DD295927A5 - IMMOBILIZED IMMUNOLOGICALLY ACTIVE REAGENT - Google Patents

Abstract

A process for preparing stabilized immobilized immunologically active compounds for immunoassays is described. The long-term stabilized immunologically active reagents are preferably used for solid-phase immunoassays for the detection of antibodies or antigens in vitro. {Immunologically active compounds; long-term stabilized reagents; Solid phase immunoassay technology; Antibody; Antigen; In vitro detection; immunoassay}

Description

Field of application of the invention

Immobilized compounds have been used in pharmacy, life sciences, biotechnology, food technology, enzymology,

Medicine, immunology a wide range of applications, eg. As for the quick and easy separation of reaction products and unwanted impurities,

Immobilized immunologically active compounds are especially for immunodiagnostic purposes, preferably for solid-phase immunoassay and in vitro detection or for the determination of an immunologically active reactant

used.

Characteristics of the known prior art

The detection or determination of antigens, haptens or antibodies is preferably carried out by immunological methods based on an antigen-antibody reaction. Because of the ease of separation of the reaction products, solid-phase techniques are preferred in which a reactant is bound to an insoluble support

 The method for the adsorptive binding of proteins to polymeric hydrocarbons developed by K. J. CATT and C. W. TREGAR (Science 15 [1967] 1570) has been widely used.

The homogeneous coating of the carrier with the immunological reactant is a prerequisite for reliable results in solid phase immunoassays, especially in the detection of medically important substances. There are attempts (DD 236400, OS 2 337 095) to achieve a more homogeneous coating with the immunoreactant by precoating with certain hydrophobic compounds. Likewise, it has been attempted to achieve better adsorptive properties of the carrier by treating the surface of the plastic carrier with organic solvents or acids (DE 2509895, DT 2509895.1.C.SHEKARCHI et al., J. Clin. Microbiol., 19 [1984] 89 et al )

The sensitive, biologically, immunologically active material bound to the one polymeric support is subject to permanent, in particular hydrophobic interactions with the support, partial structural changes of the immobilized immunoreactant and a relatively rapid loss of its immunological activity are the result.

It is known that, especially on polymeric hydrocarbons with nonpolar surface such as polystyrene u. a, bound immunologically active molecules, especially polypeptides and proteins, are subject to conformational changes by hydrophobic interactions, which may limit or prevent subsequent antigen-antibody reactions, depending on the extent of spatial alterations. In addition, a number of antigens, particularly low molecular weight immunologically active substances such as allergens, peptides, certain haptens, active amines, acting as antigenic drugs u.a. not or only to an unsatisfactory extent bind to the solid support such as polystyrene by the usual methods.

It has therefore been attempted by various authors to polystyrene by introducing functional groups, eg. As nitro groups, chemically modify (overview: G.H. PARSONS: Methods in Enzymology 73 [1981], 224) to covalently bind biologically active compounds, especially haptens. However, this procedure led to discoloration of the reaction plate and thus to disruptions of vertical photometry.

The direct interactions with the polymeric hydrophobic hydrocarbon and associated loss of activity are not suppressed.

Object of the invention

The invention has the object, a firm binding of a sufficient number of molecules of the immunoreactant to be immobilized, a reduction or inhibition of the interactions with the solid support, a coating with the immunologically active compound under standardized industrial conditions, the longest possible preservation of the immunological activity, an extension of storage time and a reduction in susceptibility to elevated temperatures and thus to achieve long-term stability of coated with sensitive immunologically active substances carrier.

Explanation of the essence of the invention

The invention has the object to achieve an improvement in the stability of immobilized immunologically active substances and at the same time to increase the binding capacity for certain immunologically active compounds to the solid support.

The object is achieved in that the immobilized immunologically active compound is bound to the Kaschierprinzip and taking advantage of the Spacereffektes to the solid support. This has the advantage that direct interactions of the sensitive immunologically active substance with the solid matrix are avoided. The suppression of these interactions takes place by application of a lower layer of an inert compound which does not adversely affect the structure of the immunologically active compound. A cover layer of the same type of compound increases the stabilizing effect.

By utilizing functional groups of the lower layer, the immunologically active compound can be firmly bound with suitable substances via chemical-covalent bonds; at the same time, the binding ability, in particular for compounds of relatively low molecular weight, is increased or, in some cases, only realized in the first place. At the same time a homogeneous bond is achieved. For the purposes of the present invention, all types of organic and inorganic polymeric carriers useful in immuno-phase solid-phase techniques are useful in medicine, pharmacy, veterinary medicine, biology, biochemistry, agriculture, etc.

Polystyrenes, their copolymers and polyvinyl chloride are particularly preferred for the purposes of the present invention in the form of microtiter plates and other reaction carriers or bars having corresponding wells with a volume of from 10 to about 1000 μΙ for carrying out the immune reactions. As a lower layer, substances of proteinogenic structure can be used according to the invention.

In particular, readily available polypeptides and proteins such as animal and human albumins (eg, bovine serum albumin, human serum albumin, rabbit, goat, sheep, horse, pig serum albumin, lactalbumin, etc.), globulins, glycoproteins (such as ovomucoid, human -Choriogonadotropin) degraded proteins, mainly degradation products of proteins of the supporting and connective tissue, polyamino acids, bacterial proteins and others

The adhesion of the backsheet to the solid support can be accomplished utilizing van der Waals forces, electrostatic or other noncovalent bonding forces, or, where appropriate, covalent bonding.

Binding to organic aliphatic and aromatic polymeric hydrocarbons such as polyethylene, polystyrene, and the like. is achieved in a known manner by adsorption, preferably by utilizing hydrophobic interactions of the carrier with the proteinogenic sub-layer. An enhancement of the hydrophobic interactions and thus a special dense and adherent proteinogenic sublayer can be advantageously achieved by prior conversion of the polypeptides or proteins "random-coil" structure.

To obtain a randam-coil structure, such known methods as heating, wherein preferably a range of 50 to 80 ° C is selected, exposure to ultrasound, ionizing radiation exposure, exposure to hydrogen bond-splitting chemical substances such as urea, guanidine or organic Solvent, pH changes.

The binding of the immunoreactant to the proteinogenic inert sublayer is covalent with known coupling reagents and conventional coupling methods.

For example, the immunoreactant may be prepared by means of p-phenylenediisothiocyanate, substituted di-sulfonic acids and other homobifunctional reagents, by means of dinitrodifluorobenzene, acrolein, methacrolein, methylgloxal, glutaraldehyde or other unsaturated aldehydes; or with bifunctional succinimide esters (such as ethylene glycol bis-succinimidyl succinate), dissuccinimidyl carboxylic acid esters, bis-Z-succinimidooxycarbonyloxyethyl sulfones; by means of heterobifunctional

Succinimide ester of maleic acid derivatives, by diazo, azide, acid chloride, anhydride, active ester method, carbodiimide WOODWARD method, activated amide method, carbodiimide method with additives such as N-hydroxybenzotriazole, N-hydroxysuccinimide, etc., sulfenyl thiocyanate method, etc. be bound to the lower layer.

It is also possible to bind haptens to the underlayer using the hemisuccinate, oxime, mixed anhydride, N-hydroxysuccinimide ester or carbodiimide ester method.

If an inert glycoprotein such as ovomucoid, ovalbumin, human ornithogonadotropin or immunoglobulin is used as the lower layer, coupling is possible by the periodate method by oxidizing the OH groups of the sugar residues of the glycoprotein to aldehyde groups and then reacting them with the immuno-lactate.

A particularly high stability of the immunologically active substance bound to the lower layer is achieved when "covering" with a medium containing aminocarboxylic acids or acid amide-linked aminocarboxylic acids and polyhydroxy compounds, respectively entering the substance with it.

As aminocarboxylic acids, natural amino acids, individually or as a mixture or as a protein hydrolyzate or else synthetic amino acids or amino acid derivatives, can be used.

Suitable acid amide-linked compounds are cheap commercially available polypeptides, such as by oxidative-thermal degradation processes from industrially obtained depolymerized hydrolysis of fibrillar scleroproteins of animal connective tissue of collagen origin or acid and enzymatically hydrolytically degraded proteins, eg. The casein,

Lactalbumins, spheroplasts, proteins of various oilseeds and legumes, the cereal seeds in a concentration of 0.006g N / 100ml to 5g N / 100ml solution preferably 0.05 to 1gN / 100ml solution.

There are also inexpensive, readily available proteins from animals and humans, such as albumins, globulins, lactalbumines and the like.

or scleroproteins, such as gelatin in question, which are used in a concentration of 0.01 to 20%, preferably 0.1 to 2%.

As polyhydroxy compounds, in particular monosaccharides and their derivatives, for. As aldoses, ketoses, deoxysugars, sugar esters, sugar acids, sugar alcohols, sugar anhydrides, anhydrous, unsaturated sugars, aldonic acids, sugar carbonyl acids, sugar acetals, sugar ethers, cyclites and acetal-linked polyhydroxycarbonyl compounds such as oligosaccharides, preferably disaccharides and polysaccharides, such as homopolysaccharides and heterosaccharides used.

Concentrations of from 0.5 to 30%, preferably from 5 to 20%, have proven to be particularly favorable.

The incubation period of the medium should be at least 20 s and can be extended to several days.

According to the invention, it is furthermore advantageous in certain cases for the "lower layer" to use protein derivatives or protein conjugates -such as proteins which are bonded to amino groups, carboxy groups, guanidino, imidazole, thioether, sulphydryl or hydroxy groups with:

a) suitable reactive with the functional groups of the protein organic groups, preferably substituted aromatic compounds, heterocyclic compounds, e.g. 2-ket-3,4-imidazoline-2-tetrahydrothiophene-n-valeric acid, and the like, which have antigenic properties or with other haptens or artificial antigens

b) peptides (homodet or heterodet-linked), peptide hormones, peptide antibiotics, peptide toxins, peptide alkaloids, peptoids (nucleo-, phospho-, lipo-, glyco-, chromopeptides), depsipeptides or proteinogenic conjugates, eg. Peptide-linked 2-ket-3,4-imidazoline-2-tetrahydrothiophene-n-valeryl-lysine,

c) proteins, in particular immunoglobulins; enzymes,

d) vitamins or vitamin derivatives

e) lectins

f) polysaccharides

g) nucleic acids, nucleotides

h) cells, cell components, bacteria or viruses or their components have been substituted or condensed.

This has the advantage that subsequently the binding of the immunoreactants used in the test takes place via protein-receptor interactions, which further reactivity losses of the reactant, which otherwise occur inevitably in chemical reactions avoided.

The preparation of the protein derivatives and protein condensates used as underlayer is carried out in a known manner with conventional coupling methods and reactions, such as by means of acid chloride, anhydride, active ester, azide, diazo, carbodiimide or other known electrophilic or nuclophilic substitution reactions ("Organikum"). , Organic Chemistry Grundpraktikum, Berlin 1985).

After binding of the immunoreactant by protein-reciprocal interactions, the "contacting" with the overlaying / embedding medium takes place according to the invention, as already described.

embodiments

example 1

Each well of a polystyrene microtitration plate (F form) is mixed with 200 μΙ a solution of bovine serum albumin (100 ng / ml) in carbonate buffer (0.1 mol / l; pH 9.5), which was heated to 7O ⁰ C, charged. It is allowed to stand for 2 h at 37 ⁰ C, filtered with suction or dumped and washed twice with water.

The wet or dried plates are incubated with a solution of methylglyoxal (1.0%) in phosphate buffer (0.1 mol / l, pH 5.8) for 4 h at 23 ± 5 ^° C. and then washed with phosphate buffer.

Subsequently, 100 μl of an immunoglobulin solution (5 μg anti-HBe IgG / ml) are pipetted into phosphate buffer (pH 7.6) and incubated for 4 hours. It is washed twice with phosphate buffer (0.01 mol / l) containing sodium chloride (0.13 mol / l).

200 μΙ of a sterile-filtered phosphate-sodium chloride solution containing horse serum albumin (0.0003 mol / l) and alpha-D-glucopyranosido-β-D-fructofuranoside (0.50 mol / l) are pipetted into each well of the plate.

After 2 h incubation at 23 ± 5 ⁰ C is filtered off with suction, dried and sealed in water-vapor-tight plastic-coated aluminum foil and stored at 37 ⁰ C.

To measure the immunological activity, the P / N quotient is calculated, which is calculated as the quotient of the extinction of the positive control and the extinction of the negative control. For this purpose, a portion of the microtiter plate with a HBe antigen-containing serum (positive control) (per well 100μΙ) and the other part with the negative control (serum containing no HBe antigen) fed. It is incubated for 2 h at 37 ⁰ C, washed as usual with phosphate-sodium chloride buffer, then incubated with anti-HBe IgG peroxidase-Knnjugat (100μΙ per well) for 2 h at 37 ° C and washed again.

100μΙ phosphate / citrate buffer pH 5.0 containing 9mmol-phenylenediamine and 7mmol hydrogen peroxide are pipetted into each well. After 30 min incubation at 23 ⁰ C is stopped with 100 μΙ sulfuric acid (1 mol / l) and evaluated at 492 nm absorbance. The P / N quotient after storage at 37 ^° C. is 10. Week 12.5; 20th week 11.8 and before storage 12.9.

For comparison, a polystyrene microtiter plate is coated directly with anti-HBe immunoglobulin in carbonate buffer, incubated for 4 h at 23 ± 5 ° C, filtered off with suction, washed with phosphate buffer, dried and sealed in plastic-coated aluminum foil.

As described, the immunological activity is measured before and after storage at 37 ^° C. and the P / N quotient is determined.

P / N quotient: before storage 10.2; 10th week after storage: 1.3

Example 2

A. Each well of an EIA reaction plate is filled with 200 μΙ of ovalbumin solution containing nitrogen

1 mg / 100 ml and additionally contains 6 mol / l urea, and stored at 23 ⁰ C. After 4 h is filtered off with suction, washed with water and dried at 37 ° C for 4 h.

Each well of the plate is charged with 100 μL of a solution of i-ethyl-S-fö-N, N-dimethylaminopropyllcarbodiimide.HCl (0.05 mol / L). After 1 h, it is washed with water. 100μΙ purified rubella virus antigen (HA titer 1: 8) is pipetted into each well. After a reaction time of 2h (37 ⁰ C) is washed. Each well is mixed with 200μΙ of a solution, the albumin from bovine serum (5.0g / l) and 0.50 mo! alpha-D-glucopyranosido-beta-D-fructofuranoside filled. After 3 hours, the product is filtered off with suction, dried, sealed in a water vapor-tight film and stored.

B. Each well of an EIA reaction plate is loaded with 100 μM of purified rubella virus antigen (HA titer 1: 8). After a reaction time of 2 h (37 ⁰ C) is washed. The plates are dried, sealed in a water vapor-tight film and stored.

C. The immunological activity is determined in the enzyme immunoassay.

For this purpose, the wells washed with phosphate / sodium chloride / Tween buffer the plate with 100μΙ of a serum containing a buffer solution (0.01 M phosphate / 0.13 M sodium chloride / 10% sheep serum, 0.05% Tween 20, pH7, 2) diluted 1: 200 and contains IgG antibodies against rubella virus, charged and incubated at 37 ⁰ C for ² h. After washing, ΙΟΟμΙ of a phosphatase-labeled anti-human Fc antibody (1 μΙ / ml) are pipetted per well. After 45min of incubation at 37 ⁰ C and quenching with 100μΙ sodium hydroxide solution (2 mol / l) is at 405 nm determined by Vertikalphotometrie the solid phase-bound enzymatic activity.

After 10 days of storage at 37 ⁰ C 93% 89% B and 0% of the immunological activity obtained after drying were measured at A and found again in B18%, after 30Tagen storage at 37 ° C at A.

Example 3

A. The surface of each well of the EIA reaction plate (F-form) is coated with bovine serum albumin as described in Example 1.

The wet or dried plates are incubated with a solution of glutaraldehyde (5 mg / ml) in phosphate buffer (0.1 mol / l, pH 6.8) for 2 h at 23 ° C ± 5 K and then washed with phosphate buffer.

In each well of the plate 200 μΙ a solution of Toxoplasma lysate antigen (25 μρ / ΓηΙ), which was prepared in a known manner according to WP G 01 N / 2307405, pipetted and 2 h at 37 ⁰ C and 1 h at 2 to 8 ° C incubated. After washing, each well is washed with 250 μΙ of solution, the partially degraded gelatin ("Gelafusal" infusion solution, 1.5 mg N / ml) and 4- (β-D-galactopyranosido) -D-glucopyranose (0.6 mol / L). contains, loaded.

After 2 h (23 ⁰ C) is filtered off with suction, dried, sealed and stored.

B. Each well of the EIA reaction plate is loaded with 200μΙ Toxoplasma Lysate antigen solution (25pg / ml). After incubation for 16 hours at 2 to 8 ⁰ C is suction filtered, washed and each well is enlhält 250μΙ with a solution containing analog "Gelafusal" solution, and 4 / beta-D-Galactopyranosido) -D-glucopyranose filled. After 2h (23 ° C) is filtered off with suction, dried and sealed in water vapor-tight packaging and stored.

C. The immunological activity is measured as described in Example 2C using instead of the rubella serum an anti-Toxoplas: na serum containing 0.2 IU of antibodies to toxoplasma.

After 20 days of storage at 37 ° C, 86% of A and 50% of B of the post-drying immunological activity were recovered.

Example 4

A. As described in Example 1, the surface of the wells of EIA reaction plate is coated with bovine serum and then washed with water. Each well of the plate is charged with 100 μΙ of an aqueous solution of 1-ethyl-3- (3-N, N-dimethylaminopropyl) carbodiimide.HCl (2 mg / ml). After 2 hours, washed with water, in each well 100μΙ of a pollen extract (500 NOON) of the grasses Phleum pratense, Poa pratensis, Lolium perenne pipetted. After a reaction time of 1 h (37 ⁰ C) is washed. Each well is filled with 200 μM of a solution containing bovine serum albumin (0.1 mmol / L) and 0.50 mol of alpha-D-glucopyranosido-beta-D-fructofuranoside. After 2 h is filtered off with suction, dried, sealed in a water vapor-tight film and stored.

B. EIA reaction plates are coated in a known manner with the 3-grass pollen extract (0.1M carbonate buffer pH 9.6, 16h, 20 ° C). After washing, analogously A is coated with a solution of albumin and alpha-glucopyranosido-beta-D-fructo-furanoside, dried, sealed, stored. ·

C. To determine immunological activity, the plates coated with allergen per well are diluted with ΙΟΟμΙ of a serum diluted with 100 parts of phosphate (0.01 MJ sodium chloride (O, 15M) buffer (pH 7.2) and specific IgE

Contains antibodies against grass pollen allergens. After 2 h (23 ⁰ C) is filtered off with suction and washed. After addition of a monoclonal mouse anti-IgE antibody (1: 600 diluted in phosphate buffered sodium chloride solution (0.15 mol / L) to which bovine serum albumin (20 mg / ml) was added); is incubated for 2 h (23 ⁰ C) and aspirated.

After reaction (2 h, 23 ⁰ C) of a peroxidase-labeled anti-mouse antibody (goat) (0.6pg / ml) (1 ΟΟμΙ per well), the solid phase-bound enzyme activity by reaction with o-phenylonediamine / hydrogen peroxide reagent, as in Example 1, determined.

After drying, A: E = 1.542 at B: E = 1.040 and after storage for 30 days at 23 ± 5 ° C, A: E = 1.420 and B: E = 0.803.

C)

Claims (9)

1. A method for producing an immobilized immunological reagent, characterized
characterized in that an immunologically active substance to one, a solid phase carrier
covering hydrophilic layer bound and then with a medium of the same kind
is overlaid. 2. The method according to claim 1, characterized in that the carrier coating layer
proteinogenic nature is. 3. The method according to claim 1 and 2, characterized in that protein derivatives or
Protein conjugates can be used as the carrier-coating layer. 4. The method according to claim 1 and 2, characterized in that water-soluble polypeptides or proteins are used with "random-coil" structure. 5. The method according to claim 1 to 4, characterized in that the immunologically active substance is covalently bound by known methods to the carrier coating layer or in the case of the use of protein derivatives or protein conjugates as a carrier-coating
Layer through protein-reciprocal interactions to the carrier coating layer
is bound. 6. The method according to claim 1, characterized in that used for overcoating
Medium contains nitrogen compounds and polyhydroxy compounds. 7. The method according to claim 1 and 6, characterized in that the nitrogen compounds
Aminocarboxylic acids or lower or higher molecular acid amide linked
Represent aminocarboxylic acids. 8. The method according to claim 1 and 6, characterized in that the polyhydroxy compound is a polyhydric alcohol, a polyhydroxy aldehyde, a polyhydroxy ketone, a derivative of
Polyhydroxycarbonyl compound, an acetal-linked polyhydroxycarbonyl compound. 9. Use of the immunological reagent prepared according to claim 1, characterized
characterized in that it is used for the qualitative detection and quantitative determination of
Antigens, such as haptens, viruses, bacteria, pharmaceuticals, proteins, allergens and antibodies
against said antigens and other biologically active compounds, and that it
also for the purification and isolation of corresponding reactants, which with the
bound immunologically active substance specific interactions, is used.