US3904367A - Contrast enhancement for immunological film detection - Google Patents

Contrast enhancement for immunological film detection Download PDF

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Publication number
US3904367A
US3904367A US388407A US38840773A US3904367A US 3904367 A US3904367 A US 3904367A US 388407 A US388407 A US 388407A US 38840773 A US38840773 A US 38840773A US 3904367 A US3904367 A US 3904367A
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United States
Prior art keywords
protein
layer
slide
antibody
antibodies
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Expired - Lifetime
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US388407A
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English (en)
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David C Golibersuch
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General Electric Co
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General Electric Co
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Application filed by General Electric Co filed Critical General Electric Co
Priority to US388407A priority Critical patent/US3904367A/en
Priority to US440712A priority patent/US3904362A/en
Priority to GB2266274A priority patent/GB1475125A/en
Priority to BR574474A priority patent/BR7405744D0/pt
Priority to DE2436010A priority patent/DE2436010A1/de
Priority to CA207,071A priority patent/CA1033294A/en
Priority to FR7428191A priority patent/FR2241073B1/fr
Priority to CH1109074A priority patent/CH601797A5/de
Priority to JP49092429A priority patent/JPS6029068B2/ja
Priority to IT2630374A priority patent/IT1025035B/it
Priority to NLAANVRAGE7410914,A priority patent/NL179003C/xx
Priority to SE7410388A priority patent/SE452066B/xx
Priority to DK436474A priority patent/DK436474A/da
Priority to AT672374A priority patent/AT331975B/de
Priority to AU72513/74A priority patent/AU490033B2/en
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Publication of US3904367A publication Critical patent/US3904367A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/26—Accessories or devices or components used for biocidal treatment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/82—Hepatitis associated antigens and antibodies

Definitions

  • This technique is used in accordance with one embodiment to build a plurality of successive antigen-antibody films on a substrate to provide for contrast between multilayer protein films and a monomolecular layer which is in excess of the contrast obtainable between monomolecular layers and bimolecular layers to thereby provide diagnostic apparatus of increased sensitivity.
  • This invention relates to the immunological detection of protein by detection of immunological complexing between a protein previously absorbed on a surface to form a test slide and the specifically immunologically reactive protein thereto. More particularly, this invention relates to the improvement of contrast observable on such immunologic diagnostic slides by the selective immunologic bonding thereto of additional protein layers.
  • the slide after immersion has only the original monomolecular protein layer on the substrate.
  • Optical, electrical, and chemical means for distinguishing between a bimolecular protein layer and a monomolecular protein layer are taught in these related copending applications of Giaever.
  • diagnostic apparatus In two embodiments of diagnostic apparatus taught in these two related copending applications of Giaever, optical distinction between monomolecular and bimolecular protein layers is made by unaided visual observation of the slide and is further taught therein to pro vide detection sensitivities which are functions of the structure of the slide and of the absolute thickness of the protein layer or layers thereon.
  • the diagnostic slide taught by Giaever to comprise a plurality of metal globules attached to a substrate and having a monomolecular protein layer thereon is taught tc have maximum sensitivity for films thinner than 200 A
  • the embodiment taught by Giaever to comprise a gold surface substrate is taught to have maximum sensitivity for films exeeding A in thickness.
  • the chemical detection embodiment taught by Giaever involves the use of a substrate having a surface of a material which forms a visible amalgam with mercury and depends for its operation on Giaevers discovery that a mercury drop placed atop a bimolecular protein layer requires approximately ten times as long a time to diffuse therethrough as a mercury drop placed atop a monomolecular protein layer.
  • the electrical detection embodiment taught by Giaever involves the use of an electri cally conductive substrate material and the placing of an electrode atop the protein layer thereon; the capacitance between the two conductive members is then measured and its value is an indication of the thickness of the dielectric protein film separating them and hence, whether such film is monomolecular or biomoleeular. It may, therefore, be seen that the electrical and chemical embodiments taught by Giaever require that the protein to be detected, if present in the solution under test,.form a complete second monomolecular layer on the substrate of the diagnostic slide.
  • the techniques and apparatus taught by Giaever provide for rapid, reliable, and inexpensive detection of immunologically reactive particles.
  • the immunologically reactive particles must exceed approximately 20 A in diameter. More significantly, some particles, whose detection is highly significant, are present in physiologic specimens only in highly dilute concentrations. In this case, only a small plurality of such particles become bonded to the diagnostic slide and their detectability is limited.
  • test slide includes a layer of an etehable metal underlying a layer of, for example, antibodies to the virus to be detected.- A thin layer of non-etchable metal is depos ited over the slide and any viruses bonded thereto. After etching, sites at which any viruses have been bonded appear as voids in the slide of sufficient size to be detected by, for example, optical microscopy.
  • antibody molecules function as antigens when introduced into the system of a vertebrate to whom they are foreign proteins. Accordingly, specifically reacting antibodies to a given antibody may be readily produced.
  • This capability has been utilized in the prior art to provide for attachment .of fluorescent molecules to immunologically complexed proteins to aid in the detection of par ticular species of microorganisms.
  • antibodies to human immunoglobulins may be prepared and, chemically combined with flourescent organic molecules such as the isothiocyanates. This socalled tagged antibody is then used to render an immunological reaction visible by ultraviolet microscopy.
  • Trcponema pallidum Trcponema pallidum
  • FTA-STS procedure a quantity of Trcponema pallidum (T. pallidum) is dried on a slide. The slide is then immersed in a blood specimen. The slide is subsequently immersed in a solution of tagged immunoglobulin. The anti-human immunoglobulin does not bond to the T. pallidum; accordingly, only if the specimen contained antibodies to T. pallidum will the slide fluoresce when observed by ultraviolet microscopy.
  • a further object of this invention is to improve such detection sensitivity by building multimolecular layers of specifically reacting proteins on such slides.
  • an immunologic test slide having as one surface thereof a monomolecular layer of protein which is immunologically reactive with the protein to be detached is immersed in a solution suspected of containing the protein to be detected.
  • the substrate After immersion in the suspected solution, the substrate has a bimolecular protein layer thereon consisting of the original monomolecular protein layer and overlying monomolecular layer of the protein to be detected if the protein to be detected was present in the suspected solution.
  • the slide after immersion therein has only the original monomolecular protein layer thereon. The slide is then immersed in a solution of protein which reacts immunologically with the protein of the second monomolecular layer.
  • the slide then has a trimolecular protein layer thereon if the suspected solution contained the protein of interest or a monomolecular layer thereon if the suspected solution did not contain the protein of interest. Accordingly, detection sensitivity of the slide is improved because of the increased contrast between a monomolecular and trimolecular protein layer as opposed to that between a monomolecular and bimolccular pr tcin layer.
  • FIG. 1 is a schematic illustration of the attachment of a plurality of protein layers to a substrate.
  • FIG. 2 is an immunologic test cross-sectional elevation view of a slide having three monomolecular protein layers thereon.
  • FIG. 3 is a sectional elevation view of a slide prepared in accordance with one embodiment of this invention illustrating the completion of a monomolecular layer on a slide.
  • FIG. 1 shows a substrate having adsorbed thereon a plurality of arbitrary protein molecules or other particles, such as, for example, viruses or hepatitis associated antigen particles 11 in accordance with the teachings of the aforementioned copending applications of Giaever.
  • a plurality of molecules or-other particles 11 are immunologically complexed with their specifically reacting antibody molecules indicated generally at 21 and 22.
  • Antibody molecules 21 and 22 are illustrated schematically to facilitate an explanation of the structure thereof.
  • the initial particle layer is referred to hereinafter as molecules", it being understood that other particles are also included in the scope of this invention.
  • the five major classes of antibodies comprise the immunoglobulins designated as IgG, IgM, IgA, IgD, and IgE.
  • the preponderant immunoglobulin is IgG, which constitutes approximately percent of the immunoglobulins in normal human sera and is found in roughly similar preponderance in the normal sera of other vertebrates.
  • An IgG particle comprises two heavy peptide chains and two light peptide chains joined by disulphide bonds to form a generally Y shaped antibody molecule.
  • IgG antibody molecules are immunologically divalent; that is to say, each molecule has two antibody active sites.
  • the active sites 2101 and 2 I112 of molecule 2] and 22111 and 2202 of molecule 22 are located at the extreme ends of the arms of the Y-shaped molecule.
  • the active sites provide for immunologic bonding of the antibody to a molecule of its associated antigen, as shown, for example, in FIG. I where active sites 21al, 22:11, and 22112 are bonded to a plurality of protein molecules 1 I.
  • antibody molecules also function as antigens when introduced into a vertebrate system to which they are a foreign protein. They accordingly stimulate the production of antibodies specific to the anitbody qua antigen.
  • An antibody to IgG antibody is another IgG antibody, and accordingly also has two active sites. These active sites bond to particular antigenically active sites on the first antibody.
  • the antigenically active sites are known alternatively as antigenic determinants or epitopes.
  • the number of antibody active sites of an IgG antibody is known to be 2.
  • the number of epitopes associated with an IgG molecule is not precisely known but the number of epitopes is known to be at least 2.
  • Epitopes are known to be present on the tail of the Y- shaped molecule and are probably also present on the arms thereof.
  • three epitopes are shown disposed along the heavy peptide chains of each antibody molecule illustrated in FIG. 1.
  • the epitopes of antibody molecule 21 are designated as 21: 1, 2Ie2, and 2le3.
  • the epitopes of antibody molecule 22 are designated as 22e1, 2262, and 22e3.
  • the adsorption of molecules 11 onto substrate 10 and the immersion of the resulting immunologic test slide into a solution of antibodies to protein 11 to immunologically bond molecules 21 and 22 thereto constitutes the practice of the aforementioned inventions of Giaever.
  • a quantity of solution of antibodies of type represented by 21 and 22 is injected into a vertebrate to stimulate the production of antibodies thereto.
  • the antibodies produced are collected by means known in the art, and a solution thereof is provided for use in the improved diagnostic procedure in accordance with this invention.
  • the slide is immersed in a solution of the anti-antibodies and if molecules 21 and 22 are bonded to molecules 11, a plurality of molecules 31, 32, 33, 34., and 35 become immunologically bonded to molecules 21 and 22.
  • the detectability of molecules 21 and 22 is improved in that their presence is determined by distinguishing between a layer of molecules 1 1 and a layer of molecules 1 1, 21., 22, 31, 32, 33, 34, and 35 as opposed to distinguishing between a layer of molecules 11 and a layer of molecules 11, 21, and 22.
  • the process may be repeated to add additional protein layers to further increase detection sensitivity, as for example, the selective bonding of molecules 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53 to molecules 31, 32, 33, 34, and 35.
  • Antibody molecules 31, 32, 3.3, 34, and 35 have respectively active sites 31:11, 31a2, 32(11, 32:12, 33u1,
  • Antibody 31 is immunologically bonded to antibody 21 by the attachment of active site 31112 to epitope 2le2.
  • Antibody 32 is immunologically bonded to antibody 21 by the attachment of active site 32611 to epitope 21a 1.
  • Antibody 33 is immunologically bonded to anitbody 22 by the at tachment of active site 3302 to epitope 2202;
  • antibody 34 is bonded to antibody 22 by the attachment of active site 34al to epitope 22el;
  • antibody 35 is bonded to antibody 32 by the attachment of active site 35111 to epitope 22e3.
  • each antibody is shown as having two active epitopes.
  • each antibody qua antigen binds two antibodies thereto.
  • antibody 21 has antibodies 31 and 32 bonded thereto which in turn have antibodies 41, 42, 43, and 44 bonded thereto. This corresponds to the known minimum number of active epitopes per IgG antibody molecule.
  • the protein chain beginning with antibody 22 is illustrated as having three active epitopes per antibody.
  • the sensitivity improvement obtained in accordance with this invention is proportional to the number of active epitopes per antibody molecule and the number of immersions of the slide into successive antibody solutions.
  • a layer of bovine serum albumin was adsorbed on a substrate.
  • the resulting slide was then immersed in a solution of rabbit anti-serum to bovine serum albumin, after which the slide contained the original bovine serum albumin layer having immunologically bonded thereto a layer of rabbit IgG immunoglobulin antibodes to bovine serum albumin.
  • the slide was next immersed in a solution of goat anti-serum to rabbit IgG, after which the slide contained a layer of bovine serum albumin, a layer of rabbit anti-BSA IgG, and a layer of goat anti-rabbit IgG antibodies.
  • the next step was the immersion of the slide in a solution of rabbit anti-serum to goat IgG to add a fourth layer of rabbit anti-goat IgG molecules to the slide.
  • the last two steps of goat antisc1um to rabbit IgG, followed by rabbit anti-serum to goat lgG may be repeated serially for as many operations as desired to thereby build an n-molecular layer protein film.
  • hepatitis associated antigen particles were adsorbed on a substrate.
  • the resulting slide was then immersed in a human serum specimen.
  • the slide was next immersed in a solution of rabbit antibodies to human hepatitis antibody. Accordingly, the slide, if the human test serum contained hepatitis antibody, has thereon hepatitis associated antigen having hepatitis antibody immunologically bonded thereto and rabbit anti-human hepatitis antibody immunologically bonded to the hepatitis antibody.
  • the serum sample were negative, the slide would have only the original hepatitis associated antigen particles thereon.
  • This technique in accordance with this invention was found to significantly improve the detectability of human hepatitis over that obtainable by the bonding of hepatitis antibody to hepatitis associated antigen alone.
  • This example points out that the technique of this invention has particular utility in cases in which the initial protein including particle deposited on the slide is substantially larger than its specifically reacting protein.
  • FIG. 2 is a sectional elevation view illustrating a multimolecular protein film on a slide.
  • Substrate 60 may, for example, in accordance with the first example cited above, initially have a monomolecular layer 61 of bovine serum albumin adsorbed thereon to constitute an immunologic diagnostic slide. Immersion in rabbit antiserum to bovine serum albumin will adhere a second monomolecular layer 62 over layer 61 by specific immunologic reaction. Subsequent immersion in goat anti-serum to rabbit IgG will immunologically bond monomolecular layer 63 to layer 62 if layer 62 is present. Similarly, layer 64 comprising rabbit anti-goat IgG is bonded to the slide by immersion in rabbit anti-scrum to goat IgG.
  • the process may be continued but since the bonding of layers is a specific immunologic reaction, the process will be irrevocably interrupted if any one layer does not form.
  • the structure of FIG. 2 has utility for high sensitivity de tection of the protein comprising layer 62.
  • Layer 61 is attached to slide 60 by adsorption which is a non specific process. All subsequent processes, however, are specific. Accordingly, if a test specimen contains the specifically reacting protein to the protein of layer 6]., layer 62 forms and an n-molccular protein layer can be formed thereover in accordance with this invention. If, on the other hand, the test specimen does not contain protein immunologically reactive with the protein of layer 61, layer 62 does not form and no subsequent layers can be formed. Therefore, a protein of interest in a specimen may be detected with any desired degree of sensitivity by distinguishing between a monomolecular protein layer on a substrate and a layer of any de sired thickness.
  • FIG. 3 is a crosssectional elevation view ofa slide illustrating another utility for the technique of this invention.
  • FIG. 3 illustrates the utility of this invention in the detection of proteins which are present in physiologic specimens in concentrations too dilute to provide for the formation of a complete layer on a slide.
  • a substrate has absorbed adsorbed a monomolecular layer of human hepatitis antibodies 71.
  • the resulting slide is immersed in a blood specimen and a plurality of hepatitis associated antigen particles 77 become immunologically bonded thereto if they are present in the specimen.
  • the slide is then immersed in a solution of monkey hepatitis antibody to bond a layer of molecules 72 thereof on such hepatitis associated antigen particles as may be bonded to the slide.
  • the slide is next immersed into a solution of rabbit anti-serum to monkey lgG to bond a layer of molecules '73 thereof over the monkey hepatitis anitbody molecules.
  • the layer of molecules 72 could be formed of human hepatitis antibody 71.
  • the next layer would have to be anti'human lgG molecules which would bond not only to those antibodies surrounding the hepatitis associated antigen particles 77 but also to the original layer of molecules 71 thereby defeating the purpose of the technique of this invention.
  • the next immersion of the slide may be, for example, into goat anti-serum to rab bit lgG to form a layer of molecules 74 thereof on the slide.
  • This process is repeated a number of times as indicated generally by the volume shown as 75 until the point is reached at which a last monomolccular protein layer 76 completely fills the spaces between particles 77 on the slide.
  • This embodiment of this invention provides for the formation of a protein layer of at least bimolecular thickness over the entirety of the slide and has particular utility to the chemical and electrical detection methods disclosed in the aforementioned copending applications of Giaever as discussed above.
  • a method for improving the detection sensitivity of immunologic diagnostic slides comprising a substrate having a layer of particles of a first protein adsorbed thereon for specifically reacting immunologically with a second protein, said second protein being the protein to be detected, said method comprising the steps of:
  • step (b) contacting said slide resulting from step (b) with said solution of antibodies.

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US388407A 1973-08-15 1973-08-15 Contrast enhancement for immunological film detection Expired - Lifetime US3904367A (en)

Priority Applications (15)

Application Number Priority Date Filing Date Title
US388407A US3904367A (en) 1973-08-15 1973-08-15 Contrast enhancement for immunological film detection
US440712A US3904362A (en) 1973-08-15 1974-02-08 Toothbrush sterilization holder and container
GB2266274A GB1475125A (en) 1973-08-15 1974-05-21 Immunological detection
BR574474A BR7405744D0 (pt) 1973-08-15 1974-07-11 Processo para aperfeicoamento da detectacao da sensibilidade de laminas de diagnostico imunologico
DE2436010A DE2436010A1 (de) 1973-08-15 1974-07-26 Kontrastverstaerkung fuer den nachweis von immunologischen filmen
JP49092429A JPS6029068B2 (ja) 1973-08-15 1974-08-14 蛋白質検出感度の改善法
FR7428191A FR2241073B1 (xx) 1973-08-15 1974-08-14
CH1109074A CH601797A5 (en) 1973-08-15 1974-08-14 Contrast strengthening for immunological film proofs
CA207,071A CA1033294A (en) 1973-08-15 1974-08-14 Contrast enhancement for immunological film detection
IT2630374A IT1025035B (it) 1973-08-15 1974-08-14 Metodo per l aumento di contrasto per la rivelazione di pel licole imminologiche
NLAANVRAGE7410914,A NL179003C (nl) 1973-08-15 1974-08-14 Werkwijze voor het verbeteren van de detectiegevoeligheid van immunologisch diagnostische plaatjes.
SE7410388A SE452066B (sv) 1973-08-15 1974-08-14 Sett att forbettra analyskensligheten vid immunologiska reaktioner pa ytor
DK436474A DK436474A (xx) 1973-08-15 1974-08-15
AT672374A AT331975B (de) 1973-08-15 1974-08-16 Verfahren zur verbesserung der nachweisempfindlichkeit von immunologischen diagnostischen proben
AU72513/74A AU490033B2 (en) 1974-08-20 Contrast enhancement for immunological film detection

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US388407A US3904367A (en) 1973-08-15 1973-08-15 Contrast enhancement for immunological film detection
US440712A US3904362A (en) 1973-08-15 1974-02-08 Toothbrush sterilization holder and container

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US440712A Expired - Lifetime US3904362A (en) 1973-08-15 1974-02-08 Toothbrush sterilization holder and container

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AT (1) AT331975B (xx)
DE (1) DE2436010A1 (xx)
FR (1) FR2241073B1 (xx)
GB (1) GB1475125A (xx)
NL (1) NL179003C (xx)

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US4034074A (en) * 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US4048298A (en) * 1975-02-25 1977-09-13 Rohm And Haas Company Solid phase double-antibody radioimmunoassay procedure
US4092408A (en) * 1975-08-28 1978-05-30 New England Nuclear Corporation Method for solid phase immunological assay of antigen
US4108972A (en) * 1974-03-15 1978-08-22 Dreyer William J Immunological reagent employing radioactive and other tracers
US4166844A (en) * 1977-04-18 1979-09-04 E. R. Squibb & Sons, Inc. Solid phase separation technique for use in radioimmunoassays
US4169138A (en) * 1974-05-29 1979-09-25 Pharmacia Diagnostics Ab Method for the detection of antibodies
US4172117A (en) * 1974-05-20 1979-10-23 Biotest-Serum-Institut Gmbh Method for the simultaneous measurement of antigens and their antibodies by solid-phase radioimmunoassay
US4189464A (en) * 1978-05-05 1980-02-19 Institute For Cancer Research Hepatitis B testing reagent and method
US4219335A (en) * 1978-09-18 1980-08-26 E. I. Du Pont De Nemours And Company Immunochemical testing using tagged reagents
US4222743A (en) * 1978-07-20 1980-09-16 Wang Wei Kung Method and apparatus for detecting biological particles by fluorescent stain
US4273756A (en) * 1978-07-28 1981-06-16 Abbott Laboratories Immunoassay for class specific antibodies
US4293538A (en) * 1979-04-20 1981-10-06 Merck & Co. Inc. Assay for hepatitis A antigen
US4328183A (en) * 1978-06-14 1982-05-04 Mt. Sinai School Of Medicine Of The City University Of New York Blood cell typing and compatibility test procedure
EP0051096A1 (en) * 1980-11-05 1982-05-12 Home Office Reference Laboratory, Inc. Method for the determination of antigens and antibodies using site-deactivating media
US4414324A (en) * 1979-05-21 1983-11-08 Bma Laboratory Services, Inc. Immunoassay method and apparatus
WO1985001354A1 (en) * 1983-09-15 1985-03-28 Immucor, Inc. Detecting an immunological reaction with activated red blood cells (erythrocytes)
US4612281A (en) * 1980-12-03 1986-09-16 Palo Alto Medical Foundation Research Institute Immunoassay for detecting immunoglobulins and test kit
US4912030A (en) * 1985-01-15 1990-03-27 Institute Of Cancer Research Viral isolates and their use in diagnosis
US5124172A (en) * 1989-04-28 1992-06-23 Alcan International Limited Thin film diagnostic device
DE19517789A1 (de) * 1995-05-15 1996-11-21 Inst Chemo Biosensorik Verfahren zum Nachweis von Antigenen mit einem Affinitätssensor
US5874216A (en) * 1996-02-23 1999-02-23 Ensys Environmental Products, Inc. Indirect label assay device for detecting small molecules and method of use thereof
US20080280310A1 (en) * 2007-05-09 2008-11-13 Louis Panagopoulos Testing for Blood Group Immunological Reaction Without the Use of Anti-Human Globulin

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GB1555142A (en) * 1975-08-14 1979-11-07 Sinai School Medicine Blood cell typing and compatibility test procedure
US4915219A (en) * 1988-10-24 1990-04-10 Anthony Ottimo Disinfecting toothbrush container
US5107987A (en) * 1988-11-18 1992-04-28 Palazzolo Frank J Toothbrush holder
EP0389301A3 (en) * 1989-03-24 1991-06-05 Biogenex Laboratories Reagent complex for immunoassay
US5566823A (en) * 1995-01-22 1996-10-22 Summers; Shirley F. Toothbrush holder
US5709301A (en) * 1996-11-01 1998-01-20 Couch; Robert Lincoln Painting implement keeper
US6135279A (en) * 1999-11-22 2000-10-24 Dryer; Richard Sanitizing toothbrush holder
US6360884B1 (en) 2000-07-31 2002-03-26 Robert James Smith Toothbrush storage container
US6601699B1 (en) 2001-05-24 2003-08-05 David Naredo Antiseptic toothbrush soak system
US6595655B2 (en) 2001-10-15 2003-07-22 Robert Neeb Mechanical dart carrier
AUPR897801A0 (en) * 2001-11-20 2001-12-13 Kerrie Lee Nominees Pty. Limited Toothbrush holder
US6702113B2 (en) 2002-06-11 2004-03-09 Anthony J. Marino Toothbrush sanitizing assembly
US20040134816A1 (en) * 2003-01-14 2004-07-15 Smargisso Joseph N. Tooth brush sanitizer and rinsing cup holder
US7708958B2 (en) * 2003-06-26 2010-05-04 Tersano Inc. System and containers for water filtration and item sanitization
US7767168B2 (en) * 2003-06-26 2010-08-03 Tersano Inc. Sanitization system and system components
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US4034074A (en) * 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
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US4166844A (en) * 1977-04-18 1979-09-04 E. R. Squibb & Sons, Inc. Solid phase separation technique for use in radioimmunoassays
US4189464A (en) * 1978-05-05 1980-02-19 Institute For Cancer Research Hepatitis B testing reagent and method
US4328183A (en) * 1978-06-14 1982-05-04 Mt. Sinai School Of Medicine Of The City University Of New York Blood cell typing and compatibility test procedure
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Also Published As

Publication number Publication date
NL179003C (nl) 1986-06-16
FR2241073A1 (xx) 1975-03-14
FR2241073B1 (xx) 1980-04-11
ATA672374A (de) 1975-12-15
GB1475125A (en) 1977-06-01
AT331975B (de) 1976-09-10
DE2436010C2 (xx) 1987-07-02
DE2436010A1 (de) 1975-02-27
US3904362A (en) 1975-09-09
AU7251374A (en) 1976-02-26
NL179003B (nl) 1986-01-16
NL7410914A (nl) 1975-02-18

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