DE2910414A1 - METHOD FOR MANUFACTURING POLYGLUTARALDEHYDE AND ITS USE - Google Patents

Description

California Institute of Technology,

A company under the laws of the state of California,

"Pasadena, California, Y.St.Ä.

"Process for the preparation of polyglutaraldehyde and its
Use".

Claimed Priority: March 17, 1978, T. St .A., JJr. 887 825.

The present invention was made in the exercise of a job under the MASA contract and is subject to the Regulations of Section 305 of the National Aeronautics and Space Act of 1958, Public law 83-568 (72 Stat'435; 42 USC 2457).

The invention relates to the production of polyglutaraldehyde, the conversion of the polymer into a fluorescent form, the binding of proteins to the polymer and the use of the polymer-protein addition products for biological and chemical research and investigation.

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The extraction and characterization of cell membranes and their components is essential to understanding the role they play Surface menebras with regard to the control of a large number play of biological and immunological effects. The current techniques used for this purpose are not completely satisfactory "

The knowledge of the type, the number and the distribution of specifically acting receptors on cell surfaces is of central importance for the understanding of the molecular basis, the biological phenomena such as cell-cell recognition during development, cell transmission and regulation by hormones and chemical vectors and underlying differences in normal and tumor cell surfaces. In previous efforts is the localization of antigens and carbohydrate moieties on the cell surfaces, especially in red blood cells, and lymphocytes, by binding antibodies or lectins (isolated from plants hämagglutinatierende substances) to such macromolecules, such as ferritin (a Crystallizing cash protein with a content of 20 to 24 percent iron and 1.2 to 2 percent phosphorus), hemocyanin or peroxidase, which have served as marking agents for transmission electron microscopy, have been determined. However, the topographical distribution of the molecular receptors on the surfaces of cell and tissue samples can be advantageous in electron microscopy with a high raster resolution in a simple manner by the same histological techniques using recently developed, by means of electron microscopy with a high raster index.

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fineness of dissolvable marking agents can be determined.

Polystyrene latex parts have recently become commercially available has been used as an immunological marker for application in electron microscopy with high screen fineness. The surface of such polystyrene particles is hydrophobic, and therefore certain types of macromolecules, such as antibodies, absorbed onto the surface under carefully controlled conditions. However, such particles adhere unspecifically Manner on numerous surfaces and molecules, and this severely limits their broad applicability.

■ The production of small, stable, spherical methacrylic acid-hydroxyethyl ester-acrylamide copolymer particles? which are biologically compatible, i.e. do not react in an unspecific manner with cells or other biological components, and which contain functional groups to which special proteins and other biochemical molecules can be covalently bound, is described in U.S. Patent 3,957,741.

Smaller and even more uniformly shaped microspheres made from acrylic polymers are described in US Pat. No. 4,138,383. Microspheres having a density different from that of cell membranes are disclosed in U.S. Patent 4,035,316 and US Pat fluorescent microspheres based on an acrylic copolymer are in U.S. patent application filed Aug. 27, 1976 No. 718 104 has been proposed.

Hydroxl groups can be activated by Bromeyan to make proteins and other chemical compounds with amino groups on Po-

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Lymerisatperlehen to bind covalently. Methacrylic acid residues, which give the particles a negative charge, are likely prevent non-specific binding to cell surfaces and provide carboxyl groups to which a "variety of biochemical Molecules covalently using the carbodiimide method can be bound. . ""

The derivatization procedure is unnecessarily complicated and requires an additional step to allow the bead surface to be covalently bonded to proteins, such as antibodies, Lectins and the like, or to other molecules such as deoxyribonucleic acid, Hormones and the like. It is for this reason that the method of derivatizing microspheres is based on acrylic polymers lengthy and limited in terms of its applicability. Monomeric glutaraldehyde is as biochemical reagent for covalent attachment of proteins, such as Immunoglobulins, to ferrite in polymer microspheres and to others small particles have been used, which are then applied to the topographical representation of receptors on cell membranes "The reaction mechanism of proteins with glutaraldehyde is difficult to determine because its structure is still unclear and has been reported to be cyclic and hydrated Forms is in balance. The reaction is not easy to be carried out and often gives unsatisfactory results,

The invention is now concerned with the direct binding of proteins to polyglutaraldehyde. In contrast to monomeric glutaraldehyde the polymer contains conjugated aldehyde groups *

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This ensures the formation of a more stable ship ^! shear bases which form after reaction with proteins, and further, since the hydrophilic polyglutaraldehyde has relatively long chains which extend from the surface into the surrounding aqueous medium, the heterogeneous reaction with protein is facilitated,

Polyglutaraldehyde can be produced in a solution that is used for pouring pollen or films or for coating surfaces of substrates such as polymer microspheres, in particular pearls substituted with amino or hydrazide groups, suitable is. - Polyglutaraldehyde microspheres can be used directly by suspension polymerization in the presence of a surface-active compound or by precipitation from a surface-active compound Compound-containing solution can be produced «Polyglutaraldehyde can be converted into fluorescent polymer with non-fluorescent compounds such as m-aminophenol. Furthermore, one can use magnetic, high density or high electron density microspheres by coating metal parts or by suspension polymerisation of glutaraldehyde in the presence of a suspension of finely divided metals or metal oxides. Higher yields and higher molecular weights can be achieved by using a mixture of water with strongly polar solvents obtain.

These and numerous other advantages of the present invention are explained in more detail below for a better understanding, whereby the drawing is to be used,

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Fig. 1 shows the rate of reaction of 60 mg of polyglutaraldehyde with 4.5 mg human immunoglobulin G- (IgG-) in 50 ml Phosphate buffered sodium chloride solution at 22 ° G:

β pH 6,

ο pH 7.4-.

Pig. 2 shows the reaction rate of beads of a vinylpyridine-acrylamide-hydrazide copolymer (hereinafter referred to as "poly-VP-AU-hydrazide") with fluorescent human IgG at 22 ^° C. and pH 7.4:

• Polyglutaraldehyde bound to poly-YP-AM hydrasid, ο Ivlonoglutaraldehyde bound to poly-VP-AM hydrazide, χ pearls made of poly-YP-AM-hydx-azide (comparison), £ 'IgG in phosphate-buffered batriurachloride solution (Comparison).

Pig. Figure 3 shows the rate of reaction of poly-VP-AM hydrazide beads with fluorescent human IgG- at 4 Ci

• Polyglutaraldehyde bound to poly-VP-AM-hydrazide, ο monoglutaraldehyde bound to poly-VP-AM-hydrazide, χ pearls over poly-VP-AM-hydrazide (comparison), £ IgG in phosphate-buffered i-sodium chloride solution ( Comparison)"

Pig. 4 shows the reaction rate of poly-VP-AM-hydrazide ^{beads with fluoresciex%} endemic anti-human goat immunoglobulin M at 4 ° C and pH 7.4: -

ο polyglutaraldehyde bound to poly-VP-AM hydrazide, Δ monoglutaraldehyde bound to poly-yp-AM hydrazide, χ Poly-VP-AM-hydrazide beads (comparison).

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■! Ig. 5 shows the reaction rate of 1.00 mg polyglutaraldehyde in 50 ml phosphate buffered Jatriumchloridlösung with 7.5 mg hemoglobin at 22 ⁰ C as a function of pH Yiertes; ο pH 6,

h pH 7.4,

χ pH 8.4,. ■ ·

• Pearls from a methacrylic acid — hydroxyethyl ester—

Acrylamide copolymer at pH 6 (comparison). Prop. 6 shows the reaction rate of beads of a methacrylic acid-hydroxyethyl ester-acrylamide copolymer with hemoglobin at pH 6 and 22 ⁰ C:

ο polyglutaraldehyde bound to the pearls, & monoglutaraldehyde bound to the pearls, Φ pearls (comparison),

Pig. 7 graphically shows the effect of concentration a surface-active compound down to the size of the microspheres (10 weight percent G-lutaraldehyde, pH 11). Fig. 8 graphically shows the effect of pH on the diameter of the microspheres. (10 weight percent glutaraldehyde, 1 percent by weight surfactant compound). 3? Ig. 9 shows the effect of concentration in a graph of glutaraldehyde to the diameter of the microspheres (1 percent by weight surface-active compound, pH 11),

The invention is described in more detail below on the basis of preferred embodiments.

Polyglutaraldehyde is made from an aqueous solution containing 5 to 50 Contains percent by weight of monomeric glutaraldehyde, by increasing

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the pH of the solution to values above 7>", preferably to values of 9 Ms 12. In the absence of a catalyst, the reaction is spontaneously exothermic Cooled from 15 to 30 ^° C. and then heated until the end of the polymerization to a temperature above 40 ^° C. It is assumed that the polymer has the following repeating unit: i

- CH ₀ - CH ₀ - CH = C-

CHO

An analysis based on the UV or IR spectrum confirms the problem of conjugated functional aldehyde groups in the polymer, die.mit molecules containing amino groups, such as proteins, form more stable bonds,

If the aqueous medium contains at least 80 percent water, the polymer precipitates and can be filtered off, washed with water and dried. Further washing with solvents such as acetone appears to reduce the yield * The polymer can be used in polar solvents such as methanol , Ethanol, or tetrahydrofuran, and especially in strongly polar solvents such as Biiaethylsulf oxide or dimethylformamide., Are redissolved. "Solution polymerization, in mixtures of water and polar solvents containing 20 to 80 percent solvent, delivers higher yields of polymer. The solution can be used to cast films or films or to coat subotrates. The polymer can be made from Solution precipitated vc: eden, Indder :: nan the Y / water content to over 80 percent

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increases and then the particle wins. *.

The examples illustrate the invention.

example 1

Commercially available aqueous solutions of glutaraldehyde are purified using activated charcoal. They are then free of polymers, as shown by the absence of the absorption band at 235 nm, measured by means of a "Cary 14" spectrophotometer. A 25 percent solution of G-lutaraldehyd is polymerized at 22 ⁰ G by adjusting the pH to 11 '5 by means of sodium hydroxide. Compartment 120 minutes the reaction mixture stirred 8 hours "at 5O ⁰ C. The precipitated polymer is filtered off, washed with water and then washed with acetone and then under reduced pressure for 24 hours dried. Yield:. 60 percent of the molar Extxnktionskoeffxzient βρ ⁷ 5 ^ ^n. Ethanol) is 1.53 x 10

- 1 - 1
Liter χ mole χ cm. The molar extinction coefficient of glutaraldehyde at 280 nm (extracted from aqueous solutions with ether and dried over magnesium perchlorate) is £ pgc ~ ^ '^ - ^ it er χ

—1 —1

Mol χ cm. The polyglutaraldehyde is soluble in dimethyl sulfoxide and has an average molecular weight of approximately 20,000 as determined by gel permeation chromatography has been on.

Example 2

The procedure of Example 1 is carried out in a mixture of water and dimethyl sulfoxide in a ratio of 1: 1 repeated. The polymer remains in solution,

Polyglutaraldehyde shows little iTuorescenz. However, it delivers

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the conversion of polyglutaraldehyde with certain compounds, which are not themselves fluorescent, a fluorescent polymer by the formation of fluorescing chromophores which are bound to the polymer. This is how anthrone reacts with acroleic units with formation of a benzanthrone fluorogen and m-aminophenol with acrolein units to form the fluorogen 7-hydroxy-quinoline ,. Aminofluorescin also reacts with Polyglutaraldehyde with formation of a fluorescent polymer "

Example 3 ■,

50 mg of the polyglutaraldehyde obtained according to Example 1 are in 10 ml of dimethyl sulfoxide dissolved. Then 20 mg · m-aminophenol is added and a (drop of 10-N hydrochloric acid and heat the solution at 50 C for 60 minutes. The solution fluoresces strongly when it is irradiated with a UY light lamp. By adding water the polymer precipitates, which is washed with Yvasser and then is dried. The isolated polymer is again dissolved in dimethyl sulfoxide solved and examined with regard to the fluorescence. A strongly fluorescing polymer is observed with a ITuorescence - eralssion maximum - at 470 to 480 nm,

Example 4

About 0.1 g of the solid polymer prepared according to Example 1 are suspended in 0.5 N sodium bicarbonate solution and mixed with an aqueous solution of 2 mg. 9 — Amino-acridine hydrochloride Room temperature. After exhausting Y / ashing with 7 / water, the polyglutaraldehyde particles show up under a suitable UY light source an intense fluorescence. After Several

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subsequent washing does not decrease the fluorescence intensity away.

Solutions of polyglutaraldehyde in strongly polar solvents, generally 0.1 to 5 percent by weight polyglutaraldehyde may be cast into clear films or coatings of varying thicknesses, usually 0.0025 to 0.125 mm by applying the solution to the surface and allowing the solvents to evaporate from the film or coating. The surfaces can be planar, particulate, spherical or bar-shaped and made of inorganic or organic substances. The inorganic substance can be a metal or a metal compound, in particular a magnetizable substance, such as iron, nickel or cobalt, or their oxides, such as magnetite, furthermore ceramic or glass beads or the like. Organic substances are usually small polymer beads like those already mentioned above Pearls made from a methacrylic acid-hydroxyäthyle'ster-acrylamide copolymer.

The polyglutaraldehyde coatings can be covalently applied to substrate surfaces which contain amino groups, such as glass beads modified with alainosilane compounds, for example Beads made from cross-linked with trimethoxy-isopropylamino-silane Acrylamide homopolymers or copolymers. The beads are preferably very small, namely 20 nm to 100 µm, generally 50 nm to 10 pm, so that the specific receptor sites can be marked on a cell surface. Pearls or beads' made of vinylpyridine or methacrylic acid-hydmxy-

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Ethyl ester-acrylamide copolymers are described in US Pat. No. 3,957 and has also been proposed in US patent application ITr, 780 007, filed March 22, 1977, on the contents thereof Is referred to. The interpolymer beads or spheres generally contain at least 50 percent vinyl pyridine (YP), 5 to 35 percent acrylamide (AM), and 0.1 to 30 percent one crosslinking agents that are copolymerizable therewith, such as bis (acrylamide) (BAM).

The adhesion of polyglutaraldehyde coatings to the pearls or spherules is further increased by converting the amino groups into hydrazide groups. The modification can be brought about by a reaction with an excess of hydrazine hydrate at a temperature of at least 40 ^° C. with subsequent separation and washing.

Example.

A 0.1 percent solution of polyglutaraldehyde in tetrahydrofuran is sprayed onto a G-Ia s subs step. After the solvent has evaporated, a transparent I ⁵ IIm is obtained, which is firmly bonded to the glass surface.

Example 6

100 mg of centrifuged 5 moist vinylpyridine-acrylamide copolymer beads, which contain 20 percent acrylamide and 20 percent bis (acrylamide) and have a diameter of 0.7 μm, are mixed with 10 ml of hydrazine for 4 hours at room temperature. hydrate stirred and then heated ^{to 120 0} G with stirring for 120 minutes. The microspheres are then washed with water. If tested with 2,4,6-ΪΎχηχΐχ-ο- ^ εηΕθΐ3 ^ ίοη8Μυτβ,

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they provide a colored product that indicates hydrazide groups. This copolymer containing hydrazide groups is described below referred to as "poly-YP-AM-hydricide".

Example 7

The poly-VP-AM hydrazide microspheres of Example 6 are with Polyglutaraldehyde encapsulated by making a 10 ml suspension (25 mg / cm) of the microspheres stirred for 240 minutes at room temperature with polyglutaraldehyde which, as in Example 1, was dissolved in diethyl sulfoxide (25 mg in 50 ml). Compartment 'to centrifugation and after thorough washing with dimethyl sulfoxide and Water, the presence of aldehyde groups on the "surface is confirmed by testing with Puchsin-Schiff'schem test reagent.

The microspheres coated with polyglutaraldehyde are suitable for clinical, diagnostic investigations, affinity chromatography, the mobilization of enzymes, the chemical separation and removal of urea and generally applicable to areas of application where a bond to amino groups Connections is useful. There are numerous areas of application found in biochemistry, biology and clinical chemistry been.

The reactivity of polyglutaraldehyde with low molecular weight Amines is investigated on a single-phase (homogeneous) system and on a two-phase (heterogeneous) system. Homogeneous reactions are 'carried out at a relatively low temperature, for example, polyglutaraldehyde with methylamine in liquid anhydrous ammonia for 60 minutes at about -40 ° C ! • s left,! Times the rise in temperature and after 7in-

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evaporate to dryness, the products are washed with water and ^{dried at 6O 0} C under reduced pressure. The products are analyzed spectrometrically and their nitrogen content is determined. The polymer is also soluble in higher-boiling amines, such as allylamine, butylamine, iithano'lamine, hydrazine and ethylenediarain, at room temperature. The reaction with these amines is carried out at 22 ° C. for 60 minutes. The analysis is carried out in the same way as in the case of the reactions at low temperature «

The heterogeneous reaction is carried out by adding 120 mg of the polymer to 20 ml of a phosphate-buffered sodium chloride solution suspended with a content of 1 g of the amine at a pH of 7.3 to 7.4 and the suspension for at least 30 hours Stir in a closed container, wash compartment with Y / ater and after drying check the nitrogen content of the product certainly. The results of the elemental analysis of the products produced are given in Table I below.

Table I.
Nitrogen fourth of polyglutaraldehyde-imine reaction products.

Amine homogeneous reaction heterogeneous reaction foE ber. ^C 0 found . <f ° E ber. ° / οΈ found Methylamine 38.1 5.6 Hydrazine 47.4 14.0 ammonia 16.8 2.9 Allylaiiiin 48.6 5.6 Butylamine 63.1 6.5 Ethanolamine ' 44.6 5.0 5.3 0.6 Diamino heptane 31.9 4.6 3.5 0.5 Hydroxylamine ' 52.1 7.5 Glycine 8.9 0.9

Average of 2 determinations;

Hydroxylaiuin-HG1 is used at pH 7.3 and 7.4; the

y
Nitrogen content

Percent"

QZ

The theoretical amount of nitrogen is calculated on the assumption that every unit -CH ₂ -CH ₂ -CH = C (GHO) - binds one molecule of amine. The spectrophotometric investigation shows that for most of the products the absorption maximum ascribed to the aldehyde group has been considerably reduced at 285 µm. Based on the results with hydroxylamine, the polyglutaraldehyde contains one aldehyde group per molecular weight of 160.

Examination of the results leads to the following conclusions:

Polyglutaraldehyde shows a high reactivity towards low molecular weight amines under mild test conditions (cf. Table I). The remarkable reactivity of hydroxylamine indicates that this compound is a better reagent over glycine used in attempts to inactivate Aldehyde groups activated with glutaraldehyde or polyglutaraldehyde Cell labeling reagents are used. The highest theoretical yields of reaction products, based on on the assumption that the repeating unit in the polymer is a substituted acrolein, amount to about 50 to 60 percent. Spectrometric studies show this Presence of unreacted aldehyde groups in the reaction products. Therefore it is likely that education by ship see bases reached a state of equilibrium and that higher yields than those given in Table I can be expected by changing the experimental conditions.

Reactions with human immunoglobulins: fluorescent human immunoglobulin G and non-human Goat immunoglobulin M are treated with diethylamine ethyl cellu-

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loosely filled columns cleaned. Your fluorescent intensity at different pH values are "at room temperature by means of a "Aminco Fluorimeter", Model SPl 125, measured.

The degree of conversion is determined after centrifuging the suspensions, which contain the fluorescent immunoglobulin G and insoluble polyglutaraldehyde. Measure the iTuorescenz of the protruding Solution determined. Two series of experiments are carried out. In the first series of tests, powdered polyglutaraldehyde is used Immediately implemented with fluorescent immunoglobulin G. and eaten the decrease in fluorescence after the implementation, so that the amount of immunoglobulin G- bound to the polymer is obtained. MikroMigelchen from a methacrylic acid-hydroxyethyl ester-acrylamide copolymer are implemented under identical conditions and are used for control and comparison purposes.

The second series of experiments consists of reactions with polyglutaraldehyde, that of the hydrazide groups on the surface of the pearls of poly-VP-AM hydrazide with a diameter of 0.7 µm is covalently bound *

The extent of the chemical bond is quietly examined for the following: 25 mg of poly-VP-AM hydrazide microspheres are at least For 5 hours with 50 ml of a solution containing 25 mg of polyglutaraldehyde Stirred solution of dimethyl sulfoxide. After centrifugation and washing with dimethyl sulfate and water, fluorescent immunoglobulins are added to 5 cm beads (Tables II, III and IV). The respective mixtures are stirred at a certain temperature, and then the loss of fluorescence is after a certain time using the centrifuged solvent

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solution in aliquot parts ".

Reaction with hemoglobin: "Polyglutaraldehyde or poly-VP-AM-hydrazide beads with a polyglutaraldehyde coating are in twice recrystallized hemoglobin in 0.01-M phosphate buffer solution is dissolved, suspended. The pH is adjusted using sodium phosphate and recorded after a certain one Time the optical density at 412 nm of filtered aliquots Shares.

Marking of lymphocytes with immune microspheres: Human lymphocytes are separated from the red blood cells by means of a "Pieoll-Hypaque" gradient ("Picoll" = a polymeric cane sugar with a molecular weight of 400,000; "Hypaque" = sodium salt of 3,5-diacetamido -2.4>'6-triiodo-benzoic acid), freed from monocytes by means of iron particles and suspended in a medium at a pH of 7.2 to 7.4 to which 20 percent serum from calf fetuses is added. The medium ("EPMl / H") consists of * "RPMI 1640" (from G-ibco, Y.St.A.) and 0.025-m F-2-hydroxyethyl piperazine-l ^l I-2-athanesulfonic acid Solution.

The microspheres attached to the immunoglobulins using polyglutaraldehyde (0.5 cur of a microsphere suspension containing 0.5 mg microspheres in phosphate buffered sodium chloride solution of pH 7.4) are 60 minutes at 4 ° C with 5x10 lymphocytes in 1 enr "RPMI / H" buffer solution with a content of 0.1 percent Incubated sodium azide. After adding 1.5 cur of serum Calf fetuses and 0.1 percent sodium azide are added to the suspension centrifuged in a centrifuge at 1500 rpm (515 g). The received Cells are again in a solution of 50 percent "RPHl / H"

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and 50 percent serum τοπ calf fetuses with a content of 0.1 percent sodium azide suspended and centrifuged again. It has been found necessary to repeat the cleaning procedure three times to remove virtually all unbound microspheres. Third centrifugation compartment
the cells are again suspended in "RPMl / H" - this time with a content of 1 percent bovine egg serum albumin and 0.1 percent sodium azide. The labeled cells are then counted under the microscope.

In a comparative experiment, the lymphocytes are 60 minutes
incubated with aggregated Iiamoglobulin G. The aggregated immunoglobulin G- is produced by dissolving human immunoglobulin G (50 mg / cm) in phosphate-buffered sodium chloride solution
at pH 7.4 and heating the solution to 63 ° C for 30 minutes. The clear supernatant solution is used to incubate the
Cells used before marking. ... *

Results ο

Reaction of polyglutaraldehyde with low molecular weight amines;
Preliminary information on the reactivity of the polymer with amino groups is obtained from carrying out reactions with a number of amines. Table I shows the results of the electrical analysis of the products. The theoretical amount of nitrogen is calculated on the assumption that each unit
-CH ₂ -CH ₂ -CH = C (CHO) - binds a molecule of amine. In the case of hydroxylamine hydrochloride, hydrazine and butalamine, the degree of conversion is in the order of 50 to 60 percent of the theoretical amount (Table I).

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Reaction of polyglutaraldehyde and methacralic acid-hydroxyethyl ester-acrylamide copolymer beads with a polyglutaraldehyde coating with immunoglobulins: Table II shows the Amount of the immunoglobulin G bound to the polyglutaraldehyde after stirring the suspensions for 135 minutes at room temperature. A constant amount of fluorescent is used in these experiments Immunoglobulin G- used.

Table II.

Reaction of powdery)! Polyglutaraldehyde with human immunoglobulin G (1 mg fluorescing immunoglobulin G in 10 ml phosphate buffer solution, pH 7.4).

PοIyglutar-

'Ζ

aldehyde mg / cm

Methacrylic acid-hydroxyethyl ester-acrylamide copolymer with a coating of polyglutaraldehyde mg / cm3

Immunoglobulin G bound to the polymer (weight / weight)

at pH 7.4 at pH 6

0.2

2.0

20.0

0.2 • 2.0 20.0

0, 160 O, 200 O, 030 O, 040 O, 004 O, 006 0 0 0 0 0 0

From Table II it can be seen that increasing amounts of the polymer a lower immunoglobulin G / polymer ratio deliver. The same results are obtained with microspheres derived from either polymeric or monomeric glutaraldehyde, obtained (see Table III).

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Table III. 2910414 Comparison of the reactivity of beads with coatings made of monomeric! or from polymeric glutaraldehyde. Effect of concentration of monomeric or polymeric glutaraldehyde (0.5 mg fluorescent Immunoglobulin G in 10 ml phosphate buffer solution, pH 7.4) In all experiments 25 mg of beads per cm are used.

comparison
ZZ
mg / cm Polyglutaraldehyde
mg / cm Glutaraldehyde
mg / cm - Weight of the bound
which immunoglobu
linsG / weight of
Polymer χ 10-5 2.5 11.0 7.5 4.0 20.0 1.4 2.5 6.0 7 ", 5 2.0 20.0 0.7 2.5. ■ • 0

Table IV.

Comparison of the realism of beads with coatings monomeric or polymeric glutaraldehyde., Effect of immunoglobulin G concentration. In all experiments 25 mg of beads per cm are used.

comparison
mg / cm Polyglu-
taraldehyde
mg / cm Glutar
aldehyde
mg / cm human.
Immunoglobulin
G / ■■ 3
mg / cm Weight of the bound
whom Iimaunglobu-
lins G / weight of
Polymer χ 10 ^ 2.5 · 0.025 5.7 2.5 0.050 ■ 9.5 2.5 0.100 14.0 ___ 2.5 0.025 5.3 2.5 0.050 4.5 2.5 0.100. ■ 9.1 2.5 . 0

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Table II illustrates that larger amounts of immunoglobulin G are bound to the polymer at pH 6 than at pH 7.4. This effect of the pH value is confirmed in the studies described below. It can also be seen that, for a given reaction time, polyglutaraldehyde is bound in larger amounts to immunoglobulin G. than to microbeads of polyglutaraldehyde. The lower amount of bound immunoglobulin G with increasing concentration of the polyglutaraldehyde is probably due to an incomplete reaction and / or an insufficient amount of immunoglobulins. Tables II and III also show that, at a given ρH value, considerably larger amounts of human immunoglobulin G are convalently bound by the polymer than by the monomer. The information for the ei ¹ - · ford variable response time is getting kinetic study. In FIG. 1, the decrease in fluorescence of the supernatant solution is plotted against time. It can be seen that at pH 7.4 the reaction is incomplete after 135 minutes (the time given in the above investigations). Furthermore, at a pH value lower than 7.4 the reaction time is faster. FIGS. 2 and 3 confirm the faster reaction time of the polymer compared to the monomer and continue to show the effect of temperature. The same results are obtained with fluorescent non-human goat immunoglobulin M (Fig. 4).

Reactions with hemoglobin: Because hemoglobin has a high extinction coefficient at'412 mn (£ "= 125> based on a

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Molecular weight of 17,000), protein immobilization is a suitable model for studies. It can be calculated from the absorption maximum at ^ 12 nm that at pH 6, 7j ^ and 8.4 polyglutaraldehyde after a reaction time of 500 minutes is 8.8 , 6.6 and 2.8 % hemoglobin, respectively, immobilized. In control tests with hydroxyethyl methaeryl ester / hemoglobin content in the order of 1 % _s based on the nitrogen analysis (calculated value: 15> 2 % N). The hemoglobin content of the polyglutaraldehyde-hemoglobin products obtained at 6, 7 »^ and 8.4 is calculated to be 10.4, 7.8 and 5.3 % based on nitrogen analysis and is essentially in good agreement with spectrophotometric results. In contrast to the heterogeneous reaction, if polyglutaraldehyde is reacted homogeneously with an excess of hemoglobin in aqueous dimethyl sulfoxide, the product obtained is

Duct contains 76 % hemoglobin (nitrogen analysis) .- The kinetics of hemoglobin bound to polyglutaraldehyde microspheres is shown in Figure 6. The trend of an increasing reaction rate at pH 6 compared to pH Ί, k is "evident .

The reactivity of the polymer with human immunoglobulin G is about twice that of the monomer (Tables II, III, IV and Figures 2, 3, * J and 6). the number of Immunoglobulin G molecules per bead 0.7 MJn in diameter after a reaction time of 10 hours at 22 ° C. (FIG. 2) the calculation is 3.8 χ 10 · ^ for polyglutaraldehyde and 2.3 χ 10 ^ for mono- "glutaraldehyde. Larger quantities and faster reaction times

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In reactions of immunoglobulins with polyglutaraldehyde than with microbeads or microspheres Polyglutaraldehyde observed (see Tables II, III and IV and Figs. 1 and 2). The same conclusions apply to the reactions with hemoglobin (Fig. 5 and 6) ..

Lowering the pH from 7.4 to 6 increases the rate of the reaction. This phenomenon applies both to human immunoglobulin G (Fig. 1) and to hemoglobin (Fig. 5) · To interpret this effect, it is believed that the reaction of polyglutaraldehyde between free amino groups of the protein, for example the lysine residues, and aldehyde groups of the polymer with the formation of S chi ff'schäl bases takes place. It can be expected that Carbonyl addition reactions by acids, more strongly catalyzed V / earth, because after attacking the oxygen atom by a proton becomes the carbon atom considerably more positive and thus more easily reactive with a nucleophilic compound:

F +
> C = 0 φ> C -OH

When the nucleophilic compound is a compound of general Formula is R-NHp, the acid converts the amine to the non-reactive type R-NH. Therefore one expects to find that the addition rate is a maximum at weakly acidic pH value with a sharp drop on both sides shows. This is actually observed in practice /

The small extent of non-specific binding of immune

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globulins and hemoglobin (Fig. 2 and 5) to substrates that are free of glutaraldehyde and polyglutaraldehyde, becomes one attributed to physical adsorption and can be reduced by lowering the reaction temperature (Fig. 3) can be reduced.

The high reactivity of polyglutaraldehyde, its stability, its easy handling and the retention of the physiological effectiveness of bound to the polymer, human iinmun globulins makes the new reagent for that Binding of proteins desirable "

Although polyglutaraldehyde coated microspheres are more reactive than glutaraldehyde, the requirement for the formation of coated beads adds to an unnecessary one Difficulty of the transfer procedure. It is on have been found, leave a suspension polymerization of glutaraldehyde in the presence of an emulsifying agent water-insoluble Polyglutaraldebyd microspheres having a diameter of 20 nm to 10 µm, usually from 50 nm to 1.5 µm. the Polymerization in the presence of a suspension of magnetic particles produces magnetic microspheres. Polyglutaraldehyde microspheres can also result from the precipitation of polyglutaraldehyde from a surface-active compound containing Form solution. Fluorescent microspheres can be polymerized by post-polymerization with a fluorochrome or by polymerization be formed in the presence of a fluorine oclirom.

-The size of the polyglutaraldehyde immune microspheres is determined by the pH value, the concentration of the surface-active substances

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binding and by the concentration of monomers! Glutaraldehyde influenced. The immune microspheres are produced by adding a suspension with 0.1 to 20 percent by weight of glutaraldehyde and 0.1 to 3 percent by weight of the surfactant compound in an aqueous medium, the pH to 7 to 13, preferably 9 to 12, and the reaction mixture is stirred for several hours. The polyglutaraldehyde microspheres are then separated and washed. The aqueous medium may be 5 to 50 percent by weight water immiscible organic liquids, such as aromatic or aliphatic organic solvents, for example benzene, toluene, Hexane, heptane or octane, or triglycerides, such as cotton seed oil, Corn oil or soybean oil.

Microspheres containing electrodense metals They give a spatially higher resolution of the cell surface. Polyglutaraldehyde immune microspheres with a content of art electron-dense metals produce a more stable marking than gold particles with physically absorbed antibodies, currently used for cell labeling. The metal-containing microspheres can be polymerized in situ of polyglutaraldehyde microspheres in the presence of a suspension of finely divided metal particles or compounds of metals, preferably by means of a colloidal dispersion of the metal. The metal will into the microspheres in an effective amount of from 0.5 to 20 percent by weight, usually from 1 to 10 percent by weight, incorporated. -

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The metal or metal compound particles are preferably finely divided, substantially equally large substances with a uniform diameter which is smaller than that of the microspheres obtained and is typically below 100 nm, usually 2.5 nm to 50 nm. The metals are preferably electron-dense heavy metals with an atomic number above 50, preferably above 75> such as lead, nickel, cobalt, platinum, gold or iron. The metal can be magnetic, such as iron, nickel, cobalt or their alloys, or an inorganic magnetic compound, such as a metal oxide. The magnetic substance is preferably magnetic iron oxide of the formula Pe ^ O _2,. Certain hard ferrite-type ceramic compounds, such as lithium ferrite, can also be used. The metal or metal compound can be brought into an easily dispersible form by suspending it in water containing a surface-active compound such as polyethyleneimine.

The polyglutaraldehyde microspheres can be made fluorescent by reacting them with a compound that interacts with Acrolein groups form fluorogens, such as Anfchron or metar jlminophenol, or fluorochromes reactive with aldehyde groups supplies, such as aminofluorescein, 9-amino-acridine, propidiuin-broniid or fluorescein isothiocyanate. Highly fluorescent microbeads can also be produced by suspension polymerization of glutaraldehyde in the presence of a fluoro οohrome with Amino groups are produced.

909839/0863 ORIGINAL BATHROOM

The surface-active compounds are water-soluble, nonionic, cationic or anionic substances in general organic type, with fluorocarbons and hydrofluorocarbons as well as silicon containing compounds are included. Cationic surfactant compounds Be amines, amine salts, sulfonium, phosphonium or quaternary ammonium compounds. The surface-active Compounds can be used alone or in the presence of 1 to 5 percent by weight of a suspending agent such as polyethylene oxide, Glycerin, polyacrylamide or polyethyleneimine, used v / earth. An example of a cationic surfactant Compound is a cationic guar sum ("Guar C13"). Water-soluble anionic surface-active compounds can .Commercially available carboxylic acids, sulfuric acid esters and alkanesulfonic acids, for example sodium dodecyl sulfate or an anionic fluorocarbon ("Zonyl PSP"). Non-ionic surface-active compounds are usually polyethylene oxide and / or polypropylene oxide derivatives of phenols, Alcohols or fatty acids. Preferred nonionic surfactants Compounds are water-soluble copolymers »

with molecular weights of 800 to 10,000 of acrylamide with 30 to 60 percent by weight of an acrylic acid ester or an alkyl- or alkoxymethyl-alkylamide, such as isobutoxymethacrylamide according to US Pat. No. H 098,987. . '.

Example8

To 100 cnr of a 5 percent by weight aqueous glutaraldehyde solution with a content of 1 percent by weight of a mixed poly

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. BADORiGiNAL

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inerisats made of 60 % acrylamide and 4.0 % isobutoxymethacrylamide

solution /
10-n sodium hydroxide / is added dropwise until a pH value of is reached. The reaction mixture is then deaerated with nitrogen, the container is tightly closed and shaken on a mechanical shaker for 2k hours at room temperature. After a reaction time of 1, 2 and h hours, the pH is adjusted to 11 by adding 10N sodium hydroxide solution. The reaction mixture is then dialyzed extensively against water and then centrifuged three times for 30 minutes each time at 2000 g. This gives 200 mg polyglutaraldehyde Kügelehen having an average diameter from 200 to 20 nm due to the scanning electron microscopy. The spheres are redispersed in 0.01 m sodium chloride solution, phosphate buffered to pH 7 ^ 1, or in distilled water. By changing the surfactant concentration, monomer content, or pH, the size of the microspheres can be changed in a predictable manner. .

The method d & s example 8 is repeated by changing the concentration of the surfactant compound, the initial monomer "content and pH to determine the effect on the microspheres diameter, as shown in FIGS. 7, _S 8 and 9 can be seen. In the absence of a surfactant In the compound, the monoglutaraldehyde is very slowly subjected to aldol condensation at pH 7. The rate of polymerization increases at pH four above 7 and less powdery products that contain aldehyde groups.

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In the presence of surface-active compounds and in the case of a basic aqueous solution, polyglutaraldehyde precipitates in the form of spherical colloidal particles. A decrease in size with increasing pH values is most likely as a result of the occurrence of a Cannizzaro see reaction which Carboxyl groups can arise. This is shown by an increased solubility at increased pH values and an increased Neutralization equivalent of the isolated polyglutaraldehyde microspheres produced at various pH values have been (Table V). The diameters of the microspheres also increase when the concentration of monomers is decreased or on surface-active compounds.

Table V

Manufacture of polyglutaraldehyde 'powder and microspheres (0.2 µm diameter) as a function of the pH value (potentiometric titration with 0.1 N sodium hydroxide solution in aqueous dimethylformamide (1: 1))

Polyglutaraldehyde Microspheres
PH - mSqüiviCarboxy IgT, Anzaj ^ l of carboxyl each micro
globules
χ 10-7 powder
PH g polyglutaraldehyde groups 10.0 <0.03 each gxlÖ " ¹⁹ 11.0 0.05 <2.0 11.5 0.10 . 3.0 12.3 0.13 6.0 13.5 * 10.5 0.95 7.8 < 1.5 11.5 <0.03 50.0 2 * 1.0 12.3 0.10 75.0 j 0.30 $ 00839 / 0863-

^X ^ soluble polymer 2910414 precipitated at pH 6

The presence of aldehyde groups is determined by means of a Fourier transform Infrared absorption spectrum reaction with hydroxylamine hydrochloride and with 2,4-dinitrophenyl hydrazine

- established. The absorption bands at 1720, 1680 and 1635 cm are previously the vibrations of non-conjugated aldehyde groups, conjugated aldehyde groups and carbon-carbon -Double bonds in ct-position "to the aldehyde group has been attributed.

From the Fourier transformed infrared absorption spectra it can be seen that all absorption bands for polyglutaraldehyde powder are characteristic of "non-magnetic" and magnetic polyglutaraldehyde microspheres available. An additional absorption band at 15 ^ 0 cm is probably based on a residual content of the used surfactant compound. The presence of iron oxide cores in the magnetic polyglutaraldehyde microspheres causes a shift in some absorption bands, for example the bands of 1680, 1360 and 1125 cm. The access

- 1 order of the absorption band at 1680 cm to the aldehyde group is confirmed by the reaction of polyglutaraldehyde with sodium bisulfite. The reaction product of polyglutaraldehyde with sodium bisulfite shows an absorption band at 1680 cm Another consideration for aldehyde functions is nitrogen analysis of polyglutaraldehyde powder, non-magnetic and magnetic microspheres made with hydroxylamine hydrochloride have been implemented. The results of the in Table VI

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The nitrogen analyzes given show that the number of aldehyde groups decreases when the pH value at which the polymers

are increased. He agrees with the all- ^; gradual increase in neutralization equivalent (Table V), that is, with increasing concentration of carboxyl groups. The latter are formed when the aldehyde groups (Table V) are consumed, as is to be expected in the case of the Cannizzarö * reaction. ·,

T a b e 1 1 e VI '

Reaction of polyglutaraldehyde powder and microspheres (0.2 µm in diameter) coated with hydroxylamine have been produced at different pH values (nitrogen analysis of the reaction products)

Polyglutaraldehyde Microspheres
pH % N in the reaction
Products Number of aldehydes per microsphere
chen xl0 ~ 7 ³ ul.vei
PH ■ ·. " 7.8
7.3
2.2 each gxlO '" ² - ¹ 10.0
11.5
12.3
13.5 10.5
11.2
11.2 ^XX 6.7
6.1.
6.7 3.1
1.8
0.9 1.2
1.0

^x corrected (the percentage nitrogen value before the reaction is 0.05 % for the powder and 1.8% for the microspheres)

Magnetic microspheres (iron content lh%, by means of atomic absorption)

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Qualitatively, the presence of aldehyde groups can be determined by means of 2,4-dinitrophenylhydrazine yellow-orange colored polyglutaraldehyde microspheres are obtained when the Implementation of hetei'Ogen in aqueous suspension at pH 2. The poly-VP-AM-hydrazide microspheres used for comparison give a negative color test. It was observed that at acidic pH and high salt concentration, polyglutaraldehyde microspheres aggregate. Since during a Cell marking microsphere aggregation must be avoided, it is in the case of magnetic ones > Requires polyglutaraldehyde microspheres containing 0.15 m sodium chloride in the phosphate buffer solution by aaa2.5 - m sucrose content to replace. However, the non-magnetic polyglutaraldehyde microspheres can be safely used as a suspension in phosphate-buffered sodium chloride solution. ''

Example 9.

The procedure of Example 8 is repeated, but with the Provided that the surfactant compound is "by 1 weight percent of an anionic surfactant compound Basis of a fluorocarbon is replaced. The mean part diameter of the Polyglutaraxdehyde microspheres is at 0.1 µm. ■ ·

Example 10

The procedure of Example 8 is repeated, but with the proviso that the surface-active compound is formed by a mixture from 1 percent by weight of a cationic interfacial

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active compound based on guar gum and 5 percent by weight a suspending agent based on a polyethylene oxide with

is replaced /

a molecular weight of 100,000 / the mean particle diameter the polyglutaraldehyde microsphere is 0.2 µm.

Example. 11

Highly fluorescent polyglutaraldehyde microspheres are obtained by adding 10N sodium hydroxide solution to an aqueous suspension with 1 percent by weight of a copolymer of 60 % acrylamide and 10% isobutoxymethacrylamide as a surface-active compound with a content of 0.05 % 9-aminoacridine as fluorochromium until pH 11 is reached. An aqueous glutaraldehyde solution is then added up to a final concentration of 5 percent by weight and the pH is again adjusted to 11. The microspheres are purified as described in Example 8. Strongly green fluorescent polyglutaraldehyde microspheres with a mean diameter of 0.15 µm are obtained.

The procedure of Example 11 is repeated with 0.05 percent by weight of various Fluoroch.romq, tf. You get the in the results given in the table below.

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Table VII

Pluorochrome

Propidium bromide

Amino fluorescein (isomer 1)

o-phthalaldehyde

5-dimethylamino-1-naphthalene sulfonyl hydrazide

Fluorescein isothioeyanate (dissolved in excess ethylenediamine)

fluorescence

Red

green green

green green

, B e i s ρ 1 e 1 12

Preserves highly fluorescent polyglutaraldehyde microspheres by shaking for 24 hours at 25 ° C of 20 mg of the after Example 8 polyglutaraldehyde microspheres prepared with 1 mg of a 0.01 prosentigen fluorescein isqthiocyanate solution

in ethylenediamine. The fluorescent microspheres will become Purified as in Example 8 and buffered in water or phosphate Sodium chloride solution redispersed.

Example 13

The addition of 2,4-dinitrophenyl hydrazine to polyglutaraldehyde microspheres of Example 8 at pH 2 has a lasting effect yellow COLOUR.

Example Ik

The procedure of Example 8 is carried out in the presence of a 1 weight rosin aqueous dispersion of Fe, O ^ with a. Repeated content of 5 weight percent iron. You get

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magnetic beads with a mean diameter of 0.1 µm. The purification of the iron-containing microspheres consists in dialysis against water and separation of diamagnetic impurities by means of a permanent magnet. The substance is finally dispersed in 0.5 M phosphate-buffered sucrose solution. The concentration of the particles in the solution is based on the dry weight analysis. A known volume of the solution is dried to constant weight ^{at HO 0 C.} . "

Example 15

Dispersible iron oxide is made by dissolving 10 grams of ferric chloride and 13.5 grams of ferric chloride in 210 ml a .l-'weight percent aqueous solution of polyethyleneimine (Molecular weight '1800). Then you give 50 percent Add sodium hydroxide solution up to pH * 7. Qas reaction mixture is refluxed for 3 hours, dialyzed thoroughly against water and three times magnetic from non-magnetic Particles' separated. The magnetic polyethyleneimine iron oxide particles are redispersed in water and then sonicated with a'klinischen sonicator for 10 minutes. Make it up magnetic particles with a diameter of 30 nm with amino groups on the surface.

Example 16 "

It becomes a 1 percent polyethyleneimine iron oxide solution as in Example 15, added to a suspension according to Example 8. Magnetic polyglutaraldehyde microspheres are obtained.

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Example 17

When a 0.01 percent fluorescein isothiocyanate solution in ethylenediamine is added to a suspension according to Example Ik , fluorescent magnetic polyglutaraldehyde microspheres with an average particle diameter of 0.1 μm are obtained.

Example 18 '

20 mg of the magnetic polyglutaraldehyde microspheres from Example Ik are shaken for 2k hours at 25 ° C. with 1 mg of fluorescein isothiocyanate dissolved in 0.02 ml of distilled ethylenediamine. The highly fluorescent magnetic polyglutaraldehyde microspheres are dialyzed thoroughly against water and then magnetically separated three times. The purified microspheres are redispersed in water or in 0.5 m phosphate-buffered sucrose solution.

Example- 19

Polyglutaraldehyde microspheres are formed in an aqueous suspension containing a water-immiscible solvent. Sodium hydroxide solution is added at 25 ° C. to a stirred aqueous solution of 5 percent by weight glutaraldehyde, 10 % toluene and / 2 % of a commercially available alkyl polyethoxyethanol ("Triton X-100") until pH 11 is reached. The solution is stirred for 3 hours and then centrifuged three times at 2000 g. There form, is polyglutaraldehyde microspheres The total to in Example 8 having an average diameter of about 0.5 _/ um, '

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-jf- ~:

cleaned processes are separated and washed.

. B is ρ ie 1 20 ■ '

Magnetic folyglutaraldehyde microspheres are made by adding a 1 weight percent aqueous dispersion of Fe-zOjj with a content of 5 weight percent iron to one Formed suspension according to Example 18, '■ then separated three times magnetically, washed and dialyzed.

Example 21,

Polyglutaraldehyde microspheres are prepared by dissolving 125 mg of polyglutaraldehyde powder according to Example 1 in 2.5 ml anhydrous dimethyl sulfoxide formed. The solution becomes vivid touched. Then 50 ml of a 2 percent aqueous solution of a polyethylene oxide with a molecular weight of 100,000 are added and stir the solution for an additional hour. Polyglutaraldehyde microspheres with a mean diameter are formed from. 0.1 µm, which are purified by centrifuging three times at 2000 g and then redispersed in water.

• Example 22.

The procedure of Example 21 is repeated, but with the proviso that the surface-active compound is a 4 percent by weight aqueous solution of an anionically substituted

Fluorocarbon used. Polyglutaraldehyde microspheres are formed. ; ■ \

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-Jtf-

Example 23

The procedure of Example 21 is repeated, but with the proviso that the surface-active compound is a 4 percent aqueous solution of a cationically substituted fluorocarbon used. Polyglutaraldehyde microspheres are formed.

Example 2k '

Fluorescent microspheres are made by dissolving 125 polyglutaraldehyde powder and 12 mg aminofluorescein under living conditions vigorous stirring in 205 ml of anhydrous dimethylsiifoxid. When adding a 2 percent aqueous poly. ethylene oxide solution gives fluorescent polyglutaraldehyde beads with an average diameter of 0.1 μm.

Example 25

The procedure of Example 21 is repeated, but with the proviso that the dimethyl sulfoxide solution is 6 percent Polyethyleneimine iron oxide solution according to Example 15 is added. Fluorescent, magnetic polyglutaraldehyde microspheres are obtained. _.

Example 26

The procedure of Example 8 is repeated using 50 mg of the water-soluble polyacrylamide instead of that in Example 8 named copolymer as a surface-active compound used. Polyglutaraldehyde microspheres are obtained with '

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a mean diameter of 0.2 µm.

Example 27

Following the procedure of Example 26 in the presence of a 1 percent by weight aqueous dispersion of Ρβ, Ο. with a Repeated iron content of 5 percent by weight gives magnetic ones Pearls. *.

Reactions of polyglutaraldehyde microbeads with human Immunoglobulin G:

To get the reaction time required to conjugate antibodies on polyglutaraldehyde microspheres is used to determine the rate of reaction using fluorescent human immunoglobulin G examined as a model case. The decrease in fluorescence intensity over time is a measure for the rate of formation of the covalent bond. The initially rapid response is with non-magnetic and with magnetic polyglutaraldehyde microspheres as well as powdered Polyglutaraldehyde investigated together with a comparative experiment, bei.dem Poly-VP-AM-hydrazide microspheres which are free of aldehyde groups can be used. The decrease in fluorescence intensity in the comparative experiment shows one physical adsorption on human immunoglobulin G.an. The powdery polyglutaraldehyde consists of particles of 20 to 80 µm in diameter and consequently has a much smaller one Area as polyglutaraldehyde microspheres of 200 nm diameter »this is probably the main reason for the

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slower reaction speed .. "" - ~ "

Marked cells and magnetic separation:

It has been found that the labeling of cell surface receptors by means of fluorescent or non-fluorescent or fluorescent magnetic polyglutaraldehyde microspheres is simple and effective, as demonstrated by numerous examinations using fixed red blood cells can be seen from humans or turkey as model substances. Preliminary experiments with living human lymphocytes for labeling immunoglobulin G molecules B cells were also successful.

Example 28

Human Red Blood Cell Labeling: An aqueous suspension of polyglutaraldehyde microspheres (0.2 cm ^ j 2 mg microspheres with a diameter of 200 nm) is mixed with 0.6 ml of a phosphate-buffered sodium chloride solution of purified goat anti-rabbit immunoglobulin M (0.2 mg in 0.2 ml of phosphate-buffered sodium chloride solution). The mixture is gently stirred at 4 ° C. for 3 hours. Then adds 20 mg of bovine serum albumin are added and stirring is continued

> ■ ■ ■

another hour away. Unbound antibodies are separated by passing the suspension through a column with a Filling of three-dimensionally crosslinked agarose can run through. The separation is monitored spectrometrically at λ = 280 nm.

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. 291Q4H

Human red blood cells from a normal donor and fixed with glutaraldehyde are sensitized with rabbit anti-human red blood cells. The red blood cells (10 '), which are suspended in 0.5 ml of a phosphate-buffered sodium chloride solution containing 0.2 mg of antiserum, are stirred for 30 minutes at room temperature. The cells are then separated and washed three times by rotating the phosphate buffered sodium chloride suspension in an international centrifuge at 500 g. The goat anti-rabbit microspheres are then added to the pellets of sensitized human red blood cells, "the mixture in 1 hour. ^ ⁰ C stirred slowly. The red blood cells are then separated from unreacted conjugated microspheres by centrifuging three times at 500 g. The marked cells are resuspended in 0.4 ml of phosphate-buffered sodium chloride solution and examined in the light of a scanning electron microscope.

Two control tests are carried out for each test:

1. Microspheres are conjugated with human immunoglobulin G. and exposed to sensitized red blood cells; and

2. Microspheres infected with goat anti-rabbit immunoglobulin G are conjugated to non-sensitized red blood cells allowed to act.

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Marking of human lymphocytes:

The labeling of living human lymphocytes using polyglutaraldehyde microspheres is examined.

Polyglutaraldehyde microspheres 200 nm in diameter are conjugated with human immunoglobulin G labeled with pluorescein isothiocyanate as described in Example 28. The number of labeled receptors are counted under the fluorescence microscope. They agree with the fourth received previously within an error limit of ί 10 % .

The separation of the magnetically marked human red blood cells is achieved in the following way:

Mixtures of human red blood cells are prepared with the following ratios of unlabeled to labeled cells: 1: 1, 7: 1 and 9: 1. 10 ml each of the mixtures are slowly stirred in a glass vessel equipped with a horseshoe magnet (0.03 T). After 2 hours, the cells that are not attracted to the vessel walls are isolated. The cells attracted by the magnet are diluted with 10 ml of a phosphate buffered sodium chloride solution and the magnetic separation is repeated. Examination under a scanning electron microscope shows that 95 % of the unlabeled cells from all three synthetic mixtures could be separated off in this way.

The degree of non-specific reaction with living human lymphocytes and the effectiveness of magnetic separation

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~ 4 ~

of the cell subpopulations by means of a magnetic separator are currently being investigated.

The new, easy-to-use immune reagent in the form of polyglutaraldehyde microspheres is available in a variety of Sizes and with a relatively narrow size distribution. The polyglutaraldehyde microspheres can During their preparation a high fluorescence intensity can be assigned, which nevertheless has a high concentration of aldehyde groups left on the surface. The functional aldehyde groups allow a covalent bond with antibodies, Enzymes and other proteins in a single step. For this reason, this immune reagent avoids those previously used Intermediate stages using a cyanobromide and carbodiimide reaction. The wide area an ion resistance and pH without the occurrence of Aggregations and the high specificity of the polyglutaraldehyde microspheres - at least as far as human red blood cells are concerned - are also desirable properties.

A small modification in the manufacturing process gives fluorescent ones magnetic polyglutaraldehyde microspheres for a wide variety of uses.

The use of magnetic parts is of great interest evoked in biochemical research and clinical medicine when used as support materials for immobilized Enzymes are used. Your easy recovery

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. i

' 291QAH

from liquids that contain colloidal and dissolved pesticides, should be of practical value. The separation of proteins and chemical compounds using affinity chromatography can be done by switching off lengthy centrifugation processes and column chromatography steps can be simplified. The magnetic particles are also recently used in radioimmunoassay techniques Hyperthermia treatment of cancer and the conduction of magnetic glow to one, vascular Malformation, such as cerebral aneurysm, has been studied with the intention of reducing the damage by inducing thrombosis to close.

Other suggested areas of application are in search of traces Bloodstream or in drug cessation vehicles. The first successful application of magnetic immune microspheres has been shown in the separation of B and T cells. The results are later using C-1300 neuroblastoma cells has been confirmed. There is little doubt that a physical segregation of cell subpopulations becomes necessary. Numerous separation methods, while useful, are limited by restricted parameter sets to which the separation can be based, and also by the fact that they are operated batchwise.

New devices for counting blood cells in the blood and new sorters allow a quantitative measurement of numerous * parameters and sorting on the basis of these measurements, but they are inso-

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far limited to the number of cells that can be separated in a given time. Magnetic cell sorters have the potential of a cell separation in a continuous Procedure. The clarity obtained in the current investigations using the model cell syste indicates polyglutaraldehyde magnetic immune microspheres of desired sizes can be conjugated with proteins in a simple and easy-to-use manner and therefore represent a potential for a broad grading of immunological cell sorting as well as other areas of application.

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Claims (45)

• 29104U patent pending 1) Process for the preparation of polyglutaraldehyde, thereby characterized that one.monomers Glutaraldehyde in an aqueous organic solvent medium containing from 5 "to 80 percent by weight of organic solvent dissolves, increases the pH of the solution to over 7 and polymerizes the glutaraldehyde. 2) Method according to claim 1, characterized in that after the polymerization, the water content of the medium is increased to over 80 percent increased and the polyglutaraldehyde precipitates, 3) Method according to claims 1 or 2, characterized in that that the organic solvent is a strongly polar solvent used in an amount of 20 to 80 percent by weight, 4) Method according to claim 3, characterized in that one as the organic solvent, dimethyl sulfoxide or dimethylformamide used, 5) Method according to .at least one of claims 1 to 4, characterized characterized in that the pH of the solution is adjusted to 9 to 12. * 6) Method according to at least one of claims 1 to 5 »thereby characterized in that one, a 0.1 to 50 percent by weight Solution of glutaraldehyde used, 7} Method according to at least one of claims 1 to 6, since 90983 ^ / 0863 ... 2310414 characterized in that the temperature of the solution is first kept at 15 to 30 ^° C. for 30 to 180 minutes and then the temperature of the solution is increased to above 40 ° C. during the polymerization. 8) Method according to at least one of claims 1, 2 and 5 to 7j, characterized in that a water-immiscible organic solvent in an amount of 5 to 50 percent by weight as the organic solvent and additionally a surface-active compound in the aqueous organic solvent medium an amount of 0.1 to 3 weight percent is used, and that the solution is stirred and _t the polyglutaraldehyde in the form of microspheres · wins and fails. 9) Method according to claim 8, characterized in that a non-ionic surface-active compound is used as the surface-active compound Connection used. 10) Method according to claims 1 and 6, characterized in that that 0.1 to 3 percent by weight of a surface-active compound is added to the solution of the polyglutaraldehyde, the resulting Stir the solution and the polyglutaraldehyde in the form of Miki-o-beads fails. 11) Method according to claim 10, characterized in that the stirred solution is additionally a suspension of a finely divided Metal adds and metal-containing polyglutaraldehyde microspheres fails. 12). Process for the production of fluorescent polyglutaraldehyde according to at least one of claims 1 to 11, characterized 909333/0363 indicates that the polyglutaraldehyde obtained is> a non-fluorescing compound converts and doing fluorogenic groups forms. 13) Method according to claim 12, characterized in that the non-fluorescent compound is reacted with the acrolein groups of the polyglutaraldehyde. 14) Method according to claims 12 or 13 »characterized in that that anthrone is used as a non-fluorescent compound, m-Amino-phenol or aminofluorescein used. 15) Method of making water-insoluble polyglutaraldehyde microspheres according to at least one of claims 1 to 14j, characterized in that an aqueous medium 0.1 up to 20 percent by weight glutaraldehyde and 0.1 to 3 percent by weight of an organic water-soluble surfactant compound adds, sets the pE- \ 7ert of the medium to 7 to 13, and/
the mixture is stirred from the reaction mixtures of polyglutaraldehyde microspheres, convoluted. 16) Method according to claim 15, characterized in that one a song with a "content of 5 to 50 percent of an organic, Solvents immiscible with water are used. 17) Method according to claims 15 or .16, characterized in that that one dera reaction mixture is a suspension of a finely divided i.Ictalls with particle diameters below 100 nm are added, 18) Method according to "claim 17, characterized in that one when iietall used one with an ordinal number above 50 BAD ORiGiNAL 19) Method according to claim 18, characterized in that there is used as Ifetal lead, nickel, cobalt, platinum, gold or iron. "- · ■ 20) Process according to claims 17 or 18, characterized in that the metal is used in the form of an oxide. 21) Method according to claim 20, characterized in that Fe ^ O is used as the metal oxide. 22) Method according to at least one of claims 17 "to 21, characterized in that a suspending agent is additionally used in an amount of 1 to 5 percent by weight. 23) Method according to claim 22, characterized in that dass.man • A polyethylene oxide is used as a suspending agent. 24) Method according to claims 15 to 23, characterized in that that the practically water-insoluble polyglutaraldehyde in the form of microspheres with a diameter of 20 nm to 10 to win. ■ 25) Method according to claim 24, characterized in that one the practically water-insoluble polyglutaraldehyde in the form of microspheres with a diameter of less than 10 nm and with a content of 0.5 to 40 percent metal wins. 26) Method according to claim 25, characterized in that one the practically water-insoluble polyglutaraldehyde in the form of magnetically attractable microsphere wins. 27) Method according to claims 12 to 26, characterized 909839 / 4B63 BATH ORIGINAL draws that the practically water-insoluble polyglutaraldehyde in the form of τοη microspheres with fluorogenic groups, 28) Process according to claim 27, characterized in that the practically water-insoluble polyglutaraldehyde is in the form of microspheres with fluorogenic groups as fluorochrome wins. 29) The method of claims 12 to 28, characterized in that reacting the polyglutaraldehyde with a reactive aldehyde groups with fluorochrome compound. JO) Process according to claim 29, characterized in that the fluorochromic compound is aminofluorescein, 9-aminophenol,
Propidium broinid or fluorescein isothioeyanate is used. 31) Method according to claim 24, characterized in that the practically water-insoluble polyglutaraldehyde in the form of. Microspheres bound to a protein wins. 32) Method according to claim 31, characterized in that one the practically water-insoluble polyglutaraldehyde in the form of microspheres of antibodies, antigens, immunoglobulins, lymphocytes and / or hemoglobin bound. wins. 33) Use of the prepared according to claims 1 to 32
Polyglutaraldehyde as a coating on a substrate surface. 34) Ver? Iendung according to claim 33, characterized in that the The thickness of the coating is 0.0025 to 0.125 mm. 35) Use according to claims 33 or 34, characterized in that that the substrate is particulate. 909839/0863 BATH ORIGINAL • 29104 H. 36) Use according to claims 33 to 35, characterized in that that the substrate is reactive with aldehyde groups functional groups on v / eist. 37) Use according to claim 36, characterized in that the substrate hydroxyl, amino or hydrazide groups as functional Has groups. 38) Use according to claims 33 ^ i * ³ 37 »characterized in that the substrate are microspheres or beads of a synthetic organic polymer with a diameter of 20 nm to 100 μm. 39) Use according to claim 38, characterized in that the polymer is an acrylamide copolymer. 40) Use according to Claim 39, characterized in that the polymer is a copolymer of at least 50 percent Vinyl pyridine or hydroxyethyl methacrylate, 5 to 35 percent Acrylamide and 0.1 to 30 percent of one copolymerizable therewith. Is crosslinking agent. 41) Use according to claim 40, characterized in that the copolymers have hydrazide groups. 42) Use according to claim 35, characterized in that the Polyglutaraldehydübersug is externally bound to an amine. 43) Use according to claim 42, characterized in that the amine is a protein. 44) Use according to claim 43> characterized in that 909839/0863 BATH ORIGINAL the protein antibodies, antigens, lymphocytes, lymphocytes and / or hemoglobin. 45) Use according to claims 33 to 44, characterized in that that the coatings are fluorescent. 909839 / G8S3