agent, wherein A may be the same or different from B.
Also provided is a method of making a gelatin-based substrate for fabricating protein arrays comprising the steps of providing a support; coating on the support a composition containing gelatin; and affixing to a surface of the gelatin a trifunctional compound A—L—B; wherein A is a functional group capable of interacting with the gelatin; L is a linking group capable if interacting with A and with B; and B is a functional group capable of interacting with a protein capture agent; wherein A may be the same or different from B.
The invention is particularly useful in fabricating protein microarrays. The invention provides a gelatin substrate with at least one surface to which certain functionalities have been affixed. Thus treated, or modified, the gelatin surface is substantially resistant to non-specific binding. Further, the functionalities are capable of interacting specifically with protein capture agents with which they come in contact. Thus, the substrate of the invention affords a high degree of specific binding between the modified gelatin surface and protein capture agents.
Gelatin substrates that have been modified according to this invention require a very low concentration of biological sample in fabricating protein microarrays when compared with unmodified gelatin substrates. Also, the gelatin substrates of the invention can be readily manufactured at low cost. The usefulness of the claimed substrate for protein attachment is demonstrated below in the examples, using several chemical modification methods and enzyme linked immunosorbent assay (ELISA).
DETAILED DESCRIPTION OF THE INVENTION
In general, a protein microarray can be prepared by first modifying a solid support, namely the protein microarray support, followed by depositing various protein capture agents onto the modified substrate at pre-defined locations. Supports of choice for protein microarray applications can be organic, inorganic or biological. Some commonly used support materials include glass, plastics, metals, semiconductors. The support can be transparent or opaque, flexible or rigid. In some cases, the support can be a porous membrane e.g. nitrocellulose and polyvinylidene difluoride, and the protein capture agents are deposited onto the membrane by physical adsorption. However, to improve robustness and reproducibility, it is more desirable to immobilize the protein capture agents onto a substrate through chemical covalent bond.
To immobilize protein capture agents onto a solid support, the support needs to be modified by certain chemical functional agents. In general, the chemically functional agent is a bi-functional molecule which can be represented as A—L—B, in which A and B are chemical functionalities that are capable of reacting or interacting with gelatin and protein capture agent molecules to be immobilized on the substrate and L is linkage group. Preferably, L is a diradical of such a length that the shortest through—bond path between the ends that connect A to B is not greater than 10 atoms.
There are two classes of bi-functional agents: 1). homofunctional agent if A=B; and 2). heterofunctional agent if A≠B. Some commonly used A and B include but are not limited to, aldehyde, epoxy, hydrazide, vinyl sulfone, succinimidyl ester, carbodiimide, maleimide, dithio, iodoacetyl, isocyanate, isothiocyanate, aziridine. The linking group L comprises any reasonable combination of relatively non-labile covalently bonded chemical units sufficient to connect the two functionalities A and B. These chemica
Chari Krishnan
Penner Thomas L.
Qiao Tiecheng A.
Yang Zhihao
Bissett Melanie
Manne Kathleen Neuner
Seidleck James J.
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