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WO1999040437A1 - Procedes et appareils ameliores d'analyse de reactifs immunitaires dans des liquides biologiques - Google Patents

Procedes et appareils ameliores d'analyse de reactifs immunitaires dans des liquides biologiques Download PDF

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Publication number
WO1999040437A1
WO1999040437A1 PCT/US1999/002624 US9902624W WO9940437A1 WO 1999040437 A1 WO1999040437 A1 WO 1999040437A1 US 9902624 W US9902624 W US 9902624W WO 9940437 A1 WO9940437 A1 WO 9940437A1
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WO
WIPO (PCT)
Prior art keywords
reaction containers
test units
fluid
allergen
test
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/002624
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English (en)
Inventor
Thomas T. Hubscher
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dexall Biomedical Labs Inc
Original Assignee
Dexall Biomedical Labs Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dexall Biomedical Labs Inc filed Critical Dexall Biomedical Labs Inc
Publication of WO1999040437A1 publication Critical patent/WO1999040437A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to improved methods and apparatus for performing determination of immune reactants in biological fluids and, more particularly, for performing enzyme-immunoassay for both qualitative and
  • extrinsic asthma or atopic eczema
  • extrinsic asthma or atopic eczema
  • reaginic antibodies belonging to the IgE class of immunoglobulins.
  • the present invention is concerned with determining the presence of circulating allergen-specific IgE antibodies in the blood, plasma or serum of an
  • Bennich et al discloses an in-vitro method for analyzing a test sample (e.g., a body fluid such as a blood
  • test sample by contacting, in vitro, the test sample with a water insoluble polymer to which a test allergen has been bound.
  • a reaction takes place between
  • test allergen on the polymer and the reagin-lgE directed against the allergen so that the reagin-lgE is bound to the test allergen on the insoluble polymer.
  • the insoluble polymer sheet is separated from the fluid, whereupon the radiation emitted from the insoluble polymer with the
  • reagin-lgE labeled reagin-lgE is bound to the insoluble phase which then emits radiation. The latter increases with increasing concentration of the reagin-lgE in the test sample.
  • the radiation of the liquid phase decreases with increasing concentration of the reagin-lgE as more labeled reagin-lgE is bound to the insoluble phase.
  • EIA enzyme-immunoassay
  • patent includes multiple test units, each taking the form of an elongated rod having
  • each rod is frictionally engaged in a respective aperture or through-hole in a support strip so that the test units are disposed in a position-identified linear array. Spacing between the test
  • test unit spacing is also selected to permit alternate test units to be removed from the support strip so that the remainino test units can be inserted into respective test tubes supported in a linear array in a test tube rack.
  • the color coding uses the combination of colors of the rod and tip, each combination being unique and identifying a particular
  • the surfaces of the tip can be smooth or can
  • the strip are inserted into respective reaction containers containing the biological sample to be analyzed (e.g., blood, serum, etc.) If there is an antibody specific to the allergen coated on any of the tips, that antibody becomes bound to the associated
  • test units are assembled without rubbing, and inserted into a second set of reaction containers in which a suitable enzyme-labeled antibody conjugate has been poured.
  • the test units are
  • test units are removed and rinsed once again and then placed into a third set of reaction containers containing chromogenic substrate that, upon positive reaction, develops a specific color. After incubation, the test units are removed, thereby stopping the
  • spectrophotometric analyzer e.g., the LabDex ASR® brand spectrophotometric analyzer from Dexall Biomedical Labs, Inc. of Gaithersburg, Maryland.
  • a reagent kit having a first color coded liquid reagent, an incubation medium in the form of a
  • a second liquid reagent is an antibody conjugate, preferably an affinity purified anti-human IgE
  • a third liquid reagent is a chromogenic substrate
  • the reagent kit When performing quantitative (i.e., instrument based) determinations, the reagent kit also includes a set of buffered and stabilized human serum solutions (calibrated in A.U./ml.), including a first blank (0 A.U./ml) solution and one or more calibrators
  • an S1 standard (2.5 A.U./ml) solution such as an S1 standard (2.5 A.U./ml) solution, an S2 (10 A.U./ml) solution and an S3 (20 A.U./ml) solution; (an A.U. is an Allerg-Ens® Unit, an arbitrarily defined unit of concentration).
  • S1 standard 2.5 A.U./ml
  • S2 10 A.U./ml
  • an S3 (20 A.U./ml) solution an A.U. is an Allerg-Ens® Unit, an arbitrarily defined unit of concentration
  • plurality of tips are also provided for use with each kit.
  • kit with predispensed reagents permitting simultaneous multiple tests for respective
  • Another aspect of the present invention is a new formulation of chromogenic substrate for use in the method of the present invention and having a stabilized and buffered solution of
  • the container can either be quantitatively analyzed for color development and intensity by the spectrophotometric analyzer (as discussed above), or, alternatively, the liquid
  • reaction containers in the reaction containers may be visually, qualitatively analyzed by classifying and grading the color for each well.
  • a reagent kit is
  • a second liquid reagent also a second liquid reagent
  • color coded is an antibody conjugate, preferably an affinity purified anti-human IgE (goat) conjugated to horseradish peroxidase, in a buffer with stabilizers and preservatives.
  • a third liquid reagent also color coded (e.g., initially colorless and so
  • the reagent kit of the present invention also includes a set of buffered and stabilized
  • an S1 standard (2.5 A.U./ml) solution an S2 (10 A.U./ml) solution and an S3 (20 A.U./ml) solution.
  • a plurality e.g.,
  • test tips are also provided in each kit; however, the tips can be separately provided.
  • the kit When used in qualitative determinations, the kit includes a color reference comparison chart including a registration template for assisting the user in
  • reaction containers positioning the reaction containers adjacent a first region displaying a continuum of increasing color saturation.
  • the continuum of color is white at one end
  • the comparison chart also includes a plurality of (e.g., six) subregions positioned in
  • each subregion including a single, distinct, level of color saturation and a written caption such as, for example "Negative" for a
  • the three incubation periods account for eighty-five minutes time.
  • the three incubation periods amount to only seventy-five minutes time.
  • FIG. 1 is an exploded view in perspective showing one embodiment of the apparatus of the present invention
  • FIG. 2 is a top view in plan of a support strip utilized the apparatus of FIG. 1 ;
  • FIG. 3 is a front view in elevation of the support strip and test units employed in the apparatus of FIG. 1 ;
  • FIG. 4 is a bottom view in plan of one embodiment of a test unit tip
  • FIG. 5 is a side view in elevation of the test unit tip of FIG. 4;
  • FIG. 6 is a bottom view in plan of an alternative embodiment of the test unit tip of the present invention.
  • FIG. 7 is a side view in elevation of the test unit tip of FIG. 6;
  • FIG. 8 is a sectional view of the tips illustrated in FIGS. 4 and 6 showing the
  • FIG. 9 is an exploded view in perspective showing how the test units and support strip of the apparatus of FIG. 1 may be employed in connection with test tubes rather than the reaction containers illustrated in FIG. 1.
  • FIGS. 10a-10i are schematic illustrations of a frame of containers having
  • predispensed reagents for use in the eight test method steps included in the qualitative or quantitative test methods of the present invention.
  • FIG. 11 illustrates a color reference comparison chart for use in qualitative
  • apparatus constructed in accordance with the principles of the present invention includes an array 10 of multiple test units 11 through 22, inclusive. Although twelve
  • Each test unit includes an elongated cylindrical rod 30 with a transversely expanded tip 31 secured to its distal end.
  • Each rod 30 may be solid or hollow and has a section of reduced diameter at both its distal and
  • the distal end is received in a suitable hole or aperture at the top of
  • a respective tip 31 is secured in place by adhesive, cement, or the like.
  • proximal end of the rod of each test unit 11-22 is removably engaged by a friction fit in a respective circular aperture or through-hole 41-52, inclusive, defined in an elongated holder strip 32.
  • the substantially identical apertures 41-52 are disposed
  • the apertures are positionally identified, as by molding the strip with position-identifying numbers (e.g., M1-M12) adjacent each aperture.
  • position-identifying numbers e.g., M1-M12
  • a printed gummed label may be attached to the strip to identify each aperture by position.
  • test units are suspended in side-by-side relation with equal spacing between each
  • Holder strip 32 has handles/support brackets 33, 34 at its opposite ends to facilitate handling/support of the strip, and the supported test units, during test procedures.
  • Tip 31 is required to project or to extend to a transversely or radially larger
  • tip 31 may take the form of three solid integrally-formed sections 35, 36, and 37.
  • Section 35 the most proximal for the three sections, in frusto-conical with its smaller
  • second frusto-conical section 37 is of substantially equal axial length and identical shape but are inversely oriented axially to provide substantial symmetry on opposite sides of section 36.
  • the surfaces of section 35, 36 and 37 may be smooth, as illustrated in Figs. 4 and 5, or
  • test unit rod 30 and tip 31 are made of a suitable water-insoluble polymeric material that is both rigid and capable of having the described allergen
  • the material should be a type which readily adsorbs the proteinaceous allergen materials. Examples of
  • suitable polymeric materials for use in manufacturing test units 11-22 are hydrocarbon polymers such as polystyrene, polyethylene, polypropylene, polybutylene, butyl rubber and other synthetic rubbers, as well as polyesters,
  • polyamides such as polyvinyl chloride and polymethyl
  • methacrylate cellulose and cellulose derivatives such as cellulose acetate.
  • cellulose methacrylate, cellulose and cellulose derivatives such as cellulose acetate.
  • the preferred substrate material for the test unit is
  • the coating on tips 31 is achieved by dipping the tips into
  • the suspended array 10 of test units 11-22 is used in conjunction with a linear array or strip 60 of reaction containers or wells 61-72, respectively, formed as an integrally-molded, one-piece, clear plastic unit.
  • Containers 61-72 are preferably positioned in abutting side-by-side relation with their
  • test units 11-22 may be simultaneously
  • pluralitural arrays 60 may be supported in
  • each of the supported arrays may receive its own array of test units 11-22 simultaneously.
  • different arrays 60 may be used successively in connection with the same array of test units during performance of the test procedure described below.
  • the test procedure described below As a further alternative, the
  • reaction containers may be formed as a one-piece unit integrally molded with tray
  • test units 11-22 in the array 10 are color coded to be visibly
  • the coding permits a technician to correlate the visible identifier for each test unit with the
  • the visible identifier may be the color of the tip 31 , the color of rod 30, the
  • the coded visible identifier is the color combination of
  • Typical color combinations employed for the test units are represented in Table 1 which is a typical example of a color-coded allergen correlation chart supplied with the apparatus of the present invention.
  • the chart includes a first column listing test unit positions 11-22 (as referenced in Figs. 1 and
  • allergen-specific IgE antibodies coated to a solid phase support on each tip 31 , reacts with allergen-specific IgE antibodies in the patient's serum during a first incubation
  • enzyme-labeled (peroxidase) anti- human IgE i.e., the antibody conjugate reagent
  • the developed color is proportional to the amount of circulating allergen-specific IgE
  • Each reaction container is identified as to its position (i.e., positions one through twelve).
  • the first calibrator standard may be buffered and stabilized human serum solution containing a known concentration (i.e., 0 A.U./ml) of antibodies.
  • the second calibrator standard may be buffered and stabilized human serum solution containing a known concentration (i.e., 10 A.U./ml) of antibodies.
  • test serum is then placed in all of the other
  • each array 60 i.e., A, B, C, D, E, F, and G is dedicated to
  • a typical incubation medium would be a buffered protein solution containing preservatives.
  • test units not be removed from the holder strip
  • Test units 11-22 are then inserted simultaneously into reaction containers 61-72, respectively.
  • the test units may be moved up and down slightly to ensure proper mixing.
  • test units 8. The inserted test units are permitted to remain in place to achieve incubation for approximately fifty minutes at room temperature.
  • test units still supported by the holder strip 32, are then transferred
  • test units are rinsed for at least one minute by filling the
  • washing solution e.g., buffered salts and detergent and/or distilled water .
  • washing solution e.g., buffered salts and detergent and/or distilled water .
  • test units are then placed on an adsorbent paper towel with care being taken not to rub the tips 31.
  • peroxidase in a buffer with stabilizers and preservatives.
  • test units may be moved up and down slightly to
  • test units 16 14 The inserted test units are permitted to remain in place to achieve a second incubation for approximately twenty minutes at room temperature.
  • test units are removed from the reaction containers and washed and
  • Each reaction container 61-72 receives two hundred micro liters of
  • TMB tetra methyl benzidine
  • test units 11-22 are gently tapped on the paper towels and placed
  • test units are gently removed from the reaction containers with care
  • reaction containers of array 60 are removed from the frame 75 and placed in a spectrophotometric analyzer such as the LabDex ASR® brand spectrophotometric analyzer sold by Dexall Biomedical Labs, Inc., of Gaithersburg,
  • allergen-specific antibodies in the patient sera expressed in allergen units per milliliter (A.U./ml). Readings can be taken immediately or within eighteen hours, in
  • test units may be employed with larger containers, such as test tubes, in
  • a rack 80 for supporting test tubes 81 , 83, 85, 87, 89 and 91 in a linear array has suitable supporting apertures having their centers spaced at twice the spacing between apertures defined in strip 32.
  • test units 11 , 13, 15, 17, 19 and 21 are positioned to permit their simultaneous insertion into test
  • test tubes 81 , 83, 85, 87, 89 and 91 The openings of the test tubes are sufficiently wide to permit the tips of the test units to be inserted and removed without scraping.
  • Holder strip 32 is 4.712 inches long, 0.250 inches
  • the twelve apertures 41-52 have diameters of 0.062 inches and are spaced, on center, by 0.355 inches.
  • Rods 30 are 3.375 inches long
  • rod 30 has an outside diameter at its widest point of 0.0925 inches.
  • the proximal end of rod 30 has a reduced diameter of 0.062 inches.
  • Tip 31 has an axial length of 0.2187 inches.
  • Cylindrical section 36 has an axial length of 0.032 inches and an outside
  • the frusto-conical sections 35 and 37 taper at a 45° angle
  • the individual containers in array 60 are cylindrical
  • strips 100 of twelve containers 102 each are arrayed in a frame or tray 104 and
  • the light transmissive plastic containers 102 are sealed within the frame 104 by a two-part, fluid-tight, resilient, adhesively applied,
  • the first and larger film membrane segment 106a is rolled back from a
  • the containers 102 of strip or row A contain fifty micro liters of predispensed incubation media.
  • the containers of row D contain one hundred twenty five micro liters of predispensed antibody conjugate.
  • containers of rows B, C, E, F and G contain two hundred micro liters of
  • test units 31 are then inserted simultaneously into reaction
  • test units 31 19 containers 102 of row A.
  • the test units 31 may be moved up and down slightly to ensure proper mixing.
  • test units are permitted to remain in place to achieve incubation for approximately fifty minutes at room temperature. 6. The test units are then removed from the containers of row A and placed on an absorbent paper towel with care being taken not to rub the tips.
  • test units still supported by the holder strip 32, are then transferred sequentially into a rinsing containers of rows B and C, the test units are rinsed for at least one minute in each row, such that two rinsing cycles are performed.
  • test units are then removed from the containers of row C and placed on an absorbent paper towel with care being taken not to rub the tips.
  • test units are then inserted simultaneously into the row D reaction containers of antibody conjugate.
  • the test units may be moved up and down slightly to ensure proper mixing.
  • test units are permitted to remain in place to achieve a second incubation for approximately twenty minutes at room temperature.
  • test units are removed from the reaction containers and blotted (not
  • test units are then sequentially dipped, rinsed and transferred in the containers of rows E, F and G, agitating and standing for one minute in the
  • test units are gently tapped on the paper towels.
  • the key is used to remove the second segment 106b of the adhesive
  • test units are placed into the reaction containers of row H, containing the chromogenic substrate.
  • test units are gently removed from the reaction containers of row H
  • reaction containers 102 of row H are removed from the frame as
  • a spectrophotometric analyzer 120 e.g., the
  • spectrophotometric analyzer 120 The quantitative results displayed by spectrophotometric analyzer 120 are direct concentrations of allergen-specific antibodies in the patient sera expressed in A.U. per milliliter (A.U./ml). Readings can be taken immediately or within eighteen hours, in which case the arrays of
  • steps 1-4, 6-9, 11-20 are performed, as above; incubation steps 5 and 10 differ slightly in length, with the incubation period of steps 5 and 10 being thirty minutes
  • step 20 the containers 102 of row H are visually, qualitatively
  • fluid in container H2 is a darker (more saturated) color than the color of the fluid in
  • FIG. 11 illustrates a color reference comparison chart 120 for use in
  • a kit includes color reference comparison chart 120 which has printed indicia including a registration template 122 for assisting the user in positioning the reaction containers (e.g., H1-H3) adjacent a
  • first region 124 displaying a continuum of increasing color saturation.
  • each subregion including a single, distinct, level of color saturation and a written caption such as, for example "Negative" for
  • subregion 130 with no color saturation e.g. , white
  • "low” for a slight level of color saturation e.g., 132
  • "Moderate” for an increased level of color saturation e.g.,
  • Comparison chart 120 preferably also
  • the invention as thus far described is specific to testing for allergen-specific antibodies in human bodily fluids. It is to be understood that the apparatus described herein, and its method of use, apply to testing other antigens, in animal and human fluids, such as bacterial, viral, or auto-antigens (i.e., lupus, DNA, etc.).
  • the invention makes available a novel apparatus for rapidly performing determinations of allergen-specific antibodies in human serum as well as a time efficient step-by-step procedure for making such determinations.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé et un appareil d'analyse des anticorps IgE spécifiques aux allergènes dans des liquides biologiques, comprenant un nécessaire contenant des blocs de test avec revêtement et codage couleur devant être introduits dans des microplateaux correspondants, renfermant le liquide à tester. Si un anticorps spécifique à l'allergène vient recouvrir l'une des extrémités des blocs de test, cet anticorps se fixe à l'allergène qui lui est associé dans le microplateau correspondant. Ces extrémités sont rincées puis réintroduites dans un second ensemble de microplateaux avec un conjugué d'anticorps à marquage enzymatique. Les blocs de test sont ensuite retirés, rincés et placés dans un troisième ensemble de microplateaux renfermant un substrat chromogène de peroxyde d'hydrogène et de tétraméthylbenzidine (TMB). Suite à une réaction positive avec l'une des extrémités, le substrat chromogène développe une couleur spécifique. Dans un autre mode de réalisation, on prévoit un nécessaire de réactifs scellé contenant des réactifs liquides à codage couleur préalablement distribués, tels qu'un milieu d'incubation d'une solution protéine tamponnée contenant des agents de conservation, un second réactif liquide pouvant être un conjugué d'anticorps dans une solution tampon avec des stabilisants et des agents de conservation, un troisième réactif liquide pouvant être un substrat chromogène, une solution stabilisée et tamponnée de peroxyde et de TMB. Le nécessaire comprend par ailleurs des solutions d'étalonnage et une pluralité d'extrémités.
PCT/US1999/002624 1998-02-06 1999-02-08 Procedes et appareils ameliores d'analyse de reactifs immunitaires dans des liquides biologiques Ceased WO1999040437A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2025298A 1998-02-06 1998-02-06
US09/020,252 1998-02-06

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WO1999040437A1 true WO1999040437A1 (fr) 1999-08-12

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016182905A1 (fr) * 2015-05-08 2016-11-17 Alpha Diagnostic International, Inc. Bandes pour le transfert quantitatif d'échantillons biochimiques
CN116008525A (zh) * 2023-02-24 2023-04-25 江苏浩欧博生物医药股份有限公司 一种过敏原特异性IgE抗体检测试剂盒

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983003677A1 (fr) * 1982-04-16 1983-10-27 Boquet, Patrice Dispositif d'analyse pour echantillons biologiques
US4458014A (en) * 1982-01-11 1984-07-03 Forsyth Dental Infirmary For Children Serological method for the identification of microorganisms
US4891321A (en) * 1987-10-21 1990-01-02 Hubscher Thomas T Apparatus for performing determinations of immune reactants in biological fluids
US5017342A (en) * 1988-06-01 1991-05-21 Ortho Diagnostic Systems Inc. Device for immunoassay determinations
US5244788A (en) * 1992-07-01 1993-09-14 Hubscher Thomas T Method and apparatus for performing determinations of immune rectants in biological fluids
US5525476A (en) * 1991-08-09 1996-06-11 Iatron Laboratories, Inc. Immunoassay, monoclonal antibody, and hybridoma

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4458014A (en) * 1982-01-11 1984-07-03 Forsyth Dental Infirmary For Children Serological method for the identification of microorganisms
WO1983003677A1 (fr) * 1982-04-16 1983-10-27 Boquet, Patrice Dispositif d'analyse pour echantillons biologiques
US4891321A (en) * 1987-10-21 1990-01-02 Hubscher Thomas T Apparatus for performing determinations of immune reactants in biological fluids
US5017342A (en) * 1988-06-01 1991-05-21 Ortho Diagnostic Systems Inc. Device for immunoassay determinations
US5525476A (en) * 1991-08-09 1996-06-11 Iatron Laboratories, Inc. Immunoassay, monoclonal antibody, and hybridoma
US5244788A (en) * 1992-07-01 1993-09-14 Hubscher Thomas T Method and apparatus for performing determinations of immune rectants in biological fluids

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016182905A1 (fr) * 2015-05-08 2016-11-17 Alpha Diagnostic International, Inc. Bandes pour le transfert quantitatif d'échantillons biochimiques
CN116008525A (zh) * 2023-02-24 2023-04-25 江苏浩欧博生物医药股份有限公司 一种过敏原特异性IgE抗体检测试剂盒

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