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
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • 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
  • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

Definitions

• the present invention relates to a device and method for the in vitro screening of human subjects for allergic sensitivity.
• serum, plasma or other bo fluid from the individual is tested in a solid phase im u assay, simultaneously in a single test apparatus, for the presence of antibodies to a variety of allergens as well for the level of Total IgE immunoglobulin in the fluid.
• This invention also relates to a method of performing mul pie tests for antibodies and/or antigens simultaneously i single device and kits therefor, which employ conventiona immunoassay techniques using radioactive-, enzyme- or fluorescent-labeled reagents.
• Antibodies of the immunoglobulin classes, IgE and Ig are known to be responsible for the allergic hypersensi- tivity in humans and animals. Individuals predisposed to allergies, either genetically or through exposure to en ⁇ vironmental allergens, by virtue of an imbalance in their immune system usually have levels of Total IgE elevated f the norm. Proper diagnosis and treatment of allergic dis orders, therefore, require not only a measure of the Tota IgE level but more so a measure of the levels of antibodi directed against specific allergens (Dockhorn, R.J., Ann. Allergy, 49, 1-7, 1982). Methods for the detection of allergen-specific anti ⁇ bodies involve both iri vivo and ;Ln vitro techniques.
• In vitro techniques consist of challenging the patient eithe directly into the skin or by inhalation of allergen aeros with aqueous extracts of the allergens (Nelson, H.S., Ann Allergy, 51, 411-417, 1983; Pelikan, Z., ibid., 51, 395-4 1983) . These iri vivo techniques are usually discomfortin to the patient and are not always applicable as in childr the elderly and patients with skin disorders. Bennich, K et al., U.S. Patent 3,720,760, disclose a radioimmunoassa for the in_ vitro detection of antibodies to specific al ⁇ lergens.
• the method comprises reacting serum from a pati with single allergens attached to a water-insoluble soli phase.
• the antibodies present in the patient's serum bi to the solid phase-allergen and are detected by subseque reaction with a radioactively-labeled second antibody re active against the patient's antibody.
• This general in vitro technique is termed "RAST" for Radio-Allergo-Sorbe Test. Modifications of the test include coupling indivi allergens to paper discs and other cellulosic materials as Sepharose or cellulose, and polymeric materials such polyvinyl or polystyrene microtiter wells.
• an enzyme-labeled second antibody to human IgE, the test ha been converted to a nonisotopic assay.
• U.S. Patent 3,941,876 to Marinkovich discloses a method for determini the allergic hypersensitivity to a large number of allerg which are coated to cellulose threads fixed within a sing test chamber.
• the chamber is filled with serum sample an incubated for 16-24 hours, then washed 3 times and filled with radioactively labeled antilgE antibody reagent. Aft another 16-24 hours incubation, the chamber is washed aga and bound radioactivity to specific allergen bands measur by exposure onto photographic film.
• a further developmen of this technique utilizes a che ilumine-scent-labeled an IgE antibody reagent (Brown, C.R.
• Acta 121: 225-230, 1982 describe a device for use in detecti or determining the presence of antigenic or haptenic sub ⁇ stances or antibodies in a sample comprising a plurality tubular or capillary elements each having antibodies, ant genic or haptenic substances attached to the internal sur face thereof, and means for causing fluids to pass simul ⁇ taneously or sequentially through the plurality of capill elements.
• a method and test kit for detecting and deter- mining the presence of antigenic or haptenic substances, e.g., snake venoms, in a sample by an enzyme-linked immun sorbent assay technique is described as using urease as t enzyme in an antibody-enzyme or antigen-enzyme conjugate, with urea being used as the enzyme substrate wherein the presence of ammonia is detected or determined using di- bromo-O-cresolsulfophthalein indicator.
• Similar capillar immunoassays have been described by Friedel in Acta Endo- crinol ⁇ Suppl.
• capillary tubin offers the advantages over the device described in U.S. Patent 3,941,876 of requiring much smaller volumes of re ⁇ agents and, by virtue of the much larger surface area to volume ratio, much faster reaction kinetics.
• This capill device described by Chandler and Hurrell (1982) utilizes glass capillary tubes which break easily in shipment and handling. In addition, assembling such a device of multi capillaries into a single tubular chain is difficult and time-consuming with the connections likely to leak reagen fluid during the test.
• the present invention describes a device and its app cation for testing a fluid sample simultaneously and rapi for the presence of antibodies to a plurality of antigens for the presence of antigens to a plurality of antibodies combinations thereof.
• the device consists of a capillar tubing encasing a series of water-insoluble segments, ea having different antigens and/or antibodies attached to surface, and a means of filling and emptying the device reagents.
• the analytes in the sample, whether antibody antigen, are immobilized through immunocomplexing with specific reagents on the segment surface and are measure subsequent reaction with labeled immunochemical reagents. Measurement of bound analyte is possible by monitoring radiation emitted through the wall of the tubing about specific segments.
• Figure 1 is a sectional view through the device (sc of 1) depicted as a particular embodiment for use in scr ing for allergies.
• a capillary tubing, 3, contains with it six reactive water-insoluble segments 4, 5, 6, 7, 8 a separated by non-reactive water-insoluble segments 10.
• syringe, 1 is connected to the capillary tubing through means of attachment, 2.
• a tubing, 11, of narrower bore is attached.
• Fig 2 is an enlarged view (scale of 4) of a portion of the device.
• Each of the six reactive segments has a differen immunochemical reagent attached to its surface as follows Segment 4 - A 1st mixture of allergens e.g.
• grasses Segment 5 A 2nd mixture of allergens e.g. mite/mou Segment 6 - A 3rd mixture of allergens e.g. weeds Segment 7 - A 4th mixture of allergens e.g. trees Segment 8 - A 5th mixture of allergens e.g. cat and allergens Segment 9 -
• Anti- IgX antibodies e.g. anti-human IgE antibodies
• the test of the present invention is particularly suited for analyzing a single biological fluid for the presence of antibodies to a panel of antigens and/or for presence of antigens to a panel or antibodies.
• Reactions are carried out within a capillary tubing chamber which, because of a high surface area to volume ratio, requires small reagent volumes and much shorter reaction times tha conventional immunoassays.
• the device is used to detect IgE antibodies to defined allergens as well as Total IgE levels in serum or plasma.
• reaction wi substrate produces color changes that can be recorded vis ually through the clear encasing capillary tubing.
• the t is ideally suited for use in a physician's office since i requires little technical skill to perform, is completed less than three hours and requires no instrumentation for testing or measurement.
• the probability of false negatives is red by utilizing a mixture of two-to-four of the major allerg in a grass mix, or weed, tree, animal or mite/mould mixes immobilized on the solid surface.
• the three floral allergen mix segments in the te device i.e., grass, weed, and tree mixes, in specialize regional allergy screens will contain the major floral allergens specific for a season and geographical region. The remaining cat, dog, mite and mould allergens would b common to regional screens across the U.S.A.
• the major allergens in any restricted region throughout world should be used in the test device for clinically diagnosing IgE antibodies and hence allergies in patient within that region.
• More specialized screens can be ass bled wherein the allergens in a single device are restri to grasses only, or weeds, trees, moulds, foods, insect stings, only. Single allergens can be coupled to each s segment as well as mixtures of allergens.
• Preparation of the device of this invention involve coupling allergens, antigens or antibodies to the surfac a water-insoluble solid phase.
• exemplary materials of t solid phase include glass, hydrocarbon polymers such as polystyrene, polyethylene, polyvinyl chloride, polypro ⁇ pylene, nylon, etc. or copolymers.
• the surfaces may be treated in such a way to form reactive functional groups such as H 2 , COOH, isocyanate, to facilitate the conjuga of reagent to the solid surface.
• the antigens or antibo may be attached to the solid phase surface by known tech niques such as passive adsorption and covalent bonding.
• allergen extracts can differ in potency from lot to lot and from one allergen to another, each extract solution should be coupled at different con ⁇ centrations to the solid phase and tested with sera con ⁇ taining antibody to the allergen to define conditions pro ducing maximum reactive capacity.
• concentra tions of individual allergenic components in the mixture should be similarly optimized. By limiting the number of components in a mixture to two-to-four major allergens, steric interference of binding of antibody to any one com ponent can be minimized.
• solid poly vinyl chloride (PVC) rod-like segments 1 cm long by 0.18 diameter are activated with a 1% (v/v) aqueous glutaralde hyde solution, washed, then reacted with allergen or anti body solution for 16-24 hours, washed, then postcoated to stabilize the immobilized allergen or antibody.
• Segments coated with different allergens and anti-IgE " antibody are inserted into a clear PVC capillary tubing of 0.23 cm inner diamet with inert segments, 10, acting as spacers separating the reactive segments.
• the inert segments may be cut from th same PVC material as the reactive segments but used un- treated as spacers.
• a narrower capillary tubing, 11 which serves to retain the segments and through which reagents are drawn into the apparatus.
• the distal end of the capillary tubi is connected to a syringe which is used to fill and empty the device of reagents.
• the cross-sectional dimension of the segments should be large enough to prevent one segmen overlapping another within the capillary tubing.
• the le of the capillary tubing should be sufficient to contain segments arranged end-to-end.
• a small inner diameter of capillary tubing, 3, is preferred for rapid kinetics of reaction and to minimize the volume of reagents required the test.
• the capillary tubing of optically clear mater is preferred to facilitate the detection of positive, spe fic changes in color or fluorescence about reactive segme within the device.
• inert spacers sepa ⁇ rating reactive segments are preferred and should be of sufficient length to restrict diffusion of colored produc from one reactive segment to another, leading to false positives within the desired time of measurement.
• 1.5 cm spacers have been used to allow measurement of specific positive signals up to at least o hour after filling the tubing with substrate.
• the lengths of reactive segments are not critical and may vary between 0.5 and 2 cm.
• the reactive segments should be of equal length and inert spacers may omitted.
• the capillary tubing, 3 is 18.5 cm long and 0.23 inner diameter, contains within six 1 x 0.18 cm reactive segments, 4-9, separated by seven 1.5 x 0.18 cm inert spacers, 10.
• the outlet tubing, 11, is 6 cm long, 0.23 c outer diameter and 0.15 cm inner diameter, and is attache by gluing to the capillary tubing, 3, to prevent leakage reagents.
• the syringe connector, 2, may also be glued in the capillary tubing to prevent leakage.
• One embodiment of the invention involves drawing the sample fluid into the capillary tubing which contains six reactive segments coated with a grass allergen mix, a weed allergen mix, a tree allergen mix, a mixture of cat and do hair allergens, a mixture of house dust mite and mould allergens and anti-Ige antibody as illustrated in Figure 1
• the apparatus stands at ambient temperature for a predeter mined time to allow IgE antibodies to react with the coate segments, forming insolubilized immune complexes on their surfaces.
• the reaction time and temperature may vary wide from 5 minutes to 24 hours and 0°C to 50°C, respectively. Most preferably, because of the rapid kinetics of reaction within capillaries (Friedel, R., Acta Endocrinol., Suppl.
• the reaction is carried out for 5 to 60 minutes at 18° to 30°C.
• the sampl fluid is then expelled and the apparatus rinsed internally with water, or buffer of neutral pH (pH 5 - 9) or detergen in water or buffer, to remove protein not specifically adsorbed to the surface of reactive segments within the device.
• Labeled second antibody, specific to IgE from the sample which is complexed to the reactive surface is next drawn into the apparatus to fill the capillary tubing.
• Reaction is allowed to proceed at ambient temperature for predetermined time, dictated principally by the affinity a concentration of the second antibody.
• High affinity anti ⁇ bodies require short reaction times of 5-30 minutes, while low affinity antibodies require longer times.
• the labeled antibody is a monoclonal antibody
• this modified procedure eliminates the need for a second reaction period with the labeled antibody.
• the apparatus is rinsed again to remove excess labele antibody in solution or nonspecifically adsorbed to sur ⁇ faces.
• the reactive segments are then remove and specifically bound radioactivity measured in an appro- -U
• the label used is an enzyme conjugated to the se antibody
• substrate reagent is drawn into the apparatus t fill the tubing.
• Enzyme-antibody complexed with IgE on reactive surface catalyses the conversion of substrate t product which in the presence of chromogen leads to a vi ble color change around reactive segments to which IgE antibody from the sample has complexed.
• enzyme- substrate systems can be utilized in the test.
• Typical examples are an antibody-horseradish peroxidase conjugat which with hydrogen peroxide (substrate) and tetramethyl benzidine (chromogen) produces a colorless to blue color change, and an antibody-urease conjugate which with urea (substrate) and bromocresol purple (chromogen) produces yellow to purple color change.
• test device In addition to i munoassay systems which produce vis ble color changes, other immunoassay systems may be emplo that yield detectable fluorescent, luminescent or colored reaction products around or upon the surface of reactive segments. Qualitative applications of the test device ca be realized where the positive signal can be recorded by visual examination, such as in the case of fluorescent or colored products. Quantitative applications of the cest device are possible with suitable instrumentation for mea- suring light energy emitted through the wall of the capil ⁇ lary tubing.
• the device of this invention can be appli to a variety of immunoassay procedures, of which the fol ⁇ lowing are some examples: 1.
• Antigens or haptens in the sample compete with labeled antigens or haptens for binding to insolu- bilized antigen- or hapten-specific antibod and are measured bv the extent of inhibitio of binding of labeled antigen or hapten t its homologous antibody on a particular segment.
• the device and method of this invention can b utilized both as a qualitative and quantitative test, no only for antibodies mediating allergies, but for other multiple analytes, such as a panel of cancer antigens o viral antigens, e.g.. Hepatitis A, B, HTLV-III, or bacte antigens, or small molecules or haptens such as drugs of abuse, as well as for the presence of antibodies to these antigens or haptens.
• the invention also contemplates a kit for testing a sample for Total IgE and IgE antibodies to allergens.
• the kit will contain the assembled capillary tubing device and syringe, labeled second antibody reagen (and substrate reagent if an EIA) , wash reagent and instr tions for use.
• This example demonstrates the preparation of the cap lary tubing device.
• the segments are washed rapidly in pH 7 buffer to remove excess glutaralde hyde, then reacted with the allergen or a mixture of alle gens at an appropriate dilution in 0.01 M pH 7 buffer for + 4 hours at ambient temperature with gentle rotation. Th segments are washed in pH 7 buffer, then reacted with 0.1% (w/v) human serum albumin (HSA) s ⁇ / 4 hours at ambient temperature to block residual glutaraldehyde-active sites their surface. Finally, the segments are treated with a 2.5% (w/v) Sucrose solution in physiological saline / ⁇ _ * 4 hours at ambient temperature. The Sucrose solution is removed and the segments allowed to dry at ambient temper ⁇ ature.
• HSA human serum albumin
• 1 cm segments are coated with monospecific anti-(human IgE) IgG antibody by activation with 1% glu ⁇ taraldehyde, reaction with the IgG antibody at an appro ⁇ priate dilution, followed by treatment with HSA and Sucros solutions, then dried.
• monospecific anti-(human IgE) IgG antibody by activation with 1% glu ⁇ taraldehyde, reaction with the IgG antibody at an appro ⁇ priate dilution, followed by treatment with HSA and Sucros solutions, then dried.
• separate lots of segments are coated with (1) a mixture of Bermuda, Kentucky Blue, Timothy and Perennial Rye grass allergens, (2) a mixture.of Short Rag ⁇ weed, English Plantain and Lamb's Quarters weed allergens, (3) a mixture of Cottonwood, Elm, Mesquite and Mountain Cedar tree allergens, (4) a mixture of D. farinae mite an Alternaria tenuis mould allergens, (5) a mixture of cat a dog
• the allergen or antibody solution is coupled to the segments at increasing concentrations, then tested wi highly allergic human sera of appropriate specificity or Total IgE level and labeled ( 125I- or urease enzyme-) mono specific anti-IgE antibody to determine the optimum coati concentration of the allergen or antibody solution.
• Optically clear, semirigid PVC capillary tubing 18.5 cm long by 0.23 cm inner diameter, is first fitted wi a syringe connector at one end. The tubing is then loade with one each of the dried reactive segments coated with grass mix, mite/mould mix, weed mix, tree mix, cat/dog mi and anti-IgE antibody. A narrower tubing, 6 cm long, is attached to the other end of the capillary tubing for in sertion into reagents. Finally, a syringe (1 ml capacit is connected to the capillary tubing device.
• the assemb apparatus is illustrated in Figure 1.
• inert untreated PVC segments 1.5 cm long and cut from the same or similar material as the reactive segmen are inserted alternately with the reactive segments to a as spacers between the segments. If the device is to be used in a radioimmunoassay, the inert spacers may be omi ted.
• This example demonstrates the application of the de as a quantitative screen for allergies in a radioimmuno- assay.
• Affinity purified goat anti-human IgE antibody (Atla t c Antibodies Scarborough, ME) is labeled with 125I usin the chloramme-T method.
• the 125I-antibody is isolated b
• Quantitative device RAST Quantitative device RAST. With the aid of the attached syringe, the sample is drawn into the device to the syringe connector. The apparatus is allowed to si at ambient temperature for 2 hours. The sample is dispel from the tubing and the syringe plunger removed completel Using a squirt bottle 1 ml of the Triton X, NaCl wash sol tion is poured into the syringe barrel and allowed to dra through the device washing the segments.
• the plunger is replaced in the syringe and the capillary tubing filled with 125I-anti-human IgE. Th apparatus is allowed to sit at ambient temperature for 16 hours. The tracer is dispelled, the syringe plunger remo and the device washed three times with 1 ml wash solution
• the segments are carefully removed from th capillary tubing in order, placed into test tubes and the bound radioactivity measured.
• the CPM bound on the reactive segments are much lowe because (1) there is a higher density of the allergens on the discs compared to the plastic segments, and (2) the volumes of reagents (sample and tracer) in the paper disc RAST are more than 6 times those involved in the reaction with each segment in the device. If the CPM bound speci ⁇ fically is expressed as a percentage of the Total CPM of
• the device of the invention can be used to quantitatively assay IgE antibod to a variety of allergens in a single test procedure, wit good correlation to the conventional RAST with respect to intensity and specificity of signals but with much less hands-on time and technical skill required by the procedu
• Monospecific anti- (human IgE) goat IgG isolated from serum by (NH. SO. precipitation and molecular sievin through an AcA34 column is coupled to Type VII Urease enzy using N-Succinimidyl 3- (2-pyridyldithio) propionate (SPDP)
• SPDP N-Succinimidyl 3- (2-pyridyldithio) propionate
• the active conjugate is purified by filtration of the re ⁇ action product on an AcA22 (1.5 x 120 cm) column.
• the conjugate is diluted in physiological saline containing protein stabilizers, detergent and EDTA to the optimum concentration required in the test.
• the substrate/indicator solution is prepared by dissolving 40 mg Bromocresol Purple indicator in 7.4 ml 0. M NaOH and diluting to 450 ml with deionized water.
• 500 m Urea substrate and 37 mg disodium EDTA are then added to t BCP solution and the
• Serum Samples Serum samples tested in the devi in an EIA have been characterized for the level of IgE antibodies to the various allergens by the quantitative paper disc RAST and for levels of Total IgE by a commer ⁇ cially available kit, the Kallestad QuantiCLONE RIA for Total IgE.
• Step 3 Dispel the conjugate and wash the device as described in Step 3 hereinabove four times with 1 ml wash solution. Replace the syrin plunger. 6. Fill the capillary tubing with the substrat indicator solution, lay the device down on the bench and observe for the occurrence of specific yellow to purple color changes in the solution about each reactive segments within the next hour.
• the quantitative results obtained in the RAST can be more easily compared with the subjective, semi quantitative results, scored visually on a scale of 0 to 4 in the Allergy Screen EIA.
• the results of the RAST are compared with those of the EIA scored after 30 minutes incubation with the substrate/indicator.

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• 1988
  • 1988-06-23 AU AU20873/88A patent/AU2087388A/en not_active Abandoned
  • 1988-06-23 WO PCT/US1988/002132 patent/WO1989000290A1/en not_active Application Discontinuation
  • 1988-06-23 JP JP50626588A patent/JPH02504188A/ja active Pending
  • 1988-06-23 EP EP19880906563 patent/EP0367794A4/en not_active Withdrawn
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