Patterning antibodies on a surface

Details Patterning antibodies on a surface Patterning antibodies on a surface

1999-05-06

2001-05-22

Chin, Christopher L. (Department: 1641)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

C430S302000, C430S304000, C430S298000, C430S297000, C430S299000, C422S051000, C422S067000, C435S033000, C435S287800, C435S287900, C435S973000, C436S527000, C427S002110, C427S002130, C427S466000, C427S470000, C427S504000, C427S534000, C427S288000, C427S286000

Reexamination Certificate

active

06235541

ABSTRACT:

BACKGROUND OF THE INVENTION
Because of their exquisite specificity, biological molecules, including antibodies, have been employed in biosensors. Biosensors are devices capable of identifying and quantifying a target chemical. Biosensors are highly sensitive to their analyte (the chemical species to be detected for an antibody-based biosensor, the analyte is the antigen to the antibody). They are able to detect quantities as small as 10
−15
gram. They are also extremely specific toward the analyte because of the unique ability of the antibodies to recognize their target species at the molecular level.
The present state of the art in antibody-based biosensors is illustrated by the various commercially available immunoassays. An immunoassay is a chemical test based on the use of antibodies to bind the molecule to be detected. In these assays, an antibody specific to the analyte (the “capture antibody”) is immobilized onto a solid surface. This surface is then exposed to the sample to be analyzed and the immobilized antibodies bind some of the analyte present in the sample. After the surface is washed, it is immersed in a solution of a second antibody (the “signal antibody”) specific to the same analyte. The signal antibody is conjugated (attached chemically) to a radioactive, fluorescent, or enzymatic label, so that it can be detected with high sensitivity. The amount of the signal antibody bound to the analyte is determined by the amount of radioactivity, intensity of fluorescence, or quantity of enzymatic reaction product, which in turn is proportional to the quantity of antigen in the sample. In the case of the enzyme label, the enzyme converts molecules of an added colorless reactant to colored reaction products. The intensity of the color change is read by a spectrophotometer. This type of assay is called enzyme-linked immunosorbent assay (ELISA). Examples of commercially available ELISA test kits are home pregnancy tests and environmental monitoring tests for BTEX (benzene, toluene, ethylbenzene, and xylene), PAH's (polynuclear aromatic hydrocarbons) or PCB's (polychlorinated biphenyls) in water. ELISA assays are also used in the military for battlefield detection of chemical and biological warfare agents. A disadvantage of these immunoassay kits is that a separate kit is required for each antigen or closely related family of antigens being tested for. Not only is this costly and labor consuming when many antigens must be tested for, but it can also result in dangerous time delays as when chemical and biological warfare agents are being tested for on the battlefield.
It would be desirable to provide a single device that could perform multiple immunoassay tests at the same time. The test results of such a device would be read and evaluated automatically. In order to achieve this, each type of antibody must be precisely and discretely located on the test surface. Cross contamination of the antibodies must be avoided. Moreover, such devices should be inexpensive and easy to manufacture.
SUMMARY OF THE INVENTION
Accordingly, an object of this invention is to provide a new device using multiple antibodies on a substrate to perform multiple immunoassay tests.
Another object of this invention is to provide a new device using multiple antibodies on a substrate to perform multiple immunoassay tests whose results can be read automatically.
A further object of this invention is to provide a new, inexpensive method of producing a device using multiple antibodies to perform multiple immunoassay tests.
Yet another object of this invention is to provide a new method of patterning multiple antibody types in discrete groups in precise locations.
These and other objects of this invention are achieved by providing a serial process for producing a multiple antibody patterned substrate by (1) coating an antibody-adsorbent substrate with an antibody-resistant material, (2) removing a portion of the antibody-resistant material by mechanical scribing to produce a bare site on the antibody-adsorbent substrate having a precise size, shape, and location on the substrate, (3) adsorbing molecules of a selected antibody on to the bare site on the antibody-adsorbent substrate, (4) rinsing the substrate to remove unadsorbed antibody molecules, (5) coating the antibody-adsorbent substrate with more of the antibody-resistant material to cover the bare surface of the substrate between the newly adsorbed antibody molecules, and (6) repeating steps (2) through (5) until each of the antibodies has been adsorbed at its specific site on the antibody-adsorbent substrate.
Alternatively, the multiple antibody patterned substrate is produced by a parallel process of (1) coating an antibody-adsorbent substrate with an antibody-resistant material that is resistant to the adsorption of antibodies, (2) simultaneously removing portions of the antibody-resistant material by mechanical scribing to produce bare sites on the antibody-adsorbent substrate having precise sizes and shapes and each site having a precise location which corresponds to a specific antibody, (3) adsorbing molecules of each antibody to its specific bare site on the antibody-adsorbent substrate, (4) rinsing the substrate to remove unadsorbed antibodies, and (5) coating the antibody-adsorbent substrate with more of the antibody-resistant material to cover the bare surface of the substrate between the adsorbed antibody molecules.
Another aspect of this invention is a biosensing device having (A) an analyte-capturing structure comprising (1) an antibody-adsorbent substrate, (2) two or more antibodies adsorbed to the substrate, wherein each antibody is located at a specific site on the substrate apart from the other antibodies, and (3) an antibody-resistant material covering the substrate between the adsorbed molecules of the antibodies for immobilization thereof at discrete locations, and (B) means for determining the types and quantities of the analytes captured by the antibodies.


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