Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1995-02-06
2001-07-24
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S007940, C435S007950, C435S970000, C435S975000, C436S518000, C436S531000, C436S809000, C422S051000, C422S051000, C427S002150, C427S002110
Reexamination Certificate
active
06265176
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the detection of components of the antigen-antibody reaction by solid phase immunoassay techniques, more particularly to enzyme immunoassay.
Many immunoassay procedures for detecting antigens, antibodies and haptens in body fluids are known in the immunoassay art. Radio-immunoassay techniques are numerous and have been shown to be highly sensitive. Numerous enzyme-immunoassay techniques are also known, including competitive, double antibody solid phase (“DASP”) and sandwich procedures. Both classes of solid phase immunoassays have been performed using various solid supports, including finely divided cellulose, solid beads or discs, polystyrene tubes and microtiter plates.
Enzyme immunoassays have included color formation as an indicator of a result. Degree of color formation has been used for quantitative determinations. Colorimeters have been used for automatic quantitative determinations based on color gradations of analytical solutions.
Dot enzyme-linked immunosorbent assays using nitrocellulose filters as the solid phase have recently been described by Hawkes et al., “A Dot-Immunobinding Assay for Mono-clonal and Other Antibodies,” Anal. Biochem. 119, 142-147 (1982). Hawkes et al. described putting antigen spots on nitrocellulose filters, cutting out areas containing spots, and either placing the cut-out portions in the wells of microtiter plates for enzyme immunoassasy or, where a range of different antigens are to be screened, using nitrocellulose strips. Similarly, Pappas et al., “Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA): a Micro Technique for the Rapid Diagnosis of Visceral Leishmaniasis,” J. Imm. Meth. 64, 205-214 (1983), have disclosed a dot assay for parasites. Both Hawkes et al. and Pappas et al. asserted advantages of nitrocellulose dot (or spot) assays, including not only a reduced need for bound reagent, but also the almost-whiteness of nitrocellulose as a background for color reading. Pappas et al. also stressed the improved binding of bound reagent to nitrocellulose as compared to microtiter plate wells. Hawkes et al. disclosed a quantitative procedure in which reflectance of spots was determined by a thin layer scanner.
Also known in the art are pregnancy tests which utilize dipsticks coated or partially coated with bound antibody to HCG, such as the PREGNASTICK pregnancy test kit sold by Monoclonal Antibodies, Inc. Enzyme immunoassay procedures are used to change the color of the coated portion of the dipstick.
The myriad assays in the prior art, while providing in some instances very high sensitivity, suffer from a variety of drawbacks. Radio-immunoassay procedures require the handling of radioactive materials, as well as their disposal, and expensive equipment. Many enzyme-immunoassay procedures produce soluble color solutions, are used with colorimeters, and require incubation periods longer than desired. Enzyme immunoassay pregnancy tests utilize single-use kits, which are thrown away after a single use. The nitrocellulose-based dot tests utilize a solid support which may absorb reactants, which can lead to background color and reduced sensitivity. Further, nitrocellulose is fragile, making it difficult to handle.
It is a principal object of this invention to provide an improved immunoassay kit and protocol which eliminates drawbacks, discussed above, of the prior art and is capable of achieving an immunoassay which is sensitive, fast, inexpensive, which does not require expensive and delicate instruments, which is usable for multiple assays, and which can be made for a qualitative or a quantitative test. This and other objects of this invention will be better understood by reference to the description which follows.
SUMMARY OF THE INVENTION
We have developed a new dot test for assaying body fluids for components (antigens, antibodies, haptens) of the antigen-antibody reaction employing an enzyme-immunoassay technique, and a kit of reagents for carrying out multiple assays. The kit includes a white opaque plastic card having identifiable small spots or dots containing thereon insolubilized binding partner of the component to be determined. The plastic card contains a preselected number of spots and may be cut into strips so that one may use only the necessary number of spots, saving the remainder for other assays. Other reagents include an enzyme conjugate, comprising a color-promoting enzyme bound to a binding partner of the component to be determined; a substrate capable of reacting with the bound enzyme to produce an insoluble colored product; and preferably control samples, specimen diluent and wash solution. The kit may include all necessary laboratory ware to perform assays. Assay instructions are included in the kit.
The spots on the card may all be one insolubilized reagent or they may be more than one reagent for testing for multiple unknowns. The latter case is particularly suited for assaying for antibodies. In that event the enzyme conjugate comprises an enzyme bound to an antibody to the class of antibodies being sought, such as, for example anti-human IgG, so that only one conjugate is required for the entire series of tests.
In the assay procedure, a small drop of body fluid which may contain the component to be determined is placed on a spot on the card containing insolubilized binding partner of that component. After a short incubation at room temperature, the spot is preferably washed and blotted dry. Next a small drop of enzyme conjugate capable of binding to the component to be determined is added to the spot. Following a short incubation at room temperature, the spot is washed to remove unbound conjugate and blotted dry. Next, a small drop of substrate is added to the spot. The substrate comprises a material which reacts with the enzyme to produce in a short time and at room temperature an insoluble colored product. The intensity of the color is proportional to the sample's concentration of component to be determined and can easily be read by the naked eye without the aid of instrumentation. No color is developed if the test sample contains none of the component to be determined.
The invention includes a process for the rapid and simple determination of the presence in a test sample of body fluid of a component to be determined of the antigen-antibody reaction, comprising:
a. applying a drop comprising concentrated test sample diluted 1:10 or less to an identifiable spot of about 1 cm diameter on a flat, opaque, inert plastic carrier, said spot comprising insolubilized first binding partner of the component to be determined;
b. incubating at room temperature for up to one-half hour;
c. removing unbound body fluid;
d. applying a drop of concentrate conjugate comprising an enzyme coupled to a second binding partner of the component to be determined;
e. incubating at room temperature for up to one-half hour;
f. washing;
g. applying a drop of enzyme substrate comprising a material capable of reacting with the enzyme at room temperature to form in a few minutes a colored deposit readable by the naked eye against the background of the opaque plastic carrier.
REFERENCES:
patent: 4376110 (1983-03-01), David et al.
patent: 4474878 (1984-10-01), Halbert et al.
patent: 4496654 (1985-01-01), Katz et al.
patent: 4594225 (1986-06-01), Arai et al.
patent: 5126276 (1992-06-01), Fish et al.
patent: 0063810 (1982-11-01), None
patent: 0125118 (1984-11-01), None
patent: 0130434 (1985-01-01), None
H. Towbin et al, “Immunoblotting and Dot Immunobinding—Current Status and Outlook,”J. of Immunol. Methods72(2):313-340, 1984.*
Shamberger, R., “Elisa on the Trail of Viral Diseases,”Diagnostic Medicine, Oct. 1984, pp 52-57.*
Pappas et al “Dot Enzyme-Linked Immunosorbent Assay (Dot-Elisa): a MicroTechnique for the Rapid Diagnosis of Visceral Leishmaniasis,”J. of Immun Meth, 64 (1983) 205-214.*
Grant & Hackh'sChemical Dictionary, Fifth Edition, (McGraw-Hill Book Company New York), 1987, p. 3.*
Pappas et al,Journal of Immunological Methods, 64 (1983) 205-214.*
Shamberger, R.,Diagnostic Med
Halbert Seymour P.
Lin Tsue-Ming
Cordis Corporation
Fish & Richardson PC
Nguyen Bao-Thuy L.
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