EP0601131A1 - Bindung von milchallergenen an eine festphase - Google Patents
Bindung von milchallergenen an eine festphaseInfo
- Publication number
- EP0601131A1 EP0601131A1 EP92923470A EP92923470A EP0601131A1 EP 0601131 A1 EP0601131 A1 EP 0601131A1 EP 92923470 A EP92923470 A EP 92923470A EP 92923470 A EP92923470 A EP 92923470A EP 0601131 A1 EP0601131 A1 EP 0601131A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- allergen
- solid phase
- composition
- milk
- casein
- 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.)
- Withdrawn
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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 the binding of antigens related to milk allergens to a solid phase material for use in a diagnostic assay for immunoglobulins that bind to said allergens.
- the invention improves the sensitivity of a diagnostic assay for immunoglobulins, suspected of being present in a blood sample, that bind to proteins found in milk.
- U.S. Patent No. 3,720,760 discloses that certain immunogenic substances, called allergens, can give rise to allergic reactions in the form of asthma, hay fever, and the like, and that the blood of a patient in whom a given allergen causes an allergic reaction usually contains low concentrations of immunoglobulins, called reagin- immunoglobulins (now usually called "IgE", which term is subsequently used herein), which are directed specifically against that allergen.
- the patent discloses a test for sensitivity to allergens which involves injecting given allergens into the skin of a patient; a skilled observer then assesses the degree of sensitivity to each of the allergens on the basis of the observed reaction (reddening or swelling of the skin) caused by each allergen.
- the patent also discloses an "in vivo" test, where the patient inhales an allergen in the form of an aerosol, and the patient is deemed to be sensitive to any allergen that causes hay fever, asthma or like symptoms.
- the patent further discloses an in vitro method for determining the presence of IgE antibodies in a body fluid.
- the method involves binding an allergen to fine particles of a copolymer, e.g., a dextran-epichlorohydrin copolymer, by treating the particles with cyanogen bromide, and suspending the particles and an allergen in an aqueous medium.
- a body fluid to be tested for the presence of IgE antibodies directed against that allergen is then contacted with the allergen bound to the copolymer.
- the product of step (2) is then brought into contact with radio-labeled antibodies which will bind to IgE antibodies, if any, that has become bound to the allergen that is bound to the copolymer.
- the radiation emitted from the solids of step (3), the liquid of step (3), or both can then be measured.
- cross-linking agents couple spontaneously in the dark to available amino groups, as in aminopropyl glass, aminophenyl glass and aminohexylagarose, and when activated by irradiation with light of a suitable wavelength, these agents also couple with a ligand such as a drug, digoxin, a steroid, or a protein.
- U.S. Patent No. 4,425,434 describes the use of biologically active substances to fill the pores of porous titania spheroids, porous calcium phosphate spheroids, porous zirconia spheroids, or similar porous support material, and that the biologically active substance can then be immobilized in the pores by precipitation and corss-linking.
- the biologically active substance can be a proteinaceous substance, such as an enzyme.
- Milk comprises a variety of proteins including ⁇ s-casein, ⁇ -casein, ⁇ -casein, ⁇ -casein, ⁇ -lactoglobulin, bovine serum albumin, ⁇ -lactalbumin, immunoglobulins, such as bovine gamma globulins, and proteose peptones.
- Milk allergy is defined as those reactions induced by the ingestion of milk or its components for which an immunological pathogenesis may be demonstrated.
- Pharmacia Diagnostics (Piscataway, New Jersey) markets in vitro radioimmunoassay and enzyme immunoassay allergy products (Phadebas RAST® and Phadezym® RAST) that detect and quantitate IgE antibodies for a variety of allergens including milk and milk proteins.
- the Pharmacia tests are performed individually utilizing a disk having the allergen of interest bound thereto. The disk is incubated with the patient sample, washed, incubated with labeled goat anti-human-lgE IgG and washed and then the amount of label on the disk is measured.
- Pharmacia markets tests for milk, ⁇ -lactalbumin, ⁇ -lactoglobulin, and casein.
- the present invention involves novel allergen compositions, using a solvent such as deionized or distilled water, containing (in milligrams of protein per milliliter, as determined by a suitable protein test): from about 0.05 to about 4.0 of Alternaria alternata allergen; from about 0.5 to about 50 Aspergilus f ⁇ migatus allergen; from about 0.8 to about 81.6 of Bermuda grass ⁇ Cynodon dactylon) allergen; from about 0.1 to about 6.0 of birch (Betula nigra) allergen; from about 0.6 to about 20.6 of cat (Felis domesticus) allergen; from about 0.04 to about 4.5 of mountain cedar (Juniperus ashei) allergen; from about 0.1 to about 20.5 of Japanese cedar (Cryptomeria japonica) allergen; from about 0.05 to about 10.0 of Cladosporium allergen; from about 1.3 to about 38.4 of dog (Canis famifiarus) allergen; from about
- farinae allergen from about 0.6 to about 84.2 of D. pteronyssinus allergen; from about 0.1 to about 10.0 of elm (Ulmus) allergen; from about 0.02 to about 2.0 of feather allergen; from about 0.2 to about 20.5 of giant ragweed (Ambrosia trifida) allergen; from about 0.4 to about 100 of house dust allergen; from about 0.05 to about 10.5 of June/Kentucky bluegrass (Poa pratensis) allergen; from about 0.2 to about 20.5 of lamb's quarters (Chenopodium album) allergen; from about 0.1 to about 11.5 of maple (Acer) allergen; from about 0.3 to about 90.4 of mugwort (Artemesia ⁇ eferop ⁇ y//a)alIergen; from about 0.1 to about 12 of mulberry (Morus) allergen; from about 0.2 to about 25.5 of oak (Quercus) allergen; from about 0.1 to
- the present invention also involves devices for detecting the presence or amount of IgE antibodies in a test sample
- the assay devices include a solid phase and an allergen immobilized upon the solid phase, wherein the allergen is typically applied as one of the above allergen compositions.
- the allergen composition is combined with a pretreatment substance such as a denaturant, organic solvent, cross-linking agent or concentrated salt solution. It has been unexpectedly found that such allergen pretreatment can enhance allergen immobilization upon the solid phase.
- the reaction or binding area of the solid phase can be optionally modified by the addition of a protein blocking reagent. Suitable blocking reagents include equine serum albumin, bovine serum albumin, fish gelatin, casein and the like.
- the present invention describes allergen compositions containing a solvent, an allergen solubilized in the solvent, thereby forming an allergen solution, and a pretreatment substance chosen from denaturants, organic solvents, cross-linking agents or concentrated salt solutions, wherein the allergen solution is combined with the pretreatment substance, and wherein the resultant composition is used for the in vitro detection of the presence or amount of IgE antibodies in a test sample.
- a pretreatment substance chosen from denaturants, organic solvents, cross-linking agents or concentrated salt solutions
- the present invention describes a composition of milk proteins which enhance the sensitivity of the in vitro detection of the presence or amount of anti-milk-protein IgE antibodies.
- In vitro detection methods can involve: providing a solid phase prepared by applying the novel allergen compositions or pretreated allergen compositions to the solid phase; contacting the sample to be tested to that solid phase, thereby immobilizing allergen-specific IgE antibody from the sample upon the solid phase by forming allergen/antibody complexes; and detecting that immobilized allergen-specific antibody to determine the presence or amount of the antibody in the test sample.
- the solid phase is contacted with an indicator reagent to determine the presence or mount of IgE antibodies in the test sample, wherein the indicator reagent includes a label conjugated to a binding member that is specific for either the allergen, IgE antibodies or an ancillary specific binding member.
- the label that is selected is not critical to the present invention and is typically chosen from chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, radioactive isotopes, colloidal metallic particles, colloidal selenium particles, dye particles, enzymes, substrates, organic polymer latex particles and liposomes or other vesicles containing signal producing components.
- the present invention also includes assay kits containing the allergen or allergens of interest immobilized upon the solid phase and a suitable indicator reagent.
- the kit can include assay buffers and wash reagents.
- FIG. 1 is an enlarged top plan view of a preferred embodiment of a reaction cartridge support having a well containing a test card which has discrete test sites with moats.
- FIG. 2 is a magnified view, partially cutaway, through lines 8-8 (FIG. 1) showing sample test sites and moats in a preferred laminate structure of the test card.
- FIG. 3 is a plot of anti-milk-protein IgE antibody containing plasma sample pools analyzed on a solid phase containing only milk protein extract and on a solid phase containing the milk protein composition of milk protein extract, casein and ⁇ -lactoglobulin.
- the relative color intensities (with background subtracted) of the solid phase test sites (measured by reflectance) are plotted against the concentration of IgE antibodies (PRU/ml) in the pools.
- the present invention is based upon the discovery that an allergen solution can be used to bind an allergen to a solid phase material without the need for covalent linkages.
- a solid phase so prepared can then be used in an in vitro diagnostic assay for IgE antibodies.
- Suitable solid phase materials include cellulose nitrate or a mixed ester cellulose.
- certain allergen concentrations are optimum insofar as the sensitivity of the assay is concerned.
- a composition containing certain milk protein concentrations are optimum insofar as the sensitivity of an assay for anti- milk-protein IgE antibodies is concerned.
- the invention is also based upon the discovery that many allergens can be pretreated to improve their adherence to the solid phase material.
- the allergen pretreatment methods of the present invention serve to enhance the binding of the allergen to the solid phase throughout the assay.
- the allergen pretreatment compositions and methods were also unexpectedly found to increase the amount of allergen which can be bound to the solid phase thereby enabling the binding of allergen in an amount that is optimal for the assay.
- the present invention involves novel allergen compositions for the preparation of solid phase devices used in binding assays.
- the allergen compositions have been unexpectedly found to enhance the binding of the allergen to the solid phase material. As a result, greater amounts of antigen may be immobilized upon the solid phase, thereby providing more antigenic sites for binding antibody during the assay.
- the present invention also involves the pretreatment of certain allergen compositions with substances such as denaturants, organic solvents, cross-linking agents, and concentrated salt solutions.
- Pretreatment of an allergen composition with one or more of these substances was unexpectedly found to enhance the adherence of the allergen to a solid phase throughout the assay procedure which may include multiple washing steps or other manipulations which could otherwise dislodge the allergen form the solid phase.
- the pretreatment of the allergen improves their binding performance at elevated temperatures often used in binding assays.
- Suitable denaturants include, but are not limited to: acids such as hydrochloric acid (HCI) and acetic acid.
- Organic solvents, such as tetrahydrofuran, are suitable for allergen pretreatment.
- Concentrated salt solutions such as concentrated solutions of sodium chloride (NaCI) are also suitable for allergen pretreatment according to the present invention.
- Suitable cross-linking agents for the pretreatment of allergens include, but are not limited to: formaldehyde, glutaraldehyde and 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC).
- Allergen compositions combined with such pretreatment substances are then used in the production of novel solid phase assay devices.
- the allergen compositions or pretreated allergen compositions are applied to a solid phase material upon which the allergen composition is dried and thereby immobilized.
- the solid phase devices can then be used in binding assays which include, but are not limited to, competitive assays, sandwich assays and indirect assays, and include both forward and reverse assay formats.
- the allergen of interest is immobilized upon a solid phase material made of nitrocellulose or a nitrocellulose derivative or compound, such as cellulose acetate/nitrate mixed ester cellulose.
- the maximum binding capacity of nitrocellulose for the protein bovine serum albumin is about 140 ⁇ g/cm 2 . This binding capacity value is converted according to the desired size of the solid phase reaction or binding area of the present invention, and a value of 2.2 mg/ml is obtained. This concentration is used as the starting protein concentration for all allergens, but the optimum allergen concentration may be above or below this value. Different concentrations of allergen solutions are pretreated, immobilized on nitrocellulose and tested with a positive test sample, as described in the specific examples which follow. The allergen concentration is adjusted such that when concentration is plotted against signal a parabolic curve is obtained, and the optimum allergen concentration can be determined from the maximum detected signal.
- anti-milk-protein IgE antibodies can be detected by the methods described herein utilizing a composition comprising a milk protein extract, casein and ⁇ -lactoglobulin immobilized upon a suitable solid phase material.
- a milk protein extract is an extract of bovine milk comprising readily water soluble proteins.
- a milk protein extract can be prepared from lyophilized milk by re-solution of lyophilized fat-free milk powder in water or buffered water and removal of undissolved material.
- a preferred bovine milk protein extract is commercially available from Greer Laboratories (Lenoir, North Carolina) under the name "Milk, Cow". Casein powder is commercially available from Greer Laboratories (Lenoir, North Carolina). ⁇ -Lactoglobulin is commercially available from Sigma Chemical Company (St. Louis, Missouri).
- a preferred method of immobilizing this milk protein composition is as follows: combining individual solutions, preferably aqueous solutions such as de-ionized water and the like, of milk protein extract, casein and ⁇ -lactoglobulin into one solution comprising a final concentration of milk protein extract within the range of about 0.5 to about 2.0 mg/ml, more preferably at about 0.7 mg/ml, of casein within the range of about 0.5 to about 2.0 mg/ml, more preferably at about 1 mg/ml, and ⁇ -lactoglobulin within the range of about 5 to about 30 mg/ml, more preferably at about 18 mg/ml; pretreating the solution preferably by acidification, such as with 6N hydrochloric acid and the like; and depositing the desired quantity of this composition solution onto the solid phase.
- An alternative method involves depositing the individual solutions, after separately pretreating each of the individual solutions, sequentially onto the same location of the solid phase.
- a further preferred embodiment of the present invention involves the inclusion of bovine gamma globulin (BGG) in the milk protein composition solution disclosed herein above.
- BGG is found in bovine milk and is known to be present in low concentrations.
- the addition of BGG to the milk protein composition solution improves the sensitivity and specificity of assays for the detection of anti-milk-protein IgE antibodies.
- the final concentration of BGG in the milk protein composition disclosed herein is within the range of 0.5 to about 16 mg/ml and more preferably at about 5 mg/ml.
- a preferred solid phase is illustrated in Figures 1 and 2.
- Test card 82 preferably contains circular depressions 89 in the binding layer material which create an array of isolated test sites 84 preferably in close proximity to each other and each composed of binding layer material encircled by a moat 99 of air space.
- Test card 82 is preferably adhered to reaction cartridge 80 using two-sided adhesive tape on the bottom of reaction well 86, which is defined by well wall 88.
- Reaction well 86 is preferably provided with a removable, preferably transparent well cover 90 which preferably includes a reagent port 92 to facilitate the delivery and removal of fluids from the reaction well 86.
- the reaction cartridge 80 also preferably includes code means 94, such as an optical bar code, adapted to be read by an optical reader (not shown) and which is attached to or printed directly on the flat surface 91.
- code means 94 such as an optical bar code
- the reaction cartridge 80 also preferably includes a panel 96 which may include information such as the expiration date of the particular reaction cartridge, the lot number of the particular panel of capture reagents or assay binding components and the like.
- FIG. 2 is a magnified view, partially cutaway, through lines 8-8 of FIG. 1 showing sample test sites and moats in a preferred laminate structure of the test card.
- the test card is preferably a laminate structure comprising a binding layer 83 adhered to a non-absorbent substrate 85 using an adhesive 87.
- the porous structure of nitrocellulose has been found to have excellent absorption and adsorption qualities for a wide variety of fluid capture reagents which may be used in connection with the invention and is therefore preferred for binding layer 83.
- Polyester film such as MYLAR plastic having a thickness of approximately 0.002 inches is suitable for non- absorbent substrate 85.
- An adhesive backed polyester film is commercially available from several sources, such as Flexcon (Spencer, MA).
- a laminate structure suitable for use in test cards is commercially available from Millipore (Bedford, MA).
- a different analyte or allergen is delivered to each test site 84 so that a single sample can be simultaneously tested for the presence of binding components specific to each of a panel of different capture reagents.
- Some test sites 84 may have analyte delivered thereto to serve as positive control sites and some may have no reagent delivered thereto, to serve as negative control or reference sites.
- each test site 84 Preferably, from about 1.1 to about 5 ⁇ L and more preferably, from about 1.25 to about 4 ⁇ L of analyte or allergen solution is delivered to each test site 84 using any number of suitable delivery methods including reagent jetting, metered air pulsing, positive displacement pump, or by capillary tube lowered to the surface of the test site 84.
- suitable delivery methods including reagent jetting, metered air pulsing, positive displacement pump, or by capillary tube lowered to the surface of the test site 84.
- test sites 84 of a test card 82 are spotted with analyte or allergen, the test sites are allowed to dry thoroughly at room temperature.
- the binding layer 83 of test card 82 is preferably "blocked" with a protein coating such as inactivated horse serum or fish gelatin. Blocking masks potential non-specific binding sites on the binding layer 83. Suitable blocking is obtained during an incubation period of about 1 hour at approximately 37°C and is preferably accomplished in tanks with agitation during incubation. Following blocking, the test card 82 is washed, such as with 10 mM Tris buffered saline, and allowed to dry overnight.
- allergen contents herein refer to the protein content of the allergen solutions, determined using a suitable protein test such as Coomasie blue or Ninhydrin as are well- known in the art.
- analyte refers to the substance to be detected in or separated from test sample.
- the analyte can be any substance for which there exists a naturally occurring specific binding member or for which a specific binding member can be prepared.
- the analyte may bind to more than one specific binding member.
- Analyte also includes any antigenic substances, haptens, antibodies, and combinations thereof.
- the main analytes to be detected or measured are IgE antibodies.
- test sample refers to virtually any liquid sample.
- the test sample can be derived from any desired source, such as a physiological fluid, for example, blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid or the like.
- the liquid test sample can be pretreated prior to use, such as preparing plasma from blood, diluting viscous liquids, or the like; methods of treatment can also involve separation, filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents.
- a solid can be used once it is modified to form a liquid medium.
- specific binding member refers to a member of a specific binding pair, i.e., two different molecules wherein one of the molecules through chemical or physical means specifically binds to the second molecule.
- other specific binding pairs include, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonuclease, S-peptide and ribonuclease S-protein), and the like.
- specific binding pairs can include members that are analogs of the original specific binding member, for example an analyte- analog.
- the specific binding member is an immunoreactant it can be, for example, an antibody, antigen, hapten, or complex thereof.
- an antibody it can be a monoclonal or polyclonal antibody, a recombinant protein or antibody, a mixture or mixtures or a fragment or fragments thereof, as well as a mixture of an antibody and other specific binding members.
- the details of the preparation of such antibodies and their suitability for use as specific binding members are well-known to those skilled-in-the-art.
- an “indicator reagent”, as used herein, refers to a label attached to a specific binding member.
- the indicator reagent produces a detectable signal at a level relative to the amount of an analyte in the test sample.
- the indicator reagent is detected or measured after it is captured on the solid phase material, but the unbound indicator reagent can also be measured to determine the result of an assay.
- the specific binding member component of the indicator reagent enables the indirect binding of the label to the analyte, to an ancillary specific binding member, to the capture reagent or to a complex thereof.
- label refers to any substance which is attached to a specific binding member and which is capable of producing a signal that is detectable by visual or instrumental means.
- Suitable labels for use in the present invention can include chromogens; catalysts; fluorescent compounds; chemiluminescent compounds; radioactive isotopes; direct visual labels including colloidal metallic and non-metallic particles, dye particles, enzymes or substrates, or organic polymer latex particles; liposomes or other vesicles containing signal producing substances; and the like.
- an enzyme/substrate signal producing system useful in the present invention is the enzyme alkaline phosphatase wherein the substrate used can be 5-bromo-4-chloro-3-indolyl phosphate or a derivative or analog thereof. If horseradish peroxidase is used, o- Phenylenediamine or 4-chloro-naphthol is added as an enzyme substrate to form a colored product which can be detected and/or measured visually or instrumentally.
- the label can be a fluorescent compound where no enzymatic manipulation of the label is required to produce a detectable signal.
- Fluorescent molecules such as fluorescein, phycobiliprotein, rhodamine and their derivatives and analogs are suitable for use as labels in this system.
- An especially preferred class of labels includes the visually detectable, colored particles which enable a direct colored readout of the presence or concentration of the analyte in the test sample without the need for using additional signal producing reagents.
- Materials for use as such particles include colloidal metals, such as gold, and dye particles as disclosed in U.S. Patent Numbers 4,313,734 and 4,373,932.
- the preparation and use of non-metallic colloids, such as colloidal selenium particles, are disclosed in co- owned and copending U.S. Patent Application Serial No. 072,084, filed July 9, 1987, which is incorporated by reference herein in its entirety.
- Organic polymer latex particles for use as labels are disclosed in co-owned and copending U.S. Patent Application Serial No.
- indicator reagents can be formed by varying either the. label or the specific binding member; it will be appreciated by one skilled-in-the-art that the choice involves consideration of the analyte to be detected and the desired means of detection.
- the selection of a particular label is not critical, so long as the label is capable of generating a detectable signal either by itself or in conjunction with one or more additional signal producing components.
- the details of the preparation of such label/specific binding member conjugates are well-known to those skilled-in-the-art.
- signal producing component refers to any substance capable of reacting with another assay reagent or the analyte to produce a reaction product or signal that indicates the presence of the analyte and that is detectable by visual or instrumental means.
- Signal production system refers to the group of assay reagents that are needed to produce the desired reaction product or signal.
- one or more signal producing components can be used to react with a label and generate the detectable signal, i.e., when the label is an enzyme, amplification of the detectable signal is obtained by reacting the enzyme with one or more substrates or additional enzymes to produce a detectable reaction product.
- capture reagent refers to a capture binding member which is attached to a solid phase material to enable the separation of the analyte or indicator reagent, that is bound thereto, from unbound analyte and assay reagents.
- attachment of the capture binding member to the solid phase material is substantially irreversible.
- a capture reagent to be used in an assay, once the capture binding member, e.g., allergen, is immobilized upon the solid phase, the remaining surface area of the solid phase is generally blocked with a suitable inactivating solution, such as bovine or equine serum albumin, casein or other proteinaceous material, to prevent non-specific binding of protein to the solid phase when the reaction mixture containing a specific binding member is contacted to the solid phase.
- a suitable inactivating solution such as bovine or equine serum albumin, casein or other proteinaceous material
- the solid phase can be used as a separation mechanism.
- the reaction mixture can be contacted to the capture reagent, and the solid phase material retains the newly formed reaction complex(es).
- Solid phase material refers to any suitable chromatographic, bibulous, porous or capillary material or other conventional solid material, well-known to those skilled-in-the-art for use in immobilizing specific binding members.
- Solid phase materials can include fiberglass, nylon or cellulose or derivatives thereof, such as cellulose nitrate or a cellulose acetate/cellulose nitrate mixed ester cellulose. The solid phase, however, is not limited to porous materials.
- the solid phase material can also include, without limitation, polymeric or glass beads, microparticles, tubes, sheets, plates, slides, magnetic beads, a microtitre plate with one or more reaction wells or a glass or plastic test tube, or the like.
- Natural, synthetic or naturally occurring materials that are synthetically modified can be used as a solid phase material including polysaccharides, e.g., cellulose materials including paper, cellulose and cellulose derivatives such as cellulose acetate, nitrocellulose and cellulose acetate/nitrate mixed ester cellulose; silica; fiberglass; inorganic materials such as deactivated alumina, diatomaceous earth or other inorganic finely divided material uniformly dispersed in a porous polymer matrix, with polymers such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloride-vinyl acetate copolymer; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon); porous gels such as silica gel,
- the specific binding member of the capture reagent can be affixed to particles, e.g., microparticles.
- microparticles can serve as the solid phase material and be retained in a column, suspended in a mixture of soluble reagents and test sample, or retained and immobilized by another solid phase base material.
- retained and immobilized is meant that the microparticles, associated with the solid phase base material, are not capable of substantial movement to positions elsewhere within that material.
- the microparticles can be selected by one skilled- in-the-art from any suitable type of particulate material including those composed of polystyrene, polymethylacrylate, polypropylene, polytetrafluoroethylene, polyacrylonitrile, polycarbonate or similar materials.
- the size of the microparticles is not critical, although it is preferred that the average diameter be smaller than the average pore size of the solid phase base material if such is used.
- ancillary specific binding member refers to a specific binding member used in addition to the specific binding members of the capture reagent and the indicator reagent.
- One or more ancillary specific binding members can be used in an assay.
- an ancillary specific binding member can be used in an assay where the specific binding member of the indicator reagent is capable of binding the ancillary specific binding member which is in turn capable of binding the analyte.
- a sandwich assay can be performed wherein a capture reagent can include an allergen which has been bound to a solid phase material.
- the capture reagent is contacted with a test sample, suspected of containing the analyte, and an indicator reagent containing an analyte-specific binding member conjugated to a label.
- the reagents can be contacted to the sample simultaneously or added sequentially.
- a binding reaction results in the formation of a capture reagent/analyte/indicator reagent complex.
- the assay may also involve a washing step to separate the resultant complex from the excess reagents and test sample. Either the unreacted indicator reagent or the complex retained upon the solid phase is then observed to detect or measure the amount of label associated therewith. If analyte is present in the sample, then label will be present on the solid phase material. The amount of label on the solid phase is proportional to the amount of analyte in the sample.
- the present invention also can be used to conduct a competitive assay.
- the capture reagent again includes a specific binding member (allergen) which has been attached to a solid phase material.
- the capture reagent is contacted with both test sample and an indicator reagent that includes an analyte or analyte analog which has been labeled with a signal generating compound.
- the indicator reagent and analyte then compete in binding to the capture reagent.
- the competitive binding reaction results in the formation of capture reagent analyte complexes or capture reagent/indicator reagent complexes.
- the capture reagent/indicator reagent complexes can be detected via the label of the indicator reagent.
- the amount of label that becomes associated with the solid phase is inversely proportional to the amount of analyte in the sample.
- the present invention can also be used in indirect immunoassays involving one or more ancillary specific binding members. For example, an indirect sandwich immunoassay with the formation of a capture reagent/analyte/anti-analyte antibody/indicator reagent complex can be performed, wherein the indicator reagent is a specific binding partner for the ancillary specific binding member which is specific for the analyte.
- the present invention can also be used in forward and reverse immunoassay protocols
- Alternaria alternate allergen was pretreated for binding to a solid phase material.
- a 37% aqueous formaldehyde solution (12.5 ⁇ L) was mixed with 100 microliters of a solution of Alternaria alternata (28.8 ⁇ g/mL) in deionized water.
- the amount of formaldehyde effective for pretreatment was found to range from about 10 ⁇ L to about 20 ⁇ L when the 37% aqueous formaldehyde solution was used.
- the resulting mixture was incubated at 4°C for about 10 hours, and the incubated composition was allowed to stand for 30 to 60 minutes at about 20°C.
- the mixture was then centrifuged, and the resultant supernatant, a pretreated Alternaria alternata allergen composition, was decanted.
- the pretreated composition was poured onto a disc of microporous cellulose nitrate (about 140 ⁇ m thick and about 3 mm in diameter) and allowed to dry. The allergen was thereby immobilized upon the solid phase material. The remaining surface of the disc was then blocked with a ten percent horse serum solution.
- the solid phase bound allergen, or Alternaria alternata capture reagent was then used in an enzyme immunoassay ("EIA").
- EIA enzyme immunoassay
- the EIA method included the following steps.
- the sample to be tested e.g., serum
- the capture reagent was then contacted to an enzyme-labeled anti-lgE antibody (indicator reagent) which bound to that IgE antibodies from the sample, if any, which had bound to the solid phase.
- the solid phase was then washed to remove unbound indicator reagent.
- the solid phase was contacted to an enzyme substrate signal producing component such that the enzyme component of the complexed indicator reagent would react with the substrate to produce a detectable signal. Prior to detection, the solid phase may undergo a third washing to remove unbound substrate. The signal which was detected was directly related to the amount of allergen-specific IgE antibodies in the test sample.
- the enzyme label was alkaline phosphatase
- the substrate was 5-bromo-4-chloro-3- ⁇ ndolylphosphate
- the detection or measurement step was performed with a reflectance spectrophotometer.
- the disc turned dark blue upon the addition of substrate to the solid phase, i.e., a positive assay result, when the serum sample contained IgE antibody specific to Alternaria alternata.
- the assay procedure was repeated using serum from different patients, and the results were found to correlate with the results obtained for the same serum samples using alternate tests, such as a radio-allergo-sorbent test (RAST) or a skin prick test as are well-known in the art.
- RAST radio-allergo-sorbent test
- a skin prick test as are well-known in the art.
- the nitrocellulose disc used as the solid phase was one of many discs on a laminate composed of a mylar sheet to which a sheet of nitrocellulose had been glued. A circular shape was embossed onto the nitrocellulose sheet to form each of the discs. The micropores in the nitrocellulose sheet had diameters of about 450 nanometers. Each individual disc had a separate allergen attached thereto. Thus, the device could be used to detect the presence of antibodies to multiple allergens.
- Example 1 The procedure of Example 1 was repeated using 100 microliter portions of solutions containing birch allergen (137 ⁇ g) or dog allergen (280 ⁇ g) which were mixed with a 37% aqueous formaldehyde solution (12.5 ⁇ L) and incubated thereby forming pretreated allergen compositions.
- the amount of formaldehyde effective for pretreatment was found to range from about 10 ⁇ L to about 15 ⁇ L when the 37% aqueous formaldehyde solution was used.
- the compositions were used substantially in accordance with the procedures described in Examples 1 and 2 to produce devices which were then used to test serum samples.
- the assay results were found to correlate with the results of testing the same serum samples by other means: the disc turned dark blue when the serum sample contained IgE antibodies specific for the allergen immobilized upon the solid phase.
- Tetrahydrofuran 25 ⁇ L was mixed with 100 microliters of a solution containing Bermuda grass allergen
- the amount of tetrahydrofuran effective for pretreatment was found to range from about 10 ⁇ L to about 50 ⁇ L.
- the resulting mixture was incubated at 4°C for about 10 hours, and the incubated composition was allowed to stand at about 20°C for 30 to 60 minutes. The solution was then centrifuged, and the resultant supernatant, a pretreated Bermuda grass allergen composition, was decanted.
- the procedure was repeated using 100 microliter- portions of solutions which contained Japanese cedar allergen (150 ⁇ g), June/Kentucky blue grass allergen (545 ⁇ g), perennial rye allergen (433 ⁇ g) or timothy allergen (43 ⁇ g) in deionized water.
- the pretreated allergen compositions were used to produce solid phase discs and were used in immunoassays substantially in accordance with the procedure described in Example 1. The assay results were found to correlate with the results of testing the same serum samples by other means: the disc turned dark blue when the serum sample contained IgE antibodies specific for the allergen immobilized upon the solid phase.
- a 37 percent aqueous formaldehyde solution (15.6 ⁇ g) was mixed with 100 microliters of a solution containing mountain cedar allergen (733 ⁇ g) in deionized water. The resulting mixture was incubated at about 20°C for approximately 30 minutes. Tetrahydrofuran (28.7 ⁇ L) was then mixed with the incubated solution. The amount of formaldehyde effective for pretreatment was found to range from about 10 ⁇ L to about 20 ⁇ L , and the amount of tetrahydrofuran was found to range from about 10 ⁇ L to about 50 ⁇ L. . The mixture was incubated for about 10 hours at 4°C and was allowed to stand at about 20°C for 30 to 60 minutes. The mixture was then centrifuged, and the resultant supernatant, a pretreated cedar allergen composition, was decanted.
- Aqueous NaCI (5 M, 12 ⁇ L) was mixed with 100 microliters of a solution containing Cladosporium (960 ⁇ g) in deionized water. The resulting mixture was incubated at about 4°C for about 10 hours, and the incubated composition was then allowed to stand at about 20°C for 30 to 60 minutes. The mixture was then centrifuged, and the resultant supernatant, a pretreated Cladosporium allergen composition, was decanted. Depending upon the molar value of the concentrated salt solution used, which value ranged from about 0.5 M to about 10 M, the amount of aqueous NaCI effective for pretreatment ranged from about 10 ⁇ L to about 20 ⁇ L.
- the procedure was repeated using 100 microliters of a solution containing feather allergen (7 ⁇ g) in deionized water.
- the pretreated allergen compositions were then used to produce assay devices and were used in immunoassays substantially in accordance with the protocol described in Example 1.
- the assay results using the compositions and devices of the present invention were found to correlate with the results of testing the same serum samples by other means: the disc turned dark blue when the serum contacted thereto contained IgE antibodies specific for the allergen immobilized upon the solid phase.
- aqueous solution of 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC, 10 ⁇ L at 50 mg/mL) was mixed with 100 microliters of a solution containing D. farinae (280 ⁇ g) in deionized water.
- the amount of EDAC effective for the first stage of pretreatment ranged from about 5.0 ⁇ L to about 15 ⁇ L.
- the resulting mixture was incubated at about 22°C for about 15 minutes.
- the amount of NaBH4 effective for the second stage of pretreatment ranged from about 1.0 ⁇ L to about 5.0 ⁇ L.
- the mixture was then allowed to stand at about 20°C for approximately 30 to 60 minutes. The mixture was centrifuged, and the resultant supernatant, a pretreated D. farinae allergen composition, was decanted.
- the procedure was repeated using 100 microliters of a solution containing D. pteronyssinus (263 ⁇ g) in deionized water.
- the pretreated allergen compositions were used substantially in accordance with the procedures described in Example 1 to produce treated discs for immunoassays.
- the EIA results were found to correlate with the results of testing the same serum samples by other means: the disc turned dark blue when the serum sample contacted thereto contained IgE antibodies specific for the allergen immobilized upon the solid phase.
- One hundred percent acetic acid (12.5 ⁇ L, with effective amounts ranging from about 5.0 ⁇ L to about 30 ⁇ L) was mixed with 100 microliters of a solution containing lamb's quarters allergen (1176 ⁇ g) in deionized water.
- the resultant mixture was incubated at about 22°C for approximately five minutes, after which time 6 N aqueous NaOH was added to adjust the pH to 7.
- the neutralized solution was incubated at about 4°C for 10 hours, and was then allowed to stand at about 20°C for 30 to 60 minutes.
- the mixture was then centrifuged, and the resultant supernatant, a pretreated lamb's quarters allergen composition, was decanted.
- the procedure was repeated using 100 microliters of a solution containing mulberry allergen (40 ⁇ g) in deionized water.
- the pretreated allergen compositions were used to produce treated discs, for enzyme immunoassays substantially in accordance with the procedures described in Example 1.
- the assay results were found to correlate with the results of testing the same serum samples by other means: the disc turned dark blue when the serum sample contacted thereto contained IgE antibodies specific for the allergen immobilized upon the solid phase.
- a solution of 6 N aqueous HCI (24 ⁇ L, with effective amounts ranging from about 6.0 ⁇ L to about 30 ⁇ L) was mixed with 100 microliters of a solution containing Penicillium (120 ⁇ g).
- the resulting mixture was incubated for approximately five minutes at about 20°C, after which time 6 N aqueous NaOH was added to adjust the pH to 7.
- the neutralized solution was incubated at 4°C for 10 hours and was then allowed to stand at about 20°C for 30 to 60 minutes.
- the mixture was then centrifuged, and the resultant supernatant, a pretreated Penicillium allergen composition, was decanted.
- the allergen pretreatment procedure was repeated with 100 microliters of a solution containing Parietaria allergen
- Example 2 400 ⁇ g in deionized water.
- the solutions were then used substantially in accordance with the procedures described in Example 1 to produce discs and to test serum samples in an EIA.
- the assay results were found to correlate with the results of testing the same serum samples by other means: the disc turned dark blue when the serum contacted thereto contained IgE antibodies specific for the allergen immobilized upon the solid phase.
- Untreated allergen compositions included from about 0.5 to about 50 Aspergillus allergen; from about 0.6 to about 20.6 of cat allergen; from about 0.1 to about 10.0 of elm allergen; from about 0.4 to about 100 of house dust allergen; from about
- maple allergen from about 0.3 to about 90.4 of mugwort allergen and from about 1.7 to about 130.4 of plantain allergen in deionized water.
- Example 1 The solutions were then used substantially in accordance with the procedures described in Example 1 to produce discs and to test serum samples in an EIA.
- the assay results were found to correlate with the results of testing the same serum samples by other means: the disc turned dark blue when the serum sample contained IgE antibodies specific for the allergen immobilized upon the solid phase.
- Pretreated allergen compositions which were produced as described in Examples 1, and 3 through 9, and which differed from one another with respect to allergen content, were used to test for IgE antibodies in a series of serum samples.
- Upper and lower allergen concentration limits were set by classifying a pretreated allergen composition as either "too dilute” if that composition failed to produce a maximum positive IgE antibodies test result with a serum sample which had tested positive with a more concentrated allergen solution, or "too concentrated” if the composition failed to produce a maximum positive IgE antibodies test result with a serum sample which had tested positive with a less concentrated allergen solution.
- the allergen concentrations tested ranged from about 0.05 milligrams of allergen per milliliter of water, prior to pretreatment, to about 170 milligrams/milliliter.
- the test results are presented in Table 1 and illustrate the most effective concentration ranges for each of the allergens tested.
- Alternaria alternata allergen from 0.05 to 4.0 mg/mL
- Aspergillus fumigatus allergen from 0.5 to 50.0 mg/mL
- Bermuda grass allergen from 0.8 to 81.6 mg/mL birch allergen from 0.1 to 6.0 mg/mL mountain cedar allergen from 0.04 to 4.5 mg/mL
- Japanese cedar allergen from 0.1 to 20.5 mg/mL
- Cladosporium allergen from 0.05 to 38.4 mg/mL cat allergen from 0.6 to 20.6 mg/mL dog allergen from 1.3 to 38.4 mg/mL
- D. pteronyssinus allergen from 0.6 to 84.2 mg/mL elm allergen from 0.1 to 146.0 mg/mL feather allergen from 0.02 to 0.2 mg/mL giant ragweed allergen from 0.2 to 148.2 mg/mL house dust allergen from 0.4 to 100 mg/mL
- Parietaria allergen from 1.0 to 40.0 mg/mL plantain allergen from 1.7 to 130.4 mg/mL
- Penicillium allergen from 0.1 to 4.8 mg/mL perennial rye allergen from 0.05 to 17.3 mg/mL short ragweed allergen from 0.2 to 151.6 mg/mL timothy allergen from 0.05 to 6.6 mg/mL
- Example 12 Penicillium allergen from 0.1 to 4.8 mg/mL perennial rye allergen from 0.05 to 17.3 mg/mL short ragweed allergen from 0.2 to 151.6 mg/mL timothy allergen from 0.05 to 6.6 mg/mL
- a milk protein composition comprising milk protein extract, casein and ⁇ -lactoglobulin was prepared as follows.
- Casein powder (Greer Laboratories, Lenoir, North Carolina) was dissolved in de-ionized water at approximately 4 mg/ml.
- ⁇ -lactoglobulin powder (Sigma Chemical Company, St. Louis, Missouri) was dissolved in de-ionized water at approximately 72 mg/ml.
- the casein solution and the ⁇ -lactoglobulin solution were combined in a 1:1 volume ratio.
- Bovine milk protein extract powder (“Milk, Cow” from Greer Laboratories, Lenoir, North Carolina) was dissolved in de-ionized water at approximately 1.4 mg/ml.
- the ⁇ -lactoglobulin-casein solution was combined with the milk solution in a 1:1 volume ratio.
- the ⁇ -lactoglobulin-casein-milk solution was acidified by the addition of 6N HCI to make a final concentration of 0.06N HCI. After 5 minutes, the solution was neutralized by the addition of 6N NaOH followed by 1M HEPPS buffer, pH 8.3, to make a final concentration of 0.1 M HEPPS buffer. This solution was stored at 2-8°C for approximately 3 days. Before use, the solution was centrifuged to provide a clear solution which may be adsorbed onto nitrocellulose.
- a milk protein composition comprising milk protein extract, casein, ⁇ -lactoglobulin and BGG was prepared as follows.
- Casein powder (Greer Laboratories, Lenoir, North Carolina) was dissolved in de-ionized water at approximately 8 mg/ml.
- ⁇ -lactoglobulin powder (Sigma Chemical Company, St. Louis, Missouri) was dissolved in de-ionized water at approximately 144 mg/ml.
- the casein solution and the ⁇ - lactoglobulin solution were combined in a 1 :1 volume ratio.
- Bovine milk protein extract powder (“Milk, Cow” from Greer Laboratories, Lenoir, North Carolina) was dissolved in de ⁇ ionized water at approximately 2.8 mg/ml.
- the ⁇ - lactoglobulin-casein solution was combined with the milk solution in a 1 :1 volume ratio.
- the ⁇ -lactoglobulin-casein- milk solution was acidified by the addition of 6N HCI to make a final concentration of 0.06N HCI. After 5 minutes, the solution was neutralized by the addition of 6N NaOH followed by 1M HEPPS buffer, pH 8.3, to make a final concentration of 0.2M HEPPS buffer.
- Bovine gamma globulin (BGG) powder (Sigma Chemical Company, St. Louis, Missouri) was dissolved in de-ionized water at approximately 10 mg/ml.
- the ⁇ - lactoglobulin-casein-milk solution was then combined with the BGG solution in a 1 :1 volume ratio. This solution was stored at 2-8°C for approximately 3 days. Before use, the solution was centrifuged to provide a clear solution which may be adsorbed onto nitrocellulose.
- First test cards 82 with a test site 84 (see Figures 1 and 2) containing a milk protein composition of only milk protein extract powder ("Milk, Cow” from Greer Laboratories, Lenoir, North Carolina) were prepared by depositing 2 microliters of approximately a 0.7 mg/ml solution (0.1 M HEPPS buffer in de-ionized water), pretreated with acid according to the method in Examples 12 and 13, onto the test site and drying the test site at room temperature overnight.
- Second test cards 82 with a test site 84 containing a milk protein composition of milk protein extract, casein and ⁇ -lactoglobulin were prepared by depositing 2 microliters of the milk protein composition solution prepared according to Example 12 onto a test site and drying the test site at room temperature overnight.
- Third test cards 82 with a test site 84 containing a milk protein composition of milk protein extract, casein, ⁇ - lactoglobulin and BGG were prepared by depositing 2 microliters of the milk protein composition solution prepared according to Example 13 onto a test site and drying the test site at room temperature overnight.
- Pools of human plasma containing high levels of anti- milk-protein IgE antibodies were prepared as follows. Human plasma samples were tested for anti-milk-protein IgE antibodies using Phadebas RAST® (Pharmacia) Milk test. Samples were combined to produce plasma pools having at least 1.1 PRU/ml (expressed in Pharmacia Radioimmunoassay Units (PRU) per milliliter) of anti-milk-protein IgE antibodies.
- the plasma poofs were then serially diluted with about 0.1 M TRIS buffer saline (pH 7.2) containing serum proteins to produce pooled plasma samples containing about 0.1 , 0.17, 0.22, 0.34 and 1.12 PRU/ml of anti-milk-protein IgE antibodies.
- the anti-milk-protein IgE antibody positive plasma pools were analyzed for the presence of detectable amounts of anti-milk- protein IgE antibodies using the first (milk protein extract only), and second (milk protein extract, casein and ⁇ - lactoglobulin) test cards prepared above.
- the presence of IgE antibodies from the sample pools captured on the solid phase was detected by incubating the solid phase with alkaline phosphatase labeled goat anti-human-lgE IgG antibodies, washing the solid phase with buffer and adding the enzyme substrate 5-bromo-4-chloro-1-indolylphophate (BCIP).
- BCIP 5-bromo-4-chloro-1-indolylphophate
- the intensity of the color developed on the solid phase was measured with a reflectance detector. After subtracting background intensity, the relative color intensity of the samples analyzed on each test card were plotted against the concentration of IgE antibodies as measured by Phadebas RAST® (PRU/ml). The plot, as shown in Figure 3, illustrates the significant improvement in sensitivity realized by the use of the milk protein composition of Example 12 on the second test cards above.
- the presence of IgE antibodies from the sample captured on the solid phase was detected by incubating the solid phase with alkaline phosphatase labeled goat anti- human-lgE IgG followed by the addition of the enzyme substrate BCIP.
- the color intensity was again measured by a reflectance detector and adjusted for the background intensity.
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US79481291A | 1991-11-15 | 1991-11-15 | |
US794812 | 1991-11-15 | ||
PCT/US1992/009281 WO1993010458A1 (en) | 1991-11-15 | 1992-10-29 | Binding of milk allergens to a solid phase |
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Publication Number | Publication Date |
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EP0601131A1 true EP0601131A1 (de) | 1994-06-15 |
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EP92923470A Withdrawn EP0601131A1 (de) | 1991-11-15 | 1992-10-29 | Bindung von milchallergenen an eine festphase |
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EP (1) | EP0601131A1 (de) |
AU (1) | AU2922292A (de) |
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EP1564556A1 (de) | 2004-02-17 | 2005-08-17 | DST Diagnostic Science & Technology GmbH | Verfahren und Vorrichtung zur Bestimmung mehrerer Analyten mit simultaner interner Kontrolle |
CN102331497A (zh) * | 2011-06-15 | 2012-01-25 | 天津医科大学 | 牛奶过敏原检测试剂盒及其制备方法 |
CN103616520B (zh) * | 2013-12-05 | 2015-09-16 | 浙江天科高新技术发展有限公司 | 高灵敏食物过敏原检测方法及其试剂盒 |
CN104931710A (zh) * | 2015-06-17 | 2015-09-23 | 深圳大学 | 一种定量检测牛乳β-乳球蛋白的双抗体夹心法 |
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1992
- 1992-10-29 AU AU29222/92A patent/AU2922292A/en not_active Abandoned
- 1992-10-29 WO PCT/US1992/009281 patent/WO1993010458A1/en not_active Application Discontinuation
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