WO1987006708A1 - Test rapide avec des recepteurs de phase solide - Google Patents

Test rapide avec des recepteurs de phase solide Download PDF

Info

Publication number
WO1987006708A1
WO1987006708A1 PCT/US1987/001018 US8701018W WO8706708A1 WO 1987006708 A1 WO1987006708 A1 WO 1987006708A1 US 8701018 W US8701018 W US 8701018W WO 8706708 A1 WO8706708 A1 WO 8706708A1
Authority
WO
WIPO (PCT)
Prior art keywords
receptor pair
ligand
solid substrate
pair member
liquid
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/US1987/001018
Other languages
English (en)
Inventor
Stephen J. Chudzik
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.)
Bio Metric Systems Inc
Original Assignee
Bio Metric Systems 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 Bio Metric Systems Inc filed Critical Bio Metric Systems Inc
Publication of WO1987006708A1 publication Critical patent/WO1987006708A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica

Definitions

  • the present invention relates to an assay kit and method for determining the presence of microor ⁇ ganisms, chemicals, and other analytes in physiologi ⁇ cal and biological fluids.
  • the traditional methods of detecting and iden ⁇ tifying microorganisms in physiological and biological fluids require cell culturing on laboratory media (sometimes followed by susceptibility testing to determine resistance to a particular antibiotic), identi ication of the organism by detection of serum antibodies against the organism, or isolation of the organism from infected tissues. These methods may be difficult to perform early enough to aid in patient management and diagnosis. In some cases the microor ⁇ ganism cannot be isolated or cultured. Development of rapid detection methods which are sensitive enough to determine if a particular microorganism is present in body fluid or biological fluids and which can be easily and rapidly performed in a clinic or doctor's office would greatly aid in the diagnosis of illnesses induced by such organisms.
  • the present invention provides an assay kit and method capable of performing an assay to detect micro ⁇ organisms or analytes in less than thirty minutes. No lab culturing of a microorganism is required, and a positive intense detectable signal is produced to alert the user to the presence of a particular micro ⁇ organism in the physiological fluid being tested. Other drugs, chemicals and the like can be rapidly detected using the invention. No additional instru ⁇ mentation is necessary for interpretation of results.
  • the present invention provides an assay kit and method for the detection of chemicals and other analytes that are members of ligand-receptor pairs.
  • the invention employs a predetermined quantity of a labeled member of a ligand-receptor pair and a first solid substrate containing a reaction zone carrying a detection system responsive to said label to produce a detectable signal.
  • the invention also employs a second solid substrate suspendable in a liquid and upon which is immobilized a member of a ligand-receptor pair, and means for binding the labeled ligand-receptor pair member to the second solid substrate in relation to the presence or quantity of analyte in the sample.
  • a predeter ⁇ mined quantity of a labeled member of a ligand- receptor pair is incubated with a sample suspected of containing an analyte that is also a member of the ligand-receptor pair in a liquid in which is suspended a solid substrate having immobilized thereon a member of the ligand-receptor pair.
  • Another solid substrate, containing a reaction zone as above described is then contacted with the reaction mixture.
  • the suspended solid substrate carries means for binding the labeled ligand-receptor pair member in relation to the quan ⁇ tity of analyte in the sample and thus, substantially physically inhibit the label from molecularly inter ⁇ acting with the detection system contained in the reaction zone.
  • the suspendable, solid substrate having immobilized there ⁇ on a member of a ligand-receptor pair is uniformly suspended in a liquid and incubated with a labeled member of the ligand-receptor pair and sample sus ⁇ pected of containing analyte.
  • Another solid substrate having a first end and carrying a reaction zone spaced from the first end of the substrate is contacted at its first end with the liquid-solid substrate admix ⁇ ture, and the liquid containing unbound labeled ligand- receptor pair member is allowed to flow upwardly through the reaction zone.
  • the reaction zone contains means for binding and thereby concentrating the labeled ligand-receptor pair member and also includes a detection system responsive to said label.
  • the invention comprises an assay kit for an analyte, the kit containing a liquid-suspendable, particulate, second solid substrate having immobilized thereon a member of a ligand-receptor pair, a predetermined quantity of a labeled member of the ligand-receptor pair, means for binding the labeled ligand-receptor pair member to the particulate substrate in relation to the quantity of analyte present in the sample, and a first solid sub ⁇ strate having a first end to be contacted with a liquid in which the second solid substrate is sus ⁇ pended to allow the liquid to flow upwardly there ⁇ through, the first substrate having a reaction zone spaced from its first end, said reaction zone in ⁇ cluding means for binding and thereby concentrating the labeled ligand-receptor pair member thereon and having a
  • Figure 1 is a schematic view, in cross-section, showing a step in the method of the invention
  • Figure 2 is an enlarged, broken away view of a solid substrate shown also in Figure 1;
  • Figure 3 is a schematic, cross-sectional view of a step in a method according to another embodiment of the invention.
  • ligand-receptor pair refers to a pair of compounds of which one, a “receptor” is capable of recognizing a particular spacial and polar organization of the other ("ligand") or portion there ⁇ of, and is capable of binding to that compound.
  • illustrative receptors forming the other half of a ligand-receptor pair include anti ⁇ bodies, enzymes, lectins, Fab fragments, complementary nucleic acids and the like. Commonly, the receptor will be an antibody and the ligand will act as an antigen or hapten.
  • the analyte or an analyte derivative will be the ligand.
  • analyte derivative means a chemical derivative of an analyte that retains the capacity to bind to the other member of a ligand-receptor pair in competition with the analyte.
  • labeled member of a ligand-receptor pair is meant a conjugate of a ligand-receptor pair member' with a chemical label such as an enzyme, a fluorescent compound or other detectable chemical species, the conjugate- retaining the capacity to bind to the other member of the ligand-receptor pair, and the enzyme or other detectable label continuing to have a capacity of being detected by a detector system (which may be a separate chemical reaction system) to provide a perceptible signal.
  • a detector system which may be a separate chemical reaction system
  • Detector refers to a chemical system that provides perceptible signals, commonly electromagnetic radiation or absorption of the same leading to perceptible fluorescense, color changes and the like, when contacted with a specific enzyme or other label.
  • the invention is useful in detecting a broad range of analytes.
  • U.S. Patents 4,374,925 and 3,817,837 set out excellent lists of analytes which are part of specific binding pairs, the teachings of which patents are incorporated herein by reference.
  • Other examples of binding pairs include nucleic acids and their complementary nucleic acids.
  • Analytes of particular interest include viruses, bacteria and fungi and macromolecules, specific products and assemblages thereof, drugs, toxins, and products of living organisms.
  • the reaction may be a competitive one in which the labeled ligand-receptor pair member competes with the analyte for binding to a limited quantity of binding sites of another member of the ligand-receptor pair, commonly antibody to the analyte.
  • the competitive reaction may be performed in several ways within the context of this invention.
  • Antibody to the analyte may be immobilized on the liquid-suspendable particulate solid substrate and labeled ligand-receptor pair member (analyte) and the analyte then may compete for binding sites on the substrate.
  • a predetermined quantity of analyte may be bound to the particulate substrate.
  • the labeled ligand-receptor pair member may be a labeled antibody to the analyte.
  • free analyte in the sample will compete with the analyte on the substrate for binding to the labeled antibody.
  • the free analyte in the sample may be pre-reacted with the labeled antibody and the resulting material then may be added to the suspended substrate.
  • a ligand-receptor reaction system that may be used with this invention is often referred to as a "sandwich" assay.
  • This system requires one ligand-receptor pair member commonly the analyte, to have at least two binding sites.
  • the labeled ligand-receptor pair member and the ligand- receptor pair member immobilized on the liquid-sus- pendable particulate solid substrate both bind to the analyte at different sites.
  • analyte-containing sample, labeled antibody, and the particulate sub ⁇ strate are incubated together, the analyte will bind to the particulate substrate and the labeled ligand- receptor pair member will then bind to the an&lyte in relation to the amount of analyte in the sample.
  • the bound label will thus be substantially physicially inhibited from reacting with the label detection system carried by the other solid substrate as described above.
  • a reaction vessel typified as a transparent hollow column of glass or the like designated (12), has an open top end (12.1 and a closed bottom end (12.2).
  • the reaction vessel contains a solid particulate substrate (20) such as particles of agarose, polyacrylamide, glass or cellulose fibers, substantially uniformly suspended in a liquid (22).
  • the liquid is compatible with the ligand-receptor reaction and the detection system contained in the reaction zone, and to be used in a particular assay.
  • the liquid is commonly phoshate buffered saline (“PBS”) or the sample itself. If the sample is a liquid the reaction vessel may contain the suspendable solid substrate and the labeled lignd- receptor pair member in a dry form.
  • PBS phoshate buffered saline
  • the solid substrate (20) is comprised of dis ⁇ crete small beads or particles (21) that have a high specific surface, that are capable of being suspended in the liquid and that have the ability to bind antibody or other substances which constitute 'members of a ligand-receptor pair.
  • FIG. 1 Shown in Figure 1 and in greater detail in Figure 2 is one embodiment of the invention that utilizes a first solid substrate (25) comprising an elongated strip having a first end (27) for contact with the ligand-solid substrate suspension (20) and having a reaction zone (30) spaced from the first end (27) a sufficient distance to avoid contact of the reaction zone with the suspension (20) when the first end (27) contacts the liquid surface (23).
  • the particles of the solid substrate (20) may contact the first end (27), as shown in Figure 2, the lower end (27) of the strip acts as a filter to restrain upward movement of the particles (21).
  • the solid substrate (25) in this embodiment is bibulous in that it allows the liquid to be drawn upwardly through the reaction zone by capillary action.
  • the solid substrate (25) may be made of filter paper or other fibrous, particulate or porous material that has the capacity to draw up liquid.
  • the substrate is a predetermined size so that a predetermined volume of liquid flows through the reaction zone.
  • the reaction zone desirably has the ability to bind antibody or other substances that constitute a member of a ligand-receptor pair and it also contains components of the detection system reactive with the label to produce a detectable signal.
  • the reaction zone contains binding means for binding the labeled ligand-receptor pair member to thus concentrate the unbound labeled ligand-receptor pair member in the reaction zone.
  • binding means may comprise an antibody or an antigen or any member of a ligand-receptor pair to which the labeled ligand- receptor pair member will bind.
  • the signal-producing means carried by the reac ⁇ tion zone is responsive to the label on the ligand- receptor pair member to produce a detectable signal.
  • the label desirably is an enzyme.
  • chromagens are available which produce visible color when added to their specific enzymes and sub ⁇ strates. For example, o-phenylenediamine and o-dianisidine have been used as peroxidase and glucose oxidase chromagen.
  • Napthol-MX-phosphate is used as a chromagen for alkaline phosphatase.
  • Yolken, R.N. "Solid-Phase Enzyme Immunoassays for the Detection of Microbial Antigens in Body Fluids," in Manual of Clinical Microbiology, pgs. 949-957 (1985) contains a more detailed description of these chromagens.
  • the entire first solid substrate (28) can be contacted with the suspension.
  • That substrate (28) includes a reaction zone (30) containing a detection system responsive to the label of the labeled ligand-receptor pair member.
  • the components of the detection system are desirably substantially insoluble in the reaction mixture.
  • substantially insoluble in this context means that the components of the detection system either remain attached to the reaction zone via chemical bonds when the solid substrate carrying the reaction zone is contacted with the liquid suspension, or the component themselves are so sparingly soluble in the liquid as to substantially remain in place in the reaction zone when the latter is contacted with the liquid suspen ⁇ sion.
  • a suspendable, solid substrate comprising cross-linked agarose gel particules (60-140 microns, 4% agarose by weight in water) (Sepharose CL-4B, a trademarked product of Pharmacia Corp.)
  • CL-4B signifies 4% agarose by weight.
  • IgG anti- phenobarbital is covalently coupled to the particles, as described below.
  • the labeled ligand-receptor pair member used is horseradish peroxidase ("HRP") - polyethylene glycol (“PEG”)-phenobarbital.
  • the other solid substrate containing a reaction zone is comprised of a strip of Whatman #17 chromatography paper 1/4" by 2 3/4" containing, midway between its ends, a 1/4" square of the same paper to which IgG anti-phenobarbital and glucose oxidase ("G.O.”) have been immobilized. (Procedure for preparation is described below.)
  • the bottom 1/2 of the strip is impregnated with a 50 microgram per milliliter 0-dianisidine solution and dried.
  • One-hundred microlitersC'uls" of a 1:1 suspension by volume of Sepharose CL-4B-IgG anti- phenobarbital particles in PBS buffer is placed in each of three test tubes.
  • 100 uls of an 18% glucose solution, and 400 uls phosphate buffered saline ("PBS") containing 25 uls of a 0.50 mg/ml HRP-PEG-Phenobarbi- tal solution is added to each test tube.
  • PBS phosphate buffered saline
  • HRP-PEG-Phenobarbi- tal solution is added to each test tube.
  • To one tube 2.5 micrograms ("ugs") of sodium phenobarbital is added as a positive control.
  • Sample suspected of containing phenobarbital is added to the second tube.
  • the third tube is the negative control.
  • the tubes are agitated and allowed to stand for two minutes, whereupon, the bottom ends of the strips are dipped into the resulting suspensions. Liquid from the suspensions is allowed to flow up the strips by capillary migration, flowing through the reaction zones and continuing to the top of the strips. When the liquid reaches the tops of the strips (about four minutes), the strips are removed and examined for color development.
  • reaction zone of the strip placed in the positive control suspension becomes dark brown, while the reaction zone of the strip placed in the other control solution remains white. Analyte present in the sample causes the reaction zone of the strip placed in the sample suspension to become brown.
  • the IgG preparation may be immobilized on the Sepharose CL-4B or Sepharose CL-6B ("CL-6B" signifies 6% agarose by weight in water) by the following method.
  • Ten mis of Sepharose CL-4B or Sepharose CL-6B gel are transferred to a scintered glass funnel and washed successively with 50% dioxane, dioxane and dry dioxane.
  • the gel is then packed into a large tube and reacted with 1 gram of carbonyldiimidazole dissolved in 20 mis of dry dioxane for 30 minutes at room temperature with agitation.
  • the thus activated gel is then transferred back to the scintered glass funnel and washed successively with dioxane, 50% dioxane and cold 0.1M borate buffer, pH 9.0.
  • the tube is rocked at 4°C for 24 hours.
  • the gel is then washed extensively with PBS and stored in PBS at 4°C.
  • the HRP-PEG-Phenobarbital conjugate was pre ⁇ pared as follows. Additional amine groups were added to HRP by first oxidizing 2 mgs HRP in .05 ml of ImM sodium acetate, pH 4.5, by the addition of 0.1 ml of a 0.1M sodium periodate solution. This reaction pro ⁇ ceeded at room temperature for 30 minutes followed by a 24 hour dialysis against ImM sodium acetate, pH 4.5. After dialysis, 0.2 mis of a PEG diamine of approximately 600 molecular weight was added and the pH adjusted to 9.5. The reaction mixture was stirred for two hours at room temperature.
  • the resulting Schiff base was reduced by the addition of 0.1 ml of a 4mg/ml sodium borohydride solution.
  • the reaction mixture was allowed to stand for 6 hours at 4°C.
  • the HRP-PEG (NH,) was dialyzed extensively against PBS.
  • the ethyl ester was saponified to the free acid by dissolving in 30 mis Tetrahydrafuran, 15 mis IN HC and 6 mis H 2 0, stirring for 20 hours at room tempera ⁇ ture and extracting the aqueous phase. .
  • the free acid was converted to the Sulfo-NHS (N-hydroxy sulfo suc- cinimide) by a N,N'-Dicyclohexylcarbodiimide (“DCC”) catalyzed reaction in DMF. The DMF is subsequently pulled off with a vacuum pump.
  • DCC N,N'-Dicyclohexylcarbodiimide
  • the IgG anti-Phenobarbital and G.O. squares were prepared as follows. 1/4" squares were cut from Whatman tl7 chromatography paper. The squares were dried at 105°C for 45 minutes, cooled in a dessicator and transferred to an appropriate vessel. The squares were activated with 1 gram of 1,1' carbonyldiimidazole dissolved in 35 mis dry dioxane per one hundred squares. The squares were activated for 45 minutes at room temperature with agitation. The squares were washed several times with cold d H-0, and again sev ⁇ eral times with 0.1M borate buffer, pH 9.0.
  • the suspendable, solid sub ⁇ strate is comprised of Sepharose CL-6B particles (particle size 45-165 microns) to which monoclonal antibodies against T 2 toxin have been covalently coupled, as described below.
  • the labeled ligand- receptor pair member used is G.O. labeled T 2 toxin.
  • the other solid substrate is comprised of a strip of Whatman #17 chromatography paper 1/4" by 4 1/4", divided into three contiguous segments, the middle segment comprising a reaction zone.
  • the end segments are of equal dimension (1/4" x 2"), the lower segment being impregnated with a 50ug/ml 0-dianisidine solu ⁇ tion and then dried.
  • the reaction zone is a 1/4" square of the same paper to which T 2 antibodies and HRP have been covalently coupled, and to which an 18% glucose solution is dried.
  • the 2 antibody/HRP squares were prepared as follows. A stable HRP-PEG NH 2 conjugated was pre ⁇ pared as described in Example 1. 1/4" squares of filter paper were cut and activated in the same manner as described in Example 1. After the squares had been activated and washed, they were reacted with a solu ⁇ tion consisting of 35 mis 0.1M borate buffer, pH 9.0, 20 mgs IgG and 2 mgs HRP-PEG (NH.,) for every 100 squares. The reaction was allowed to proceed for 20 hours at 4°C with rocking. After the reaction was completed, the disks were washed extensively with cold PBS and stored in PBS at 4"C.
  • the G.0.-T 2 toxin conjugate can be prepared in the following manner. Twenty mgs of T 2 toxin are dissolved in 10 mis of pyridine. Five mgs succinic anhydride are added and the reaction is stirred at room temperature for 24 hours. The pyridine solution is evaporated to dryness vacuo. The resulting hemi-succinate is converted to the sulfo-NHS by a DCC catalyzed reaction in DMF. The DMF solution is evapo ⁇ rated to dryness in vacuo and the resulting T 2 - sulfo NHS compound is used for coupling to G.O.
  • the suspendable, solid sub ⁇ strate is comprised of Sepharose CL-4B particles to which choleragenoid antibodies have been covalently immobilized, as described below.
  • the labeled ligand- receptor pair member used is a HRP labeled cholerage ⁇ noid.
  • the other solid substrate is comprised of a strip of Whatman #17 chromatography paper 1/4" by 4 1/4", divided into three contiguous segments, the middle segment of which constitutes the reaction zone.
  • the end segments are of equal dimension (1/4" x 2"), the lower segment being impregnated with a 50 ug/ml 0-dianisidine solution and dried.
  • the reaction zone comprises.a 1/4" square of the same paper to which choleragenoid antibodies and G.O. have been covalently coupled.
  • each of three test tubes 100 uls Sepharose 4B-IgG anti-choleragenoid gel. 800 uls PBS, 100 uls of an 18% glucose solution and 15 uls of a 0.10 mg/ml ' HRP-choleragenoid are added to each tube.
  • l ug Cholera toxin beta subunit is added as a positive control.
  • Sample suspected of•.containing analyte is- added to the second tube.
  • the third tube is untreated and is the negative control.
  • the reac ⁇ tion tubes -ire agitated and allowed to stand for 2 minutes, whereupon the bottem ends of the strips are dipped into the resulting suspension.
  • the HRP-Choleragenoid conjugate was prepared as follows. The carbohydrate portion of HRP is oxidized by sodium periodate, as described in Example 1. Cholera toxin beta subunit was purchased and repuri- fied by chromatographing on a G-75 column. To 2 mgs of the oxidized HRP is added 2.9 mgs of the purified cholera toxin beta sub-unit. The pH is adjusted to 9.5 by the addition of sodium carbonate. After two hours of stirring at room temperature, the resulting Schiff base is reduced by the addition of 0.1 ml of a 4 mg/ l sodium borohydride solution. The reaction mixture is left to stand for 6 hour at 4°C. The crude conjugate is chromatographed on a G-75 column with fraction absorbances monitored at A 2 o 8 n 0 and A4. n 03.
  • the suspendable, solid sub ⁇ strate is comprised of Controlled Pore Glass-N-hydroxy succinimide (CPG-NHS) glycophase glass beads (125-177 uM particle size) to which monoclonal antibodies against Chlamydia have been covalently coupled, as described below.
  • the labeled ligand-receptor pair member is HRP-labeled Chlamydia.
  • the other solid sub ⁇ strate is comprised of a strip of Whatman #17 chro ato- graphy paper, 0.5 cm x 5 cm, divided into three contiguous segments, the middle segment of which is comprised by a reaction zone.
  • the end segments are of equal dimension, the lower segment being impregnated with a 50 ug/ml 0-dianisidine solution and dried.
  • the reaction zone is comprised of a 0.5 cm x 1 cm segment of the same paper to which G.O. and Chlamydia anti ⁇ bodies have been covalently coupled.
  • each of three test tubes 40 uls CPG-IgG anti-Chlamydia beads.
  • 350 uls PBS, 100 uls of an 18% glucose solution and 10 uls of an 0.040 mg/ml HRP-chlamydia solution is added to each tube.
  • To one tube 1 x 10 9 Chlamydial organisms are added to act as a positive control.
  • Sample suspected of containing analyte is added to the second tube.
  • Nothing else is added to the third tube and it provides a negative control.
  • the reaction tubes are agitated and left to stand for two minutes, whereupon the bottera ends of the strips are dipped into the resulting suspensions. Liquid from the suspension flows up the strips, pass ⁇ ing through the reaction zones.
  • the Chlamydial antigen was prepared as follows. 12 mis of a frozen chlamydia sample were placed in a flask along with 12 mis of PBS. The Chlamydia sample was heat-killed at 50°C with gentle agitation for 2 hours. The sample was sonicated using a Braunsonic 1510 sonicator, with 10, 200 watt bursts of 15 seconds, with a 30 second cool down between bursts. The sample was centrifuged for 15 minutes at 15,000RPM. 10 mis of supernatant were collected con ⁇ taining 1.5 mg protein as determined by a Bio-Rad protein assay using a BSA standard. This protein from the supernatant was used as the Chlamydial antigen.
  • the HRP-Chla ydia conjugate was prepared as follows.
  • the carbohydrate portion of HRP was oxidized by sodium periodate as described in Example 1.
  • 1.5 mgs of the Chlamydia antigen preparation were adjusted to pH 9.0 by the addition of 0.3M sodium carbonate. This solution was added to 3 mg of the oxidized HRP and the reaction mixture was stirred at room tempera ⁇ ture for 2 hours.
  • the Schiff base was reduced by the addition of 0.2 ml of a 4 mg/ l sodium borohydride solution. The reaction mixture was left to stand for 6 hours at 4°C.
  • the crude conjugate was dialyzed against PBS and then concentrated to 2.5 mis by ultra-centrifuga- tion.
  • the conjugate was chromatographed on a G-150 column with fraction absorbances monitored at A 2go and A 4(J3 .
  • the IgG was immobilized on the solid substrate by the following method.
  • An IgG preparation was dialyzed against 0.1M borate buffer, pH 9.0, for 24 hours at 4°C. After dialysis, the IgG was added to 0.4 gram CPG-NHS (5A pore diameter, 125-177 uM par ⁇ ticle size). Two mis 0.1M Borate buffer, pH 9.0 was added and the reaction mixture rotated at 4°C for 36 hours. When the reaction was completed, the beads were washed extensively with cold PBS and stored in PBS at 4°C. For many of the applications of this invention, the segment of the strip which contacts the reaction
  • the suspendable solid sub ⁇ strate is comprised of Sepharose CL-4B particles to which phenobarbital antibodies had been covalently immobilized, as described in Example 1.
  • the labeled ligand-receptor pair member is an HRP-PEG-Phenobarbital conjugate.
  • the other solid substrate is an individual reaction disk comprised of 1/4" diameter #17 Whatman chromatography paper which contains covalently im ⁇ mobilized IgG anti- henobarbital and glucose oxidase and has been impregnated with a 100 ug/ml 0-dianisi- dine solution and dried.
  • each of four test tubes 25 uls Sepharose 4B-IgG anti-phenobarbital gel.
  • 350 uls PBS 100 uls of an 18% glucose soution and 30 uls of a 0.010 mg/ml HRP-PEG-phenobarbital solution is added to each tube.
  • 0.5 ug Phenobarbital is also added to the second tube.
  • To the third tube is added 1.0 ug phenobarbital.
  • 2.5 ugs phenobarbital are added to the fourth tube.
  • the reaction tubes are agitated and left to stand for 2 minutes, whereupon reaction disks are added to the reaction solutions.
  • the reaction tubes are reagitated and allowed to stand for an additional three minutes.
  • reaction disks are removed and examined for color development.
  • the reaction disk from the first tube remains white.
  • the reaction disk from the second tube becomes light brown.
  • the reaction disk from the third tube becomes a darker brown and the reaction disk from the fourth tube exhibits the darkest color.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Inorganic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On peut utiliser des procédés et des kits de test pour déterminer rapidement la présence ou l'absence de quantités minimes d'analyte. Dans ces kits et ces procédés, on utilise une quantité prédéterminée d'un élément marqué une paire ligand-récepteur, un premier substrat solide (25) portant une zone de réaction (30) qui contient un système de détection sensible à la substance marquée et un deuxième substrat solide (20) soluble dans un liquide sur lequel est immobilisé un élément d'une paire ligand-récepteur et ayant des moyens susceptibles de lier l'élément marqué d'une paire ligand-récepteur en fonction de la quantité d'analyte présente dans un échantillon. La substance marquée ainsi liée est physiquement empêchée de réagir avec le système de détection porté par le premier substrat solide (25).
PCT/US1987/001018 1986-04-28 1987-04-24 Test rapide avec des recepteurs de phase solide Ceased WO1987006708A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US85694286A 1986-04-28 1986-04-28
US856,942 1986-04-28

Publications (1)

Publication Number Publication Date
WO1987006708A1 true WO1987006708A1 (fr) 1987-11-05

Family

ID=25324819

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1987/001018 Ceased WO1987006708A1 (fr) 1986-04-28 1987-04-24 Test rapide avec des recepteurs de phase solide

Country Status (3)

Country Link
AU (1) AU7439787A (fr)
IL (1) IL82339A0 (fr)
WO (1) WO1987006708A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0727038A4 (fr) * 1992-07-31 1998-03-18 Biostar Inc Dispositifs et procedes pour la detection d'un analyte, bases sur l'interference de la lumiere
US7736689B2 (en) 2001-12-21 2010-06-15 Surmodics, Inc. Reagent and method for providing coatings on surfaces

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3654090A (en) * 1968-09-24 1972-04-04 Organon Method for the determination of antigens and antibodies
US4094647A (en) * 1976-07-02 1978-06-13 Thyroid Diagnostics, Inc. Test device
US4447526A (en) * 1981-04-20 1984-05-08 Miles Laboratories, Inc. Homogeneous specific binding assay with carrier matrix incorporating specific binding partner
US4587102A (en) * 1981-06-05 1986-05-06 Fuji Photo Film Co., Ltd. Multi-layer analysis element utilizing specific binding reaction
US4594327A (en) * 1983-11-02 1986-06-10 Syntex (U.S.A.) Inc. Assay method for whole blood samples

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3654090A (en) * 1968-09-24 1972-04-04 Organon Method for the determination of antigens and antibodies
US3654090B1 (fr) * 1968-09-24 1982-07-20
US4094647A (en) * 1976-07-02 1978-06-13 Thyroid Diagnostics, Inc. Test device
US4447526A (en) * 1981-04-20 1984-05-08 Miles Laboratories, Inc. Homogeneous specific binding assay with carrier matrix incorporating specific binding partner
US4587102A (en) * 1981-06-05 1986-05-06 Fuji Photo Film Co., Ltd. Multi-layer analysis element utilizing specific binding reaction
US4594327A (en) * 1983-11-02 1986-06-10 Syntex (U.S.A.) Inc. Assay method for whole blood samples

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0727038A4 (fr) * 1992-07-31 1998-03-18 Biostar Inc Dispositifs et procedes pour la detection d'un analyte, bases sur l'interference de la lumiere
US7736689B2 (en) 2001-12-21 2010-06-15 Surmodics, Inc. Reagent and method for providing coatings on surfaces
US8039524B2 (en) 2001-12-21 2011-10-18 Surmodics, Inc. Reagent and method for providing coatings on surfaces

Also Published As

Publication number Publication date
AU7439787A (en) 1987-11-24
IL82339A0 (en) 1987-10-30

Similar Documents

Publication Publication Date Title
US4298685A (en) Diagnostic reagent
JP3333755B2 (ja) 免疫化学的標識およびこれを含有する水性懸濁液、ならびに該免疫化学的標識の製造方法
EP0586590B1 (fr) Reactifs contenant un inhibiteur de fixation non specifique pour analyses de fixation avec capture d'ions
US4853335A (en) Colloidal gold particle concentration immunoassay
AU619231B2 (en) Chromatographic binding assay devices and methods
US4948726A (en) Enzyme immunoassay based on membrane separation of antigen-antibody complexes
US5629164A (en) Enzyme immunoassay device
JPH0566547B2 (fr)
WO1984002193A1 (fr) Analyse immunologique a support chromogene
EP0468585B1 (fr) Elément analytique incorporé avec un réactif biologiquement actif et méthodes pour utiliser le réactif
JPS62191765A (ja) 凝集性結合試薬を用いた不均一系特異的結合試験方法及び不均一系イムノアツセイ法並びにその試験キツト
EP0641442B1 (fr) Dispositifs permettant d'effectuer des analyses de fixation avec capture d'ions
JP2579972B2 (ja) 膜親和濃縮免疫測定方法
JP3267614B2 (ja) カルボキシメチルアミロースに結合させた結合メンバーを使用するイオン捕獲アッセイ
CA2064972A1 (fr) Essai immunologique pour immunoglobulines
WO1990015328A1 (fr) Essai immunitaire ameliore
US5990274A (en) Cyclosporine derivatives and uses thereof
EP0152254A2 (fr) Immunoessai à base d'un support chromogénique utilisant des composants complémentaires marqués
JP2001305139A (ja) 特異的結合体
WO1987006708A1 (fr) Test rapide avec des recepteurs de phase solide
JPS60500548A (ja) 発色性支持体免疫測定法
JP3102827B2 (ja) 特異的結合アッセイ試薬及びそれを使用した測定方法
EP0206779A1 (fr) Détection des anticorps antinucléaires
US5206178A (en) Membrane affinity concentration immunoassay
JP2001013145A (ja) 標識複合体とその製造方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE