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United States Patent (19) 11 Patent Number: 5,066,582 Tsuruta Et Al

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United States Patent (19) 11 Patent Number: 5,066,582 Tsuruta Et Al United States Patent (19) 11 Patent Number: 5,066,582 Tsuruta et al. (45) Date of Patent: Nov. 19, 1991 54 METHOD AND APPARATUS FOR THE mination of Human Chorionic Gonadotropin' & Anal. MEASUREMENT OF ANALYTE Lett. 1982 5(B2), 101-113. SUBSTANCES Chemical Abstracts, vol. 100, 1984, p. 270 abstract No. 75 Inventors: Hitoshi Tsuruta; Hideaki Yamada, 153448k, Columbus, Ohio, U.S., & JP-A-59 28 648 both of Kurashiki, Michihiro Kuraray Co. Ltd.) 15-02-84. Nakamura, Soja, all of Japan Chemical Abstracts, vol. 90, 1979, p. 244, abstract No. 164334w, Columbus, Ohio, U.S., & JP-A-78 149 392 73 Assignee: Kuraray Co., Ltd., Kurashiki, Japan (Kuraray Co., Ltd.) 26-12-1978. 21 Appl. No.: 311,779 Chemical Abstracts, vol. 103, 1985, p. 394, abstract No. 209457u, Columbus, Ohio, U.S.; R. R. Premier et al.: (22) Filed: Feb. 17, 1989 “An Evaluation of the Use of a pH Indicator for the 30 Foreign Application Priority Data Detection of Beta-Lactamase in Enzyme Immunoas Feb. 19, 1988 JP Japan .................................. 63-38274 say", & J. Immunol. Methods 1985, 83(2) 371-377. Boitieux, J-L, and Thomas, D; Clinical Biochemistry, (51 int. Cl. ........................................... G01N 33/543 17, 151-156. 52 U.S. C. ..................................... 435/7.1; 436/163; 204/403; 204/433; 422/56 Primary Examiner-David L. Lacey 58) Field of Search ..................... 436/163; 422/82.03, Assistant Examiner-Thomas E. Daley 422/82.04, 56-58; 204/403, 433; 435/7.1 Attorney, Agent, or Firm-Oblon, Spivak, McClelland, (56) References Cited Maier & Neustadt U.S. PATENT DOCUMENTS 57 ABSTRACT 4,318,709 3/1982 Fab ....................................... 23/230 A method for measuring trace amounts of analyte sub 4,73,165 12/1987 Conover .............................. 2O4/403 stance(s), which utilize a pH electrode is disclosed. This method is by far simpler than conventional optical de FOREIGN PATENT DOCUMENTS tecting systems. The apparatus for practicing the 0170375 6/1985 European Pat. Off. method is also disclosed. By the use of pH electrode, the O328380 10/1988 European Pat. Off. apparatus is compact, inexpensive and easy to operate, OTHER PUBLICATIONS and hence usable in small-size and medium-size hospi Chemical Abstracts, vol. 96, 1982 p. 96, abstract No. tals or clinical laboratories and by patient's bedside. 155710n, Columbus, Ohio, U.S.; M. Mascini et al.: "pH Eiectrode-Based Enzyme Immunoassay for the Deter 6 Claims, 8 Drawing Sheets -daV/dt (m V/SEC) -AW (mW) 60 40 20 1.2 00 .0 80 0.8 60 0.6 40 0.4 20 0.2 4. 6 8 TIME (MINUTE) U.S. Patent Nov. 19, 1991 Sheet 1 of 8 5,066,582 F. G. 4. F. G.2 U.S. Patent Nov. 19, 1991 Sheet 2 of 8 5,066,582 FG3 F G4 F. G.5 U.S. Patent Nov. 19, 1991 Sheet 3 of 8 5,066,582 U.S. Patent Nov. 19, 1991 Sheet 4 of 8 5,066,582 CN C. 3 S : 3 S. s S Al-Al-Aswar aZAZAZeze-de-SSa C Q U.S. Patent Nov. 19, 1991 Sheet 5 of 8 5,066,582 Ss3 3 É s O O C. e. Sp S O E se1 a U.S. Patent Nov. 19, 1991 Sheet 6 of 8 5,066,582 O C O t CN L SY S. S. 5 cd us 2. O an Cld - No 3, CD s L- CO L U.S. Patent Nov. 19, 1991 Sheet 7 of 8 5,066,582 S.3s c S S. E Na1 Dg U.S. Patent Nov. 19, 1991 Sheet 8 of 8 5,066,582 O 9. O No E s S CO El O Li L- C CN L SN O O es N5O c\O S. O arts DC S.E- N3S2 Sl 5,066,582 1. 2 an enzyme. After being washed, the solid phase is con METHOD AND APPARATUS FOR THE tacted with a substrate solution for the above-men MEASUREMENT OF ANALYTE SUBSTANCES tioned enzyme used as the label and, if necessary, a chromophore, whereby the optical property of the sys BACKGROUND OF THE INVENTION 5 tem is changed with the proceeding of the decomposi i. Field of the Invention tion reaction of the substrate, which change is then The present invention relates to a method for measur observed. ing analyte substance(s) and the apparatus therefor, and Several methods have been used for the purpose of in particular to a method and apparatus suited for the observing the change of the optical property of the quantitative immunoassay of specific substances con 10 substrate solution. Among them, there are methods tained in trace amounts in a multi-component solution utilizing optical instruments such as optical absorption such as specimens from biological sources and the appa instruments fluorophotometer, and chemiluminescence ratus therefor. While the present invention will herein photometer (see for example Ishikawa, Kawai and below be illustrated only for measurement of biosub Miyai, ENZYME IMMUNOASSAYS, 1982, published stances in clinical examination, the method and appara 15 from Igaku Shoin, Tokyo). tus of the present invention are applicable to a wide There is also a method which comprises comparing a variety of fields such as pharmacy, biology, zoology, substrate solution with a control solution and observing botany, agriculture and chemistry. visually the difference in the color to judge the presence 2. Description of the Prior Art of a trace amount of an analyte substance (see for exam Substances participating in bioactivity of a living 20 ple Japanese Patent Application Laid-Open No. body are generally present in trace amounts, and still 128369/1985). many of them play very important roles for the living The above-mentioned optical measuring systems uti body. The quantitative determination of such bioactive lizing optical instruments, however, require expensive substances therefore is important in fields associated and large and complex equipment because they gener with living organisms, such as medical science and bio 25 ally need a stable light source, a photometer having chemistry. Various methods for that purpose have been high sensitivity, a photomultiplier circuit with high proposed and put to practical use, among which a deter mination method of analytereceptor system utilizing precision, and the like. Further, specialized technicians enzymes, radioactive isotopes, chemiluminescent sub must attend to the measurement since special technique stances or the like as a label has been widely used. The 30 is required for the measurement. analyte-receptor-type measurement comprises first con On the other hand, the direct visual observation tacting a solid phase having fixed a first receptor which method is a qualitative one. It is liable to personal varia can specifically bind an analyte which is the substance tion in judgement of color change and to the observer's to be determined, with a sample solution and a labelled subject. Further, judgement itself is sometimes difficult second receptor or a labelled analyte (hereinafter these 35 for measurement of an extreme trace substance where labelled substances are referred to as "the conjugates') the color change is very small. simultaneously or successively, thereby effecting an SUMMARY OF THE INVENTION analyte-receptor reaction, washing the reaction mix ture, and thereafter determining the amount of the con Accordingly, an object of the present invention is to jugates remaining on the solid phase to measure the provide a method for the measurement of an analyte amount of the analyte in the sample solution. In this case substance(s) in a sample by measuring the decomposi substances having a large amplification effect such as tion reaction of a substrate solution objectively and radioisotopes and enzymes are used as the label. As with high detection accuracy, with the method being regards the receptor, for an antigen or a hapten, an free from the indefiniteness of judgement which de antibody which can specifically bind it is used; for an 45 pends on the observer's subject; and the apparatus antibody, its antigen is used; for a DNA or RNA, an therefor. other DNA or RNA complementary thereto is used; Another object of the present invention is to provide and for a ligand, its receptor is used. The heterogeneous a method for the measurement of an analyte substan EIA, so-called Enzyme Linked Immuno Sorbent Assay ce(s), which has a measurement system which is far (ELISA) is known as a representative example of such 50 simpler from the optical measurement systems, and the determination. apparatus therefor. In ELISA, a solid phase obtained by fixing a receptor Still another object of the present invention is to capable of specifically binding the analyte to be deter provide an apparatus for measuring analyte substances, mined, on a test tube, microplate or the like is used for which can be operated by an untrained operator or capturing the substance to be determined in the sample 55 which is suited for use, when a quick decision is re solution. An enzyme is used as the label for signal ampli quired for diagnosis, by a person in charge of medical fication. For example where the analyte to be deter care or of other medical field. mined is an antigen, the sandwich-ELISA uses an en Yet another object of the present invention is to pro zyme-labelled second antibody (a second receptor) vide a compact and inexpensive apparatus for measur bindable to the antigen, and the competitive-ELISA ing analyte substances, which can be used in hospitals of uses an enzyme-labelled antigen as the conjugate. On small or medium size, clinical laboratories or at a pa the other hand, where the analyte to be determined is an tient's bed side.
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