JPS59193356A - Fluorescent luminous intensity analysis for total ige concentration and reagent therefor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is disclosed in Patent Application No. 4764 filed on March 17, 1983.
No. 51, Patent Application No. 4625 filed on January 31, 1983
No. 85, Patent Application No. 444 filed on November 26, 1982
．． No. 622 and a continuation in part of Application No. 434,061 filed October 13, 1982.

The present invention relates to methods and reagents for analyzing serum of patients exhibiting allergic syndromes to determine total IgEtff1 degree in serum. In particular, the present invention relates to methods and reagents for measuring total ICIEI'1 degrees with improved speed and accuracy, the results of which are useful for the diagnosis of allergic diseases and for determining the sensitivity of individuals. It can be used with high confidence in the identification of different possible allergens.

Radiometric and fluorometric methods for identifying and quantifying allergy-specific IgE concentrations in patient serum are commercially available and are known, for example, as RAS technology. US Patent No. RE-29/17 April: 3555143
@;3648346@;3720760 and 3966
No. 898 deals with these methods d1 and reagents therefor.

Enzymatic immunological methods for identifying and quantifying antigens and antibodies in fluids are also widely used, e.g.
It is known as LISA and EfA.

Basic techniques and reagents for enzymatic analysis methods are described, for example, in US Pat. No. RE-29169:36540.
No. 90; No. 3791932, No. 3839153, 386
No. 7517@, No. 4016043 and No. 4098876. For example, PHADEBAS □II
The IE PRIS engineering labeled immunoassay test (Pharmacia) and the TANDEM labeled immunoassay test (Hybritic) are known. U.S. Pat. relates to a two-site sandwich method using monoclonal antibodies from a single clone or, if the antigen has two non-identical binding sites, unmixed monoclonal antibodies from two clones. .

An editorial on the circularity of techniques for immunoassays for the detection of proteins in solution can be found in R. Rose et al.
Handbook of Clinical Immunology, 2nd edition, American Society for Microbiology, Washington, DC, pp. 327-429, 775-84.9 (
1980> and A., Otter et al., “8o
8G Immunoassay" University Bark Breath, Baltimore (1981), and the references cited therein, the entire contents of both of these publications are hereby incorporated by reference. T, A. , E., Platts-Mil Is, et al., Chapter 2, “Discriminatory Immunoassay Methods in Parallelopathy A5” 2
Pages 89-311 and the references therein provide a review of the field of the invention.

Methods for binding proteins to insoluble carriers are primarily described. U.S. Patent No. 35515 b b
Fr, 35533108, 'to/18298 and [
(Antibodies can also be covalently attached to insoluble carriers, as described in U.S. Pat. No. 3,646-46 and U.S. Pat. No. 108 and U.S. Pat. No. 3,720,760j;
There is a covalent bond between the body and the body.

A. Polson et al., Biochim.

B 1oplrys, A ctaJ vol. 82, '16
pp. 3-415 (1964) and A. Bolson et al., Vo.
x3ang, Volume 23, pp. 107-118 (197
As noted by 2), polyethylene glycol has been used in protein fractionation methods.

The present invention relates to a method for determining the total IQ E1 degree in the serum of a patient. The invention involves first contacting the insoluble carrier to which the anti-IgE antibody is attached with the patient's serum for a sufficient period of time to allow conjugation of the anti-IgE antibody to JgE in the patient's serum. The patient's serum is then removed from the carrier. Second, the insoluble carrier is contacted with the enzyme-labeled anti-IgE antibody for a period of time sufficient to allow conjugation of serum 1gE bound to the insoluble carrier with the enzyme-labeled anti-IgE antibody used in the present invention. A fluorogenic enzyme is defined as an enzyme by which a suitable substrate undergoes a chemical reaction to produce a fluorescent product.

Unconjugated enzyme labeling then removes the anti-1gE antibody from the carrier. Third, the insoluble carrier is contacted with a solution of a substrate that, in the presence of fluorogenic enzymes, undergoes a chemical reaction to yield a fluorescent product; The intensity of the fluorescence in the solution is then measured.

In the insoluble anti-1qE antibody reagent of the present invention, anti-IGE
Antibodies can be attached to insoluble carriers by covalent bonds or non-covalent bonds, such as by absorption or adsorption. The anti-IgE antibody that adheres to an insoluble carrier can be prepared using a polysaccharide-animal protein complex prepared by treating the surface of the insoluble carrier to which the anti-IgE antibody is to be bound with an aqueous solution of polysaccharide and animal protein before drying. It is preferable to use the stabilizer. Preferably, the anti-ICE antibody is attached to an insoluble carrier by non-covalent bonding.

In some preferred embodiments of the invention, the insoluble carrier has a plurality of test compartments separated by an opaque material, the enzyme is alkaline bosphatase, and the enzyme labeled anti-1gE is 1 to 8 weight percent. 1000 to 1
0,000 molecular weight and a nonionic surfactant, and the substrate is phosphorus m4-methylumbelliferyl. The anti-I (IE antibody is an affinity chromatography-purified polyclonal antibody, a monoclonal antibody or preferably a combination of different monoclonal antibodies from several different clones) attached to an insoluble carrier and/or coupled to an enzyme. It can be a polyclonal antibody that is a mixture.

The key to effective treatment of allergic reactions is accurate identification of the offending allergen or allergens and titration of the patient to determine the desensitizing dose. Generally, the reduced allergen extract is injected in an amount sufficient to cause significant production of antigen specificity 1 (IG (blocking antibody)) and significant production and/or activation of Zabrethusa T lymphocytes. However, , the m should not be sufficient to cause a significant allergic reaction.

In order to produce increased concentrations of antigen-specific IoE, it is essential that IqG and Zapretusa IqE production be in a balance that prevents allergic reactions.

The concentration and duration of the desensitizing medication depends on many factors specific to the patient experiencing the allergic reaction. Therefore, it is necessary to titrate the patient to determine the appropriate dosage.

Various standard methods can be used to accomplish this procedure. An example of a commonly practiced method is Remington's Pharmacology.
ceutical science) 1344
It is described on pages 1352 to 1352. However, the methods used prior to the present invention lack specificity and precision to be more than a rough approximation of an appropriate starting dose range. Moreover, conventional methods do not provide an effective procedure for determining the total IE1 degree in a subject's serum, and as a result, allergen specificity
(Does not provide an effective strategy for correlating IE1 degree determinations to total [+ Eilif1 degrees.) The PRIST test takes several days to complete and the TANDEM test takes a full day.

In contrast, the test of the present invention can be completed in as little as two hours with great accuracy.

The method of the invention provides specificity for determining appropriate desensitization doses, especially when the allergen used for desensitization and the allergen component of the diagnostic method have the same allergen profile and specificity. and provide accuracy. In addition to identifying the offending allergen or allergens, desensitization immunotherapy procedures are used. A commonly used procedure involves administering gradually increasing doses of the allergen at different intervals until protection against the agent develops, usually up to the maximum tolerated dose (the dose that does not produce a significant allergic response).゛Includes injection. In the method of the present invention,
A more accurate assessment of the appropriate desensitization dose can be determined initially, making the skilled procedures previously required unnecessary. The exact mechanism of this process (f4) is not completely clear. Booster injections to maintain allergic responsiveness are required at intervals of 1 to 4 weeks. W is substantially higher than the maximum dose required for suppression of the initial lvl allergic reaction.

The method of the present invention involves contacting an insoluble carrier to which an anti-9E antibody is attached with a patient's face for a sufficient period of time without causing conjugation of the anti-IgF antibody with 19E in the patient's serum; The method includes the step of removing the fool's serum from the carrier.

At step 5 of this procedure, the patient's serum is preferably not diluted prior to contact with the loaded anti-IgE antibodies. The incubation time must be sufficient to effect substantial conjugation and is dependent on humidity. The appropriate heat retention time is 15-15℃ with a temperature within the range of 155-50℃.
180 minutes, and the preferred welding time is 30 to 120 minutes at a temperature within the range of 20 to 40''C.

The insoluble carrier to which the anti-1gE antibody is attached is an important aspect of the invention. Anti-[E antibodies are available from many sources, and the methodology for producing them is known and described in numerous patents and publications.

Preferred anti-1gE antibodies are affinity purified polyclonal antibodies obtained from the serum of an immunized mammal or from multiple hybridoma clones.

Monoclonal antibodies derived from a single clone can also be used or have the disadvantage of site specificity. Any structural form or steric hindrance that blocks the binding site will prevent the binding of all antibodies, possibly leading to unpredictable changes.

Polyclonal antibodies purified by affinity chromatography by standard procedures using antigen-binding gels can bind to one non-blocking site. Therefore, a polyclonal antibody mixture of monoclonal antibodies derived from different clones and binding to a variety of different binding sites is preferred, and is more suitable than the two-site assay method of U.S. Pat. No. 4,376,110. It has advantages.

A method for producing monoclonal antibodies has been developed rapidly, and a suitable method for producing monoclonal anti-IfIFF antibodies is described by J. D., Catty et al., "Human Immunoglobulins". An immunoassay method specifically related to monoclonal antibodies directed against d-3 (antisera against d-3) is described in the 80-year-old immunoassay method, pp. 133-153 and references cited therein. , the entire contents of which are included herein for reference.

Although a wide variety of different compounds can be used as the solid support, the main considerations are the binding of the anti-IF antibody to the surface, the absence of interference from the anti-1gE antibody reagent with the enzyme label, and the ease with which the substrate reacts with the enzyme. and the fluorescence of the enzymatic reaction products. Both natural and synthetic inorganic materials can be used as solid supports. Examples of suitable polymers are polyethylene, boria 1"pyrene, polyethylene, poly(4-methylbutylene), butyl rubber. and its related synthetic rubbers, silicone combs and silicone resins, polynisdel, polyamides, cellulose and I? /Relose derivatives such as cellulose acetate, di[・[]cell 1
]-su, etc.), acrylic ester, methacrylic acid]1
Step 1, vinyl polymers (such as polyvinyl acetate, polyvinyl chloride, polyvinyl chloride, polyvinyl fluoride, etc.), polystyrene and styrene graph 1 ~ copolymers, styrene-acrylic copolymers , rayon, J iron, polyvinyl butyral, polystyrene, rayon, J iron, polyvinyl butyral, polystyrene, etc. Used as an insoluble carrier
Other possible materials include silica gel, silicon wafer, glass, paper, insoluble proteins, metals, metalloids, metal oxides, magnetic abrasives, semiconductor materials, Celmet, and the like.

Furthermore, gel-forming substances such as proteins such as gelatin, lipopolysaccharides, silicates, acarose, polyacrylamides or polymers forming some aqueous phase, such as for example Textran, polyalkylene glycols. (
alkylene having 2 to 3 carbon atoms) or the field ij'+i
activators, such as amphiphilic A compounds such as phospholipids;
Long chain (12-24 carbons 1ヰ(child)j'ru4-ruan■
Also included is a 3-surface, if desired, a covalent bond between a small number of reagents and the surface. sensuality fl
It's good to be familiar with riJ Noh (a).

Functional groups that can be used for 7:, y, and y should not be present on the surface: carboxylic acid, rededide, amine group,
Methods for binding antibodies to a variety of surfaces are well known in the art and are available in the literature, e.g. Kako Mebi°, Chihata Ijibi, Halsdet Press,
New York, 1978 and A, -] = 1 ~ Reka
] Nos (Cuajrecasas, J.
B io, Ct+em.

245.30b9 (1970).

The size of the bonding group can vary widely depending on the elongation of the compound to be bonded, the effect of the distance between the bonded compound and the surface on the properties of the bonded compound, the crosslinkability of the bonded compound, etc. The linking group may be a single bond, or there may be up to about 12, usually 10, in the chain.
It may have up to 100 atoms. The linking group can be aliphatic, cycloaliphatic, aromatic, heterocyclic, or combinations thereof. The total number of atoms in the bonding group is approximately 201, excluding hydrogen.
within 1,000 yen, usually no more than about 16 atoms, which atoms include carbon, oxy or oxo, oxygen as both oxo-carbonyl and non-oxo-carbonyl, nitrogen as amino or amide, or thiocarbonyl. or sulfur as thiono. Representative groups include methylenecarbonyl, succinimidyl, alphahaloacetyl, thiomethylene, glycyl or polyglycyl, succindioyl, maledioyl, glutardialkyrite, methylenephenyldiazo and ureido.

A preferred diagnostic carrier of the present invention is polystyrene, such as a styrene-acrylonitrile copolymer.
Styrene copolymer containing (vinyl monomer) copolymer,
Examples include polyolefins such as polystyrene and polypyrene, as well as acrylates and methacrylates.
1. Includes copolymers and copolymers. Can anti-IgE antibodies be attached to carriers by adsorption, ionic bonding, i.n. der Waals adsorption, electrostatic bonding, or other non-covalent bonds? 2 thick. It is also possible to attach it to the carrier by covalent bonding. A particularly advantageous support for the method of the invention consists of a microtiter plate with a plurality of wells. A plastic cup insert on the surface or therein can represent an insoluble carrier. The microtiter plate or well insert is protected from light so that the excitation light applied to it or the fluorescence produced in response thereto does not reach or affect the surrounding contents. The most advantageous thing is to be transparent and opaque. In this system, each of the 71 houses can be used as a test system unrelated to other wells.

In the method for the non-covalent attachment of anti-Iq[E antibodies to the surface of an insoluble carrier, C. The insert may be applied to the surface of a carrier such as a well. The surface is first cleaned with a cleaning liquid, such as methanol, and then dried. Buffered anti-1gE antibody solution is placed in a well or insert cup and incubated at room temperature until adsorption occurs, e.g.
Insulate at d5 for 8 hours, preferably 16-18 hours, at a temperature of 4-40°C, preferably 20-26°C.

The well is then washed with dilute saline and dried.

The procedure described in US Pat. No. 4,210,418 may be used to coat the surface with protein and use, for example, curcuraldehyde as a coupling agent to avoid binding to anti-I9F antibodies. In yet another method, the wells may be coated with a layer having free isocyanate groups, such as polyether isocyanate, and then treated with an anti-1gE antibody in an aqueous solution. Application brings about the necessary bond.

In yet another l) method, U.S. Patent No. 372,076
The anti-1 (IE antibody) can be attached to the [1-roxylated residue using cyanogen bromide as described in No. 0.

In another procedure, the surface of the support can be first coated with an inert protein, 6"n. This procedure will be described for polyethylene or boris-dilene tubes, but it can also be used for single cells, beads, etc. are also suitable. Following this method, one avoids attaching 1 to the reactive base with an inert protein. Proteins that can be used are known in the art, such as blood) albumin or globulins from various animal species. There may be other homogeneous materials containing proteinaceous substances or other homogeneous materials.Particularly suitable are bovine gamma macropurine and gelatin, which are readily available. The protein material to be used must be sufficiently homogeneous so that an essentially continuous surface can be obtained through its use.The anti-1qE antibody is then bound to the inert protein.

More specifically, a plastic surface is coated (a) by adsorption with an inert protein under adsorption conditions;
b) attaching the anti-[IE antibody to an inert protein coating; (C) contacting the bound moiety with a stabilizer to stabilize the anti-1gE antibody against denaturation; and (d) attaching the anti-1gE antibody to an inert protein coating; The process consists of drying the reactive part under substantially non-denaturing drying conditions.

The amount of inert protein needed to give the best results is
It depends on the extent of the inactive protein, the surface and the nature of the anti-1gE antibody. This proviso can be easily determined by those skilled in the art. In general, a thin coat of protein (
For example, at least one layer of molecules thick> is bonded to the surface. Generally, this will be an amount sufficient to provide a uniform coating capable of binding biologically active substances.

The inert protein is applied to the surface to form a coating by spraying, dipping, or preferably by immersing the surface under coating conditions in an aqueous solution of the inert protein, such as an aqueous buffer solution. In this way, proteins are adsorbed to the surface. It is advantageous to use an aqueous phosphate buffer solution. Such buffers are described in the United States Pharmacopeia XIX and are generally Hydrogen phosphate nia J
Adjust using potassium dihydrogen phosphate and potassium dihydrogen phosphate.

The inactive ↑11 protein is coated under the adsorption strip 4 that does not lead to protein denaturation. The specific 1)1'' and temperature conditions depend on the inactive protein in question. The adsorption conditions are the usual 1)
1'', such as from 3 to 10 degrees Celsius, and the usual temperatures, such as from about 20 to 30 degrees Celsius. For example, lower temperatures such as a low temperature of 4℃ and a high temperature of about 50℃1.
Marks and high temperatures can be used, but there is no right handedness. In fact, at temperatures above 50°C, proteins are generally denatured. At temperatures lower than 4°C, it is difficult to avoid protein deposition. For example, bovine gamma globulin generally has a concentration of 5 to 7 (6, 4 is optimal).
Coating is carried out at air temperature at a pH of .

To facilitate the attachment of inert proteins, the surface of the relatively less reactive portions may be treated with a solvent, surfactant, acid or base prior to attachment.

A surfactant, preferably sodium dotesyl sulfate, is used as a cleaning agent to clean the surface and make it wettable. If the polymer contains carboxyl groups on its surface, it can be treated with a salt-forming base, such as KOH, to convert it to the salt form, thus increasing its electrical attraction and increasing its adsorption. It is often desirable to provide a charge.

J-bases also aid in surface cleaning. In another aspect, it is advantageous to have a surface charge distribution approximately equal to that of the inert protein to which it is attached.

This is accomplished by washing the surface prior to coating with an aqueous buffer solution having approximately the same pH as the coating solution containing the inert protein.

Anti-IUE antibodies can be attached by any suitable means. Known such means include adsorption, covalent bonding, ionic bonding and uptake. A method for covalently linking anti-1 (IE antibodies to inert proteins is described in U.S. Pat. No. 355).
Nos. 3310 and 36395588 J5, which are hereby incorporated by reference. A preferred method to avoid covalent binding to inactive proteins is to first treat the protein with an aldehyde coupling agent and then apply the aldehyde under conditions that allow covalent binding of the aldehyde to both the inactive protein and the anti-1 (IE antibody). , consisting of adding an anti-1gE antibody.Suitable aldehyde tuberifying agents include:
Ethylenically unsaturated compounds, such as acrolein, methacrolein, and 2-butinal, can contain either or both aldehyde groups.

For example, dialdehydes such as glutaraldehyde, propane dial and phthanedial can be used.

When one of these aldehydes is brought into contact with the surface of an inert protein, the protein is stabilized and polymerized by cross-linking, and the aldehyde active moieties are immobilized on the surface. These moieties appear to be carbonyl groups, which are highly reactive towards the amine groups of anti-HE antibodies since they form a covalent bond between the protein particle and the antibody.

Aldehyde or ethylenically unsaturated coupling methods can also be used to covalently attach anti-1gE antibodies to other surfaces containing primary amino groups. example is,
Polylysine coated polystyrene is glutaraltehyde 1
can be conjugated to an anti-I (IE antibody) by

Instead of aldehydes, other coupling agents can also be used, such as compounds having two or more of the reactive groups listed below. An active chloro group attached to a ring with a resonance structure. These reactive groups can react with the primary amino, sulfurylhydryl, and carboxyl groups in the inactive protein and the anti-10E antibody to which it is attached.

A representative list of this J-unakablinking agent is hiscyazohenzacin, disulfonic acid, tetrazo-di-diamine, cyfluorodi-lohenzene, difluorodinitrophenyl sulfone, carbodiimide,
Toluene diisocyanol-1, dicyamel dihydride, dichlorol-lyazine, and N-1-phthyl-5-methylisoA-gi-1-nozolium perchlorate. Carbodiimides that can be used are N,N-dicyclohexyl-sulfodiimyl', 1-ethyl-3(3-dimethylaminopropyl)carbosiimyl-hydrochloride, and 1-cyclohexyl-3(2-morpholinyl( 4)-ethylcarbodiimide)-me]-1-ruenesulfoner I~.

Alternatively, anti-IQE antibodies can be used in U.S. Patent No. 3,551,555
J: can be attached to the adsorption according to one method. The solid support surface may be coated with a polyether isocyanate coating such as that provided by a polyether isocyanate coating (Hypolu 2000, W.R., Brace End Company, Lexington, Mass.).
It can also be coated with a material that binds to anti-IgF antibodies by conjugation.

Methods for attaching reagents to glass surfaces are known and are described, for example, in US Pat. No. 4,280,992. Binding anti-IqE antibody to glass 1
The method used for c is not limited. Matte crow is preferred. Anti-IgF antibodies can be attached to the crow surface by physical or chemical methods. large amount of anti-1
(Chemical methods are preferred if it is desired to firmly and permanently bind the IE antibody to the crow.

Bonding by physical methods can be achieved primarily by physical adsorption (van der Waals adsorption).

Thus, the glass may be immersed in a solution of anti-1 (IE antibody) and then kept warm or allowed to stand for a suitable period of time to form a physical bond.
1 to 400 g/l, preferably t;jo, 1 to 1.0 (
It has a concentration of 1/l.

Immersion treatment, e.g. 1-48M at a temperature of 0-45℃
Just take your time.

A suitable chemical method involves combining the anti-1oF antibody with the aid of a silane coupling agent and, if necessary, a cross-linking agent.
C. It can be bonded to a glass surface, preferably a frosted glass surface. Functional brackets and anti-I that react with glass
Any silane coupling agent containing in its molecule both the gE antibody and/or a functional group that reacts with the crosslinking agent may be used.

*Examples of suitable functional groups include clouds that react with silanols of glass, such as alkoxysilyl groups (e.g. methoxy or silyl groups imitated)
etc. Examples of suitable functional groups that react with anti-1qE antibodies and/or crosslinkers are those that react with amines, capoxyls and/or thiol groups, such as carboxyl, epoxy, haloalkyl (e.g. chloroethyl and chloropropyl), Aldehydes, primary and secondary amino acids, thiols, incyanates, carboxylates 1
~, imino and nitrile (or cyano) groups, etc. More specifically, examples of suitable functional groups that react with amino groups are carboxyl, epoxy, haloalkyl and aldehyde groups. Examples of functional groups include primary and secondary amino groups, and epoxy groups.

Suitable functional groups that react with thiol groups include thiol, epoxy, haloalkyl, and aldehyde groups.

When bonding anti-1gE antibodies to glass, a silane coupling agent can be used with or without a crosslinking agent. The crosslinking agent can be selected according to the type of silane coupling agent and the type of anti-1aE antibody to be bound. Any cross-linking agent capable of cross-linking the silane coupling agent and the anti-1 (IE antibody) may be used. Such cross-linking agents include immunologically Mention may be made of compounds which are capable of forming cross-links with amine, carboxyl or thiol groups of the active substance, for example between thiol groups or between amino and thiol groups. Examples of suitable compounds capable of cross-linking between amino groups are aliphatic dialdehydes (
For example, cryoki->boule, malonaltehyde, succinaldehy 1-, glutaraltehyde 1-) and dichlorotriazine (e.g. 2-amino-4,6-cycloS-1-lyazine). Suitable crosslinking agents between thiol groups are, for example, simalimide compounds (eg N,N'--0-phenylene dimaleimide, N,N-
--m-phenylene dimaleimide). Suitable crosslinking agents between amino groups and thiol groups are, for example, maleimidocarboxyl-N-hydroxysuccinimide esters (eg nl-maleimidobenzoyl-N-hydroxysuccinimide ester, and hydroxysuccinimide ester).

Adsorbents useful in the methods of the invention as solid supports for anti-I (IE antibodies) are known. Suitable materials include: Adsorbents and Absorbents Non-ionic celluloses such as Wotman (Clifton, Nicol Jersey State, USA> Variety - CF-1, long fiber powder CF-11, medium woven cord powder CC-31'E), fine particulate powder CC-4,1'
O, Fine Powder Example: Hyorad (Richmond Calipornia, USA) Variety - Selex oN 1. Powder Selex 4" 10, Powdered silica gel, Wot 1~Man variety-8G81, Filled paper:
Or Bio-Rad variety - Hi-A-Gino δA or Bio-Sil HA Hi1~]21xyl U7 Patai 1-・<Hio-Rad)
Alumina: Acidic, basic or neutral varieties (for example, alumina C-gamma gel (Bio-Rad) calcium phosphate) - Macia (Viscota, New Jersey) , USA) I, 87 Aketsu, Yu ■ l-H20 Dex 1-lan (Pharmacia) UN M Dextran (Pharmacia) Alkyl analogy is Pharmacia variety - Octyl Sepharose ■
C1-4B or phenyl-cephalol C1-4
B For example, Miles Research Products (El Kart, Indiana, USA) variety - ω-aminoalkyl agarose requin agarose (Miles Research Products),
Poly-L-lysine agarose (Miles Research Production Plastics, such as polystyrene, polyethylene and polypropylene pores) ion exchanger diethylaminoethyl (CEAE) Cellulose I DE-1 10, Powder DE-2 20, Fibrous DE-2 3 (io, Fibrous DE-32■, Dry, Particulate o E-5 2(E), Wet, Particulate DE-8
10. Paper, for example, Hiorado variety - Selex D.

Fibrous diethylaminoethyl (DEAE> Acarose, for example, Diorad variety - DEAE bioglu ■△ Diethylaminoethyl < 1) E A E ) Tekisu [~ Orchid analogy (J, Pharmacia variety - DEAE Senoadex ■, Beet-like a, 7,,.Yatsu,.Waa.-Yu ■48 (7A7Sia) Ecteolarerulose analogy is Wot 1~Man variety-ET'-1 1■, Powder E 1'-4 1■ , 1 ball of powder (high purity) l −
8 1■, 謬(For example, HiAara 1~item (Tohirex■[.

Fibrous 1 to Liedylaminoethyl (TEAE) cellulose For example, Biorat variety - Selex ■-1
−.

Fibrous diethyl-(2-hydroxypropyl)-amino (QA
I yo), cellulose tc oe (yo 1.3 oats F' 1m 't'fi-
(QAE), 1-fibrous diethyl-(2-hydroxypropyl)-amino (QA
E) Dextran For example, Pharmacia variety - QAE - Sephadecyu ■ Henzoylated diethylamine ethylcellulose For example, Bio-Rad variety - Selex ■ BD, fibrous cation exchange vJ material Phosphoric cellulose For example, Wotman variety - P-1 ■, Flock P-110, Powder P-410, Powder (high purity) P-81 ■ 1 Paper carboxymethyl cellulose For example, Wood 1 Heman variety - CM-1 ■, Flock CM-11 ■, Powder CM-22 ■, fibrous i = 1 − 2 3'P', bud%fIilf
4 people CM-320, dry, powdery CM-52■,! ! i! Jun, particulate CM-ε32
■9 paper For example, HiA-Rat breed-SELEX[F]C to 4
．． Loss rate (A talented calho 1 dimethyl text) ~ run For example, Pharmacia variety - CM - Sephadecmeri fibrous carboni 1-dimethyl acarose For example, Hi A - Rat variety - CM Hyogno ρ For example, Pharmacia variety - 〇 H-Ceno10-su” 4
B Sulfopropyl dextran such as Pharmacia variety-8P-Sephadex [
F] Reagents produced by chemically coupling or conjugating anti-1 pE antibodies to polymeric carrier particles of different sizes are described, for example, in U.S. Pat. No. 3,882,222.
5M: No. 3957931; No. 3825525; 3629
No. 558: No. 3565987; No. 3553310;
No. 3407076; No. 3236732: 309625
No. 0: No. 4092114: No. 4140662; /1.
No. 210723; 4226747@; 4.259313
No.;3088875;3766013+36193
No. 71; No. 3809613 + No. 3853987; 39
No. 63441: No. 3551555; and 3 (34934
No. 6, Dutch Patent No. 7201308: as well as British Patent No. 1257263.

Polymerization of anti-1 to Ebeide (if covalent attachment of the IE antibody is desired, the anti-1gE antibody can be reacted with an amine, amide, or sulfone group on the anti-1gE antibody to which it is attached). Preference is given to using monomers which retain the radicals, for example chloropentyl, ri[alpha]loacetyl, chloroethylcarbonyl, ly[]loethylsulfonyl, acryloyl or vinylylsulfonyl groups, after this formation.

Surface groups can be attached to anti-IgE antibodies by reactive heat surface groups and bifunctional linking groups that react with anti-19F antibodies. .

Heath is usually prepared by polymerizing one or more vinyl heptamers by standard methods. Suitable vinyl heptanones which can be copolymerized with the undercarriage and/or with any other combination to obtain the desired performance are, for example, Monohynylidene carbocyclic E-1, for example, subuurene, alpha methylstyrene, ar-(t-butyl)styrene, a
l'-methylstyrene, ar, al'-dimethylstyrene, 81'-chlorostyrene, ar-(t-amyl)
Styrene, al'-)' lomostyrene, ar-fluorostyrene, ar-(t-amyl)styrene, ar-nolomostyrene, at'-fufureostyrene, old'-cyanostyrene, ar-methoxystyrene, ar-ethylstyrene , ar-hydroxymethylstyrene, ar-oxystyrene, ar-chloro-ar-methylstyrene, a:・, a1゛-dichlorostyrene, ar, ar
- difluorostyrene, vinylnaphthalene, and such emulsion-polymerizable septamers thereof having up to 26 carbon atoms;
Esters of heterethylenic unsaturated carboxylic acids, such as (J, methacrylic 0)methyl, chloroethyl methacrylate, 1)-butyl methacrylate, ethyl methacrylate, isobutyl methacrylate, isopropyl methacrylate, phenyl methacrylate, chloroac 1-nor (Pt butyl, cyclohexyl chloroacrylate, ethyl chloroacrylate, methyl chloroacrylate, isobrovir chloroacrylate and other such substances which can be polymerized to produce hard r'1 polymers) - sprue; Alpha, peta-edylenically unsaturated esters of polymerizable carboxylic acids, such as vinyl benzoate, vinyl 1-rulyate, vinyl former ゛-ethylbenzoate, allyl ar-ethylbenzoate, i-vinyl dimethylacetate, vinyl piperate,
Vinyl trichloroacetate and other such monomers in which the unsaturated moiety has 2 to 14 carbon atoms and the acid moiety has 2 to 12 carbon atoms; for example, nitriles having 12 or more carbon atoms; Other polymerizable vinyl monomers are included, such as alpha, heteric, tyrenically unsaturated di-1-lyl;

In the preferred method, an anti-1gE antibody is placed on an insoluble carrier, such as a well surface, and an animal protein is placed on the well surface. 1
- Stabilize with an aqueous buffer solution forming a saccharide complex. A preferred My1 solution contains 0.05 to 1.0 weight of water-soluble animal protein. Suitable water-soluble proteins include bovine serum albumin (BSA), human (H3A), rabbit (
R8A), Yaqui (GSA), Sheep (SSA), Horse (
+-103A); serum gamma-globulin of the above-mentioned animals; and ovalbumin, such as
It includes its relative animal proteins such as fibrinogen, thrombin, i-transferrin, and glycoproteins.

A preferred water-soluble animal protein is bovine serum albumin. The solution also contains 0.1-5% by weight of one or more water-soluble saccharides. Suitable saccharides include sorbitol, sucrose, maltose, robiose, lactose, cocoon, anilose, and the like. 78′a, is 0°01-0.5fv11. with a pH of 6-8. J
Sufficient phosphate and e.g. 0.01 to 0.1 wt.
Nosu (contains preservatives such as helium azide).

Preferably, the first step of the method of the invention is preceded by a pre-washing step. In the pre-washing step, the carrier surface is brought into contact with an aqueous cleansing solution containing 0.0001-0.5% by weight of water-soluble protein. This pre-IM1 wash step is particularly advantageous when the water-soluble protein is a water-soluble animal protein. The preferred cleaning solution of the present invention is 0.000
Phosphorus i′l from 1 to 0.05! Salt molar degree, 6 to 8 +>
Aqueous phosphorus C! having H and containing o, ooi~0.1% by weight of nonionic surfactant and 0.0001~0.5% by weight of water-soluble protein! Salt buffer solution. Suitable non-ionic surfactants include, for example, lauryl, cetyl,
oleyl, stearyl, and polyoxyethylene such as oleyl, stearyl, and
(BRI.[F], :, 3e(yopo.

Polyoxyethylene sorbitans (D-WEENC') such as oxyebrensolhitan monolaure 1, monopalmizo-1-, monostearate I-, monoolea-1 and 1-heliolae 1; and other polyoxyethylene ethers. A preferred nonionic surfactant is octylphenoxypolyethoxyethanol having 40 ethylnonoxide units (Triton X-405, Rohm End Haas).
It is. Suitable water-soluble proteins include bovine<SSA), human1~
(1”sA), rabbit (RS△), A7gi (GS△),
Serum albumin of sheep (SSA), horses < 1-108 A), serum casimer globulin of the above animals: and
-thrombin, (-transferrin, including other animal proteins such as glycoproteins.

The buffer solution contains 0.005-2.5% animal protein, 0.5-5% nonionic surfactant, 10-20%
A reagent concentrate of the invention is prepared from a reagent concentrate of the invention consisting of 0.5% to 5% by weight of a stabilizer and sufficient phosphate to give a 0.02-0.05M phosphate solution. That is advantageous. l) H can be 6-8. A suitable thick liquid contains about 0.5% animal protein by weight;
0.1% by weight of 1-Lium X-405 non-ionic surfactant, 1.7% by weight of 1-lium chloride, and 2% by weight of 1-lium acid, 0.01-1% by weight of Contains phosphate and has 7.4 1) 1''.

Blood f' with anti-[F antibody attached to insoluble 113 body
After JIQE conjugation occurred, the patient's serum was then 11! Remove. After removing excess liquid, the solid surface is
For example, wash with a suitable cleaning solution C, such as those mentioned above.

The second step of the method of the invention consists of contacting the insoluble 111 bodies with an anti-IqF antibody that has been labeled with norologone. Serum conjugated with anti-IgE antibody (if present)
's fermentation label i' () anti 1! Conjugation with E antibody continues for a sufficient 4ζ hours. After moisturizing, excess liquid is removed and the surface of the insoluble carrier is washed with a mild saline solution as described above for the first step to remove unconjugated antibody.

It is preferred to wash the carrier with a suitable washing solution of the present invention as described above.

The anti-gE antibody may be the same as or different from those described above, but is preferably a monoclonal antibody.

Fluorogon enzymes and methods for binding them to anti-IgE antibodies and selectively conjugating 9F without compromising the ability of the antibodies are known in the art. Suitable enzymes and methods for conjugating them to antibodies are described, for example, in US Pat. No. 4,190,496, the contents of which are incorporated herein by reference. Preferred full-A logon enzymes and correspondingly suitable substrates are horseradish peroxide, beluogysidase and small mohanillic acid or 4-hydroxy-3- as suitable substrates therefor.
Methoxyphenylacetic acid, beta-galac 1-cytase and a suitable NIM for -C4-methylumbelliferyl-beta-D-galac 1-cyto, alkaline small sphatase and phosphate as a suitable substrate for it. 4-methylumbelliferyl and, for example, 4-carboxy-umbelliferyl phosphate and umbelliferyl phosphate, 4-carbogysif-7-rukyl nystyl.

An example of a suitable procedure for enzymatically labeling an anti-101 antibody is as described in J3 for the covalent attachment of an anti-19F antibody to protein 71 with calphodiimide, dialdehyde, and
Including the use of r273'-capable linking agents. Cutting agent (Jl, for example 1-King Chiru 3-(3
-N,N-dimethylaminopropylbodiimide acid]
Preferably, the carbodiimide is a carbodiimide such as 1-cyclo·'\=inijiru-33 (2-mole small ethyl)carbodiimide 1-hemethyl-but-ru-tama-unsurbo-bu-1. Other suitable coupling agents may include
J. or altehyde with a small number of aldehyde groups, such as glutaralachite, b[1panedial or phthanedial]. (-includes coupling agents. Examples of such coupling agents include Su/＼disuccinimidyl phosphate, imidyl, disuccinimidyl tartrate, his-[
2-succinimidoxy(sulfonyloxy)J-delj
sulfone, cissuccinimidyl (N,N''-cyacetylbo-1'-cysdine, didiΔbis-(succinimidylpropyl A+-1~), ethylene glycol bis-(succinimidyl sufcin-1) difunctional NH3- such as
I-methyl, for example N-5-acid-2-2-[]]
Penzoyasis succinimitobromide (]-azidophenacyl, I)-azidophenylclioquinol, 4-fluoro-3-21-[J)tinylic acid, N-human 1doxysuccinimidyl- 4-azidohenso I -1-
lm-Maleimitohenzoyl N-hydro 4-cissuccinimide 1-king ester, methyl-4-azi1-penzoimite-1-.1-IC1, p-nide 1'-phenyl 2-diazo-3,3.3- 1-Linololopiropiota-1-1
N-succinimidyl-6=-(4-~ando-2-1 ditrophenylamino)hexanoate, succinimidyl 4~<N--maleimidomethyl)-1-carbexylate, succinimidyl 4-(p
-maleimido-noconyl)notyle 1~, N-succinimidyl (/1-7shi1-)1nylditiΔ)propyl: 4
t-1~, N-succinimidyl 3-<2-pyridyldithi A) zo[jbio J--1, N-, -< 4-azido-)tinylthio)notalimii~ of J: ')',
(: to 1 [12 functional test; Tang; for example 1,5-cinol-o-2./1-dinyl-IJ /\nUun, 4,4'
-difluoroD-3゜3'-sinoenylsulfoshi, 4. ．． 4'-Shiisosaocyano2,2'-
disulfonic acid sulfonate I\n, ])-]no-dilene diisodiosilane f-1, carbonyl hiss (L-medionine 1)
)-2 I-mouth noenyl esunru), = 1.4”-sibuA
Homobifunctional assays such as hisphenyl azide, -Li1-Li-rubiscalchoner; and, for example, dimethyl? dipimide-1-2+-IC1, dimethylschelimide-1, dimethyl 3,3--dioxybisbrobinimide 211CI, 2-iminothiolane HCI
It includes difunctional imide esters such as. Covalent attachment of the enzyme to the anti-I (IE antibody) can be carried out by conventional known reactions using the reagents described above, e.g., in an aqueous solution at neutral pH at a temperature below 10° C. for 18 hours or overnight. .

The enzyme-labeled anti-1 (IE antibody) is applied to the insoluble carrier in an aqueous solution. The solution contains appropriate salts and buffers to preserve the reactants and facilitate the conjugation reaction. For example, the solution may contain bovine serum albumin (BSΔ), phosphate buffered saline (PBS), and a mild interface, such as polyoxyethylene sorbitan, which is used in the cleaning solution described above. Activators may be included.Washing solutions as described herein may also be used.

Preferred solutions of the invention have a phosphate molar concentration of from 0.0001 to 0.1, preferably from 0.005 to 0.05 and from 6.0 to
0.1 to 5 micrograms per ml, preferably 11 to 2 micrograms per ml in an aqueous phosphate buffer solution with a p+-+ of 8.0, preferably 7.2 to 7°6. Includes anti-fgE antibodies.

anti-1 g [single row component in E solution is 1 to 8, preferably 2
Borinylene glycol having a molecular width of 1000 to 800, preferably 200 to 100, at a concentration of 6% by weight. Polyethylene glycol significantly increases the speed and sensitivity of the reaction. Another important component is 0.001~0.5, preferably 0.02~
It is a nonionic surfactant at a temperature of 0.1% by weight.

Suitable non-ionic surfactants include, for example, those mentioned above for washing fluids. A preferred non-ionic surfactant is X-405. The surfactant significantly reduces the non-specific background fluorescence signal for analysis.

For preferred anti-1pE solutions of the present invention, the amount of time the solution is incubated with the insoluble carrier is temperature dependent.

J5 at temperatures between 15 and 40'C, at least 15
Incubation times of ˜180 minutes can be used. The preferred temperature is within the range of 20 to 30°C, and 45°C to these temperatures.
It is possible to use a heat retention time of 120 minutes to R30. It is clear that at any stage of the present invention, long heat retention times will reduce the efficiency of the culm.
There is. Since rapid analysis is the purpose of this enlightenment, is it desirable? The minimum time required to increase the temperature is preferably C.

The solid support is then washed to remove any remaining unconjugated enzyme-labeled anti-1gE antibody. The washing solutions described above are suitable.

The third step in method b of the present invention involves placing the solid support in the presence of a norofluorocene enzyme with a solution of the substrate that undergoes a chemical reaction and for a sufficient period of time to form a fluorescent compound.
It consists of making contact with each other.

Suitable substrates and enzymes to be reacted are known and described, for example, in U.S. Pat. No. 4,190,496. Examples of substrates are as previously described for the corresponding fluorogenic enzymes .

The solid is 10-2 to 10-side mole'a degree, preferably 10
-'-10-5 mol. Ha?-77 mino'
-2-Mef-Rule-1,,,-7 f2J Panol fgt
Contains anti-inflammatory drugs and anti-inflammatory agents.

base solution; 1k was added to (with a ζ-soluble carrier) [live reaction mixture]
) i9 ql; +, keep warm for a light minute I time to generate I.

An incubation time of 1240 minutes can be used for -U5- at a temperature of 1 B-/l()°C. A hot stone within the range of 20 to 26°C and a heat retention time of 30 to 90 minutes are preferred.

Next, the fluorescence intensity in the C solution is measured. , the apparatus and h-method for the measurement of the intensity of fluorescence in substrate solutions are common in the art. Fluorescence intensity (is a function of the enzyme port of the insoluble carrier, 15.F1a is the patient's blood7)
By comparing the intensity of this fluorescence with the intensity measured by performing the procedure with a control solution containing IgE antibodies at a known temperature, the Exactly 4 of the corresponding F antibodies in the serum of
9 degrees can be determined.

Appropriate fluorophotometers (Perkin-Elmer, American Instruments Inc., and Turner Tesins);
Allage Energy, Mountain Pico, Calipornia) are preferred.

The invention is further illustrated by the following specific but non-limiting examples. Temperatures are in degrees Celsius and concentrations are expressed in % unless otherwise noted.

111 M on a microtiter plate made of opaque polystyrene.
, O-Sulliva 7 (N4. O-5ulliva11)
et al., Analytical 3 iochem, 10
0.100 (1979) of different French human antibodies prepared according to the modified procedure of
．． It is used in a 0° 1M phosphate buffer solution, pH 8.5, containing 1% by weight of sourium acid preservative.

Cover]”+7! Listen to it at room temperature for 2 o'clock (or Hiiragi). At the end of each culm, the liquid in each ID is removed with suction and the carrier is air-dried for 1 hour at room temperature.

Next, Tsukito, 2.5 weight? 6 Souf [course, 0゜2
5 layers L) 1 head) c) Bovine serum albumin, 0.05
% by weight of helium azide and 1-1-1-X405
0, 1N 1 phosphate buffer solution containing <f)
After removing excess solution by avoiding contact with B S ) for 30 minutes, the wells are air-dried and sealed. The coated wells can then be used to assay for total IgF antibodies.

Example 2 The Microtiku plate Tsukido product of Example 1, which has anti-Ig1 antibodies attached to fq-C, was brought into contact with patient serum containing IgE antibodies and kept warm for 1 hour. Remove serum and reduce l0 to 0.
0.85% by weight of sodium chloride, 0.05% by weight of sodium chloride, 0.05% by weight of sodium chloride, and 0.1% by weight of sodium chloride in 01M phosphate buffer solution (pH 7,2). Wash three times with buffered wash solution containing preservative. Serum IgE-specific antibodies are conjugated to the surface of the microtiter plate wells.

Next, microtiter wells, M, 4th Norivan et al.
A analytic [31ocl]em,,
100, 100 (1979) and conjugated to alkaline phosphatase for 30 minutes. Zeru. The antibody was prepared in a 0.01M phosphate buffer containing 4% by weight of polyethylene glycol with a molecular weight of 4,000, 0.05% by weight of 1-ridone X-405 and 0.1% by weight of a sodium azide anti-inflammatory agent. Add to the solution of saline solution (1) l-17,2>. The alkaline bosphatase-conjugated anti-H1-IgE antibody solution is removed from the microtiter plate wells and washed three times with the buffered wash solution described above.

Microtiter plate wells were then injected with 0.125 mM magnesium chloride and 0.1 wt% helium 7 sills.
Contains wo! I-Ru II! Io') Underwater CD 1,
25 M (7) Add 100 microliters of a substrate solution containing 10@-4 M 4-methylanyl phosphate in 2-noamino-2-methylpropanol (1-19,5). After 30 minutes, increase the fluorescence intensity to 365
Measurements are made using fluorophotometry with excitation at 45 nm and reading at 45 Q nm.

By comparing this reading with the fluorescence intensity measured by repeating this procedure using a control solution with total IgE antibodies of known hydration, the total IgE antibody in the serum of the patient was determined.
Determine 1 degree.

Example 3 The microtiter plate crystals of Example 1 (on which the anti-1gF antibody was attached) were added to a 0.01M phosphate buffer aqueous solution <1)H7,2), 0.85% by weight. Sour chloride
-Rium, 0.05 wt 0% 1~1hen X/105.
After washing for 5 minutes with a buffered wash solution containing 0.01% by weight BSA and 0.1% Ogawa preservative, excess liquid is removed. The prewashed gummy titer plates are then contacted with patient serum containing IE antibodies and incubated for 1 hour. The serum is removed and the wells are washed three times with buffered wash solution.

A buffered wash solution is prepared by diluting the following Fuchi ν solution with 50 parts by volume of distilled or deionized water to 1 part by volume of agricultural F7 solution. Serum albumin 0.5% by weight Non-ionic surfactant (Tri-1-X-405) 0.1% by weight Chloride ~ Lium 17 m% Soluium acid 2 Monomer % Helium phosphate 0 .05 MpH 7
．． 4 and control serum I9 [antibody is conjugated to the surface of a microtiter plate.

Microtiter wells were then prepared by M. O'Sallivan et al., △
analytic [3iocbem, , 10
0, 100 (1979) (1979) (1979), a mixture of anti-H1-1 (IE only) antibodies from different clones prepared according to the modified procedure (100 microliters of conjugated alkaline, phosphorylated phosphatase) 1 to 30 liters of solution and 30
Leave in contact for minutes. The antibody was 4% by weight/1. Polyethylene glycol (PEG) that determines the molecular weight of OOO
4000), 0.05wt 0%] edge 1~nX-4
BS△ of 05,0゜01 weight^) (lε), and 0.1
Weight % of 1 to 1 gnome reed 1 to 0 containing anti-inflammatory agent,
01tvl phosphate mild tjj saline solution (pl-17,2
＞Used inside. The alkaline phosphatase-conjugated anti-H-111F antibody solution is removed from the MIG1-] tartar plate wells and the mouths are washed three times with the buffered wash solution described above.

Then 0.125m for the Migml titer plate well
M magnesium chloride and 0. 1.25N 42-amino-2-methylpropertool in deionized water containing ~lium azide, pl-19,5,
Add 100 microliters of substrate solution containing 0-''M 4-methylumbelifol phosphate.
After 3 minutes, the fluorescence intensity was measured with a fluorometer at 3 (35 nm).
Measurements were made using 5 µm excitation and readings at 45 Q nm. By comparing this reading with the intensity measured by repeating this procedure using a control solution with total IgE antibodies of known hydration, total IgE antibodies in the serum of loyalists were determined.
Determine the Eie degree.

On a microtiter plate made of opaque polystyrene, M
, Osariuan et al., A analytical bio
-cl>em,, 100.100 (1979)
100 microliters prepared according to a modified procedure of
Add a solution of all anti-human antibodies to IgE alone.

Monoclonal antibodies are used in 0.1M phosphate buffer solution containing 0.11% by weight sodium azide preservative.

The coating process is allowed to proceed at air temperature for 2 hours or overnight. At the end of the coating process, the liquid in each well is removed by suction and the support is air-dried for 1 hour at room temperature.

One unit was then treated with 2.5% sucrose, 0°25,000% bovine serum albumin, and 0.05% P1.
0,1-1 phosphoric acid containing ~lium acid 1~ and l-rean Riri. The coated wells were thus coated with total Ig [antibodies (-
It can be used to test the serum of thoughts.

Example 5 The microtiter plate well product of Example 4 to which anti-IQ antibodies are attached is contacted with serum containing IglE antibodies and incubated for 1 hour. Remove serum, pl-l 7
, 2 O,,01 NI + 0 in phosphate slow rain aqueous solution
．． Wash the surface three times with a buffered wash solution containing 851 Old 6 Jn, 0.05% by weight of Jnx405, and 0.1% sodium azide preservative. Igf = specific antibody conjugated to the surface of the microtiter plate well.

Next (“Microtiter wells, N11. Ozariuan et al., A analytical Biochem,
100, 100 (1979).
I3 Prepared alkaline small sphatase-conjugated anti-human]q
30 minutes of contact with a 100 microliter solution of monoclonal antibody, monoclonal antibody was 4fffffi%
Polyethylene glycol (PEG40) has a molecular weight of 4000
00), 0.05% by weight of Triton X-405 and 0.05% by weight of Triton X-405.
I) containing 1% by weight of solu~thuliumzide preservative
H/, 2 O: -C in 1M phosphate buffered saline solution is used. I9F to alkaline small sphatase conjugated anti-human 1
The monoclonal antithermal fluid is removed from the microtiter plate wells and washed three times with the buffer wash solution described above.

Microtiter plate wells were then injected with 10-10- in 1.25 M 2-amino-2-methylpropanol (pH 9,5) in deionized water containing 0.125 mM magnesium chloride and 0.1 wt% sodium azide. ' Add 100 microliters of substrate solution containing M umbelliferyl phosphate.

Every 30 minutes, the fluorescence intensity is measured by excitation at 365 nm and reading at 450 nm. Determine the total 19 degrees of EilB in the serum of the thought by comparing this intensity reading with the intensity measured by repeating this procedure using a total of 1 (control solution containing the IE antibody) of known moisture. do.

Example 6 The microtiter plate of Example 4 (on which the anti-1gE antibody is attached) was added in 1 volume (0.01M phosphate buffer solution) (0.85% by weight in pt-17,2>). triurium chloride\, 0.05% of Juichi% 1 ~ Lydone X405.0.01
Wash for 5 minutes in buffered wash solution C containing 1% BSΔ, and 1% helium acid preservative. The prewashed microtiter plates were then contacted with patient serum containing gE antibodies. Keep warm for 1 hour. The serum is removed and the wells are washed three times with buffered wash solution.

Buffered washing) 1 is prepared by diluting and covering 1 part of the sanitary solution with 50 parts of distilled water or deionized water per volume. : Bovine serum albumin 0.5ml 1% ゛Nonionic surfactant (Trihen X-405>(), 1% by weight Helium chloride 17
Heliud, aji 1mi 211゜phosphoric acid ol~lium Q, 05 MpH
Adjust serum 1qE antibody to 7.4 and conjugate it to the surface of the microtiter plate well.
Ru.

Microquiter wells were then prepared using NII Osariuan et al., Analytical Biochem, 1
00. 100 (1979), alkaline small staphaase-conjugated anti-human IfJE alone [
] - Contact with a solution of 100 microliters of body temperature for 30 minutes. The monoclonal antibody is 4 m ffl
', 'i, molecules m4000 polyethylene glycol (F'E G4000>, 0.05 wt 0% trione 0.01M containing Nosu 1 helium azide protection agent
Use in phosphate J buffered saline solution (+)H/, 2>. Alkaline t'l-small sphatase conjugated anti-history l-1 (I
Remove the antibody solution from the microtiter well and wash it three times with buffered wash solution C described above.

Then, it was placed in a temporary microtiter door and was heated to 0.125 m.
10% in 1.25M 2-amino-2-methylpropanol (pH 9.5) in ionized water containing magnesium j7A and 0.1% by weight of sodium acid.
100 microliters of substrate solution containing 11.1 l of 4-methyllanhydriferyl phosphate
I can understand. After 30 minutes, using a fluorescence intensity of 365 n
The fluorescence intensity is measured by excitation at 63C:j and reading at 450 nm. Take this reading for all 1g of known temperature.
By comparing the intensity measured by repeating this procedure with a control solution containing F antibody, 19
Determine the serum total 1 g' E 8 degrees in the serum of a person.

Continued from page 1 Priority claim @February 3, 1984 ■United States (US) 576586 0 Inventor Myron A. Beigler - Sualtos Hills, California 940220, United States of America.

Manuela Kuchido 14780

Claims (1)

[Claims] 1.8) contacting the insoluble carrier to which the anti-IQE antibody is attached with the patient's serum for a sufficient period of time to cause conjugation and removing the patient's serum therefrom; b) contacting the insoluble carrier with a fluorogen C) contacting the insoluble carrier with an anti-19E antibody labeled with a fluorogenic enzyme and removing the unconjugated enzyme-labeled anti-19E antibody therefrom; C) reacting the insoluble carrier in the presence of a fluorogenic enzyme to generate fluorescence; 1. A method for identifying and quantifying total ICE depth in serum of a patient, comprising: , the anti-1gE antibody on the insoluble carrier and the labeled anti-1gE antibody are each selected from the group consisting of an affinity-purified polyclonal antibody or a mixture of monoclonal antibodies derived from a plurality of different clones; Law. 2. The method according to claim 1, wherein the insoluble support has a plurality of reaction wells separated by an opaque material. 3. The method according to claim 2, wherein the insoluble carrier is a microtiter plate made of opaque material F4. 4. The method of claim 1, wherein the anti]oE antibody is a mixture of monoclonal antibodies from multiple different clones. 5. The method according to claim 1, in which the anti-19E antibody is labeled (=l) with alkaline bosphatase. 6. The method according to claim 5, wherein the substrate is 4-methyllan helifuryl phosphate. 7. The microtiter plate is Borisjiren or Sujiren (
2. The method according to claim 1, wherein the method is a vinyl monomer) co-Q combination. 8. In the step (1)), the insoluble carrier is
Claim 1: Contact with an enzyme-labeled anti-I (IE antibody) in an aqueous solution containing 1 to 8% by weight of polyethylene glycol having a molecular weight within the range of 10,000.
The method described in section. 9. The method according to claim 8, wherein the aqueous solution contains 0.01=0.1% by weight of nonionic surfactant. 10. The method according to claim 9, wherein the nonionic surfactant is octylphenoxyborea-1-oxyethanol. 11. In step (1)), add the insoluble carrier to 0.01
2. The method of claim 1, wherein the enzyme-labeled anti-1gE antibody is contacted in an aqueous solution containing ~0.1% by weight of a nonionic surfactant. 12. The patient's serum is freed from insoluble carriers in step (a) by washing with a phosphate buffer solution having 1) l-1 in the range of 6 to 8 and containing a nonionic surfactant. The method of claim 1 for removing. 13. Conjugate the anti-1qE antibody without conjugating it to step (1))
2. Method B according to claim 1, wherein the insoluble carrier is removed using a phosphate buffer solution having a DH within the range of 6 to 8 and containing a nonionic surfactant. 14. Before contacting the insoluble carrier with the patient's serum,
2. A method according to claim 1, comprising prewashing with an aqueous buffer solution containing 0.001 to 0.5% by weight of animal protein. 15. The method of claim 14, wherein the 15° soluble animal protein is bovine serum albumin. 1(3,a) The anti-1gE antibody is deposited on an opaque polystyrene or styrene-(hinyl monomer) copolymer carrier for a time sufficient to allow conjugation of the patient's serum with that of the serum 19F antibody to occur. Contact: 1)) removing residual patient serum from the carrier; C) removing the carrier with an anti-1gE antibody labeled with a fluorogenic enzyme in an aqueous solution containing polyethylene glycol and a nonionic surfactant; Allergen specificity I conjugated to a carrier
(1) remove residual patient serum from the carrier; e) subject the carrier to a reaction in the presence of a fluorogenic enzyme; 2. The method of claim 1, comprising: contacting a solution of a substrate to provide a fluorescent product; and f) measuring the intensity of fluorescence of the solution. 17. The method according to claim 1, wherein the insoluble carrier to which the anti-I-E antibody is attached is treated with an aqueous buffer solution containing water-soluble lactic acid and a water-soluble animal protein. 18. An insoluble carrier to which an anti-tE antibody is attached and has a complex of water-soluble saccharide and water-soluble animal protein on its surface. 19. The insoluble carrier according to claim 18, wherein the anti-1 (IE antibody) is non-covalently bonded (thus attached to the surface of the insoluble carrier). 20. The insoluble carrier is a styrene-(vinyl 7-mer) copolymer. Claim 1, which is polystyrene with a combination of
Insoluble carrier according to item 9. 21. The insoluble carrier according to claim 20, wherein the insoluble carrier is a Miku[] titer well. 22. The water-soluble complex is produced by treating an insoluble carrier to which the anti-IgE antibody is attached with an aqueous slow J solution containing water-soluble leetucalide and a water-soluble animal protein.Claim 20. 111 insoluble bodies described. 23. The buffer solution has a pH of 6-8, with a pH of 0.05-1.
0.01-0.5M phosphorus containing 0% by weight of water-soluble animal protein and 0.1-5.0% by weight of water-soluble saccharide.
'I! 23. The insoluble carrier according to claim 22, which is a salt buffer solution. 24. The insoluble carrier with attached protein has a pH of 6-8, 0.05-1.0% by weight of water-soluble animal protein and 0.1-5.0% by weight of water-soluble saccharide. A method for stabilizing proteins attached to an insoluble carrier by contacting with a 0.01-0.5M phosphate buffer solution containing. 25. The method according to claim 24, wherein the protein is an antibody. 26. The method according to claim 25, wherein the 2G protein is an anti-1qE antibody bound to an insoluble carrier by a non-covalent bond.