One-step all-in-one dry reagent immunoassay

Details One-step all-in-one dry reagent immunoassay One-step all-in-one dry reagent immunoassay

1996-04-08

2002-08-06

Graser, Jennifer (Department: 1645)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

C436S169000, C436S546000, C436S800000, C436S531000, C436S532000, C436S809000, C435S004000, C435S007920, C435S007940, C435S810000, C435S962000, C435S287200, C435S287900, C435S969000

Reexamination Certificate

active

06429026

ABSTRACT:

This invention relates to a novel immunoassay in which all the necessary assay specific components are beforehand added to the reaction well and dried before the sample is added. The invention further relates to a device useful in carrying out said assay.
BACKGROUND OF THE INVENTION
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference. Lanthanide chelate labels and time-resolved fluorometry were introduced in the field of immunoassays already more than 10 years ago (Siitari et al. Nature 301, 258-60, 1983). The availability of monoclonal antibodies in combination with the high specific activity of the label enabled non-competitive immunoassays with superior sensitivity and dynamic range to be carried out (Ekins and Dakubu, Pure Appl Chem 57,473-82, 1985, Lövgren and Petterssson in: Van Dyke K and Van Dyke R eds. Luminescece Immunoassays and Molecular Applications. Boca Raton, U.S.A: CRC Press Inc., 233-50,1990). Several different label alternatives with high specific activities have since appeared (Kricka Clin Chem 40,347-57,1994, Price and Newman eds., Principles and Practice of Immunoassays. New York: Stockton Press, 650pp,1991, Dickson et al. Pharmac Ther 66,207-35,1995). Monoclonal antibodies have extensively replaced polyclonal antibodies and a non-competitive assay design is preferred whenever possible. Especially the usefulness of the new non-isotopic labels contributed to the present level of automation of field of immunoassays.
Alternative strategies can be selected to improve the sensitivity and reliability of immunoassays (Kricka Clin Chem 40,347-57,1994, Price and Newman eds., Principles and Practice of Immunoassays. New York: Stockton Press, 650pp,1991, Dickson et al. Pharmac Ther 66,207-35,1995, Ekins and Chu Clin Chem 37,1955-67,1991). As the potential sensitivity of a non-competitive immunoassay can be realized only by using labels with a high specific activity together with high-affinity antibodies (Ekins and Dakubu, Pure Appl Chem 57,473-82, 1985, Ekins and Chu Clin Chem 37,1955-67,1991) considerable effort is spent on the search for more sensitive detection technologies. Besides factors such as detector noice, interference in the sample causing a nonspecific signal, and nonspecific binding of the label reagent reduce the overall assay sensitivity (Ekins and Dakubu, Pure Appl Chem 57,473-82, 1985, Lövgren and Petterssson in: Van Dyke K and Van Dyke R eds. Luminescece Immunoassays and Molecular Applications. Boca Raton, U.S.A: CRC Press Inc., 233-50,1990). Other reasons to design novel immunoassay strategies are to improve the reliability by high quality key reagents and to simplify the assay by eliminating unnecessary steps to obtain more direct procedures.
The time-resolved fluorometry and lanthinide chelate label based immunoassay technology was originally based upon the dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) concept (Hemmilä et al. Anal Biochem 137,335-43,1984). The high specific activity of the label that could be covalently bound to monoclonal antibodies without any effects on the affinity and specificity (Lövgren and Petterssson in: Van Dyke K and Van Dyke R eds. Luminescece Immunoassays and Molecular Applications. Boca Raton, U.S.A: CRC Press Inc., 233-50,1990) proved to be very useful in the design of several non-competitive assays with improved sensitivity (Pettersson et al. Clin Chem 29,60-4,1983, Näntö et al. Isr J Clin Biochem Lab Sci 4,36,1985, Suonpää et al. Clin Chem Acta 145,341-8,1985, Hemmiläin:Winefordner ed. Chemical Analysis, Vol 117. Toronto: Wiley & Sons, 343pp,1991, Xu et al. Clin Chem 38,2038-43,1992). Furthermore competitive assays (Bertoft et al. FEBS Lett 173,313-6,1984, Eskola et al. Clin Chem 31,1731-4,1985, Näntö et al. Isr J Clin Biochem Lab Sci 4,52,1985, Lövgren Steroid Biochem 27,47-51,1987) with good performance chracteristics were carried out although the label does not essentially contribute to the sensitivity in this assay design (Ekins and Dakubu, Pure Appl Chem 57,473-82, 1985, Ekins and Chu Clin Chem 37,1955-67,1991).
The immunoassays carried out in a routine laboratory have been subject to extensive automation. The analyzers should operate in a fast random and continuous access mode. The throughput capacity should be similar to that of general chemistry analyzers and a large on-board supply of reagents should reduce the operators hands-on time. The automation requirements have turned out to be difficult for most label technologies and the performance characteristics of the analytes in the panels have suffered because of compromises that had to be made. In the present invention we describe a relatively fast immunoassasy technology suitable for automation in which all assays are performed in one-step procedures in microtitration wells containing all the assay specific reagents necessary for one test in dry form. An intrinsically fluorescent stable lanthanide chelate with a high specific activity is used as the label. The performance of the model assays has been tested in quantitative measurements biochemical cardiac markers in whole blood for the early detection of acute myocardial infarction (AMI).
Early confirmation or exclusion of AMI is essential for the correct treatment decision (Rozenman Y et al., Annu Rev Med 1994;45:31-44 and L Kristein Newby et al., Clin Chem 1995;41:1263-1265). Because half of all AMI patients have nondiagnostic ECGs at the time of admission, biochemical markers are often required to confirm diagnosis. Unfortunately laboratory results on biochemical markers are usually not available until several hours after the first diagnosis and eventual admission of the patient. More than 50% of the deaths occur within the first 2 hours after the onset of symptoms. In addition, the availability of thrombolytic therapy and the clear benefit of early intervention emphasize the need for rapid diagnosis. Optimally results on biochemical markers should be available simultaneously with the ECG data. Still the sensitivity of the creatine kinase MB isoenzyme (CK-MB) activity measurement, the most accepted protein marker, is too low to accurately rule in or rule out myocardial injury during these early hours.
Myoglobin (Mb) is a 17 800 kDa heme protein present in cardiac and skeletal muscle. Due to its low molecular mass, Mb is realesed early (2-4 h) from the injured cardiac muscle, and transferred to the serum. The fast rise of Mb in patients with AMI makes it an ideal marker for early confirmation or exclusion of AMI and for monitoring cardiac reperfusion (Ohman EM et al., Br Heart J 1990;63:335-338). However, the lack of rapid availability of quantitative results has limited its clinical application.
The optimal advantage of Mb and other biochemical cardiac marker would be gained with quantitative assays with a turn-around time short enough to offer results simultaneously with the ECG to be analyzed by the clinician. One of the rate-limiting steps in the assays of most early biochemical markers includes manual separation of plasma or serum from whole blood. The possibility to use a direct assay on whole blood samples would shorten the total time from the blood collection to the interpretation of the result notably. Our aim was therefore to optimize a rapid time-resolved immunofluorometric assay suitable for simple automatic processing of whole blood samples to yield highly reproducible quantitative results on Mb and other biochemical cardiac markers in order to make results available for early diagnosis of AMI.
SUMMARY OF THE INVENTION
An immunoassay has been invented in which all assay specific components are beforehand added to the reaction well and dried before the well is used in immunoassays to quantitatively measure analytes in e.g. serum, plasma or whole blood samples. According to the invention biochemical markers, especially biochemical cardiac markers, can be measured directly from whole blood samples for the early detection or

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