DE2542258A1 - METHOD OF IMMUNOCHEMICAL METHOD OF ANALYSIS - Google Patents

Description

MCHTSANWÄITE DR. JUR. DJPL-CHSM. WAITER ΒΕΑ 2 Sep. J975

ALFRED HOtPPSiNCR

DR. JUR. DIPL-CM5 / Λ R-J. WOLFP

DR. JUR. HANS Chi ?. Ax

62S FRANKFURT AM MAIN-HOCHSf

Our no. 20 152 Ka / La

Pharmacia Diagnostics AB Uppsala / Sweden

Process for performing immunochemical analysis methods

The present invention relates to an improvement in the general method of performing immunochemicals Analysis methods using a water-insoluble but water-absorbing polymer material, the hydrophilic Groups, preferably hydroxyl groups and / or amide groups contains, wherein the polymeric material a) an antibody (I), which with an antigen (A) against which the antibody (I) is directed, is reacted or has been reacted, or b) an antigen (II), which with an antibody (B), which is directed against the antigen (II), is converted or has been converted, is bound, and where the polymeric material against from the immunohemical analysis method included; * antibodies ^ and antigens ^ 'immunochemical is inert. The improvement is that the polymeric material used is a material which is hydrophobic Has groups which are able to the antibody (I) or the antigen (II) by so-called hydrophobic binding bind, wherein the antibody (I) or the antigen (II) is bound to the polymeric material by means of such a hydrophobic bond are.

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A large number of immunochemical analysis methods are known in which a water-insoluble polymeric material is used to which an antibody or an antigen, preferably containing polypeptide, is bound. (The word "antigen" as used herein and in the claims _{; also} includes haptens; the word "polypeptide" also includes proteins). So it is, for example, from the Swedish patent documents 343 949 and 341 239, from Biochim. Biophys. Acta 130 (1966), page 257 and Radioimmuno as say Methods (KE Kirkham and WM Hunter, Churchil Livingstone, London 1971). For example, pages 405 to 412 of the article "Solid Phase Antigen-Antibody Systems" by L. Wide, it is known to use a water-insoluble polymeric material to which an antibody or an antigen is bound by means of bonds of a covalent nature. An immunochemical analysis method using antibodies adsorbed on the inner surface of a plastic test tube is known from US Pat. No. 3,646,346.

If in this immuno before mi see methods of analysis of the antibodies is bound directly to the polymeric material, the antibody is directed against an antigen, which is covered by the ÄnaJLysenverfahren and which immuno ehe mi sch is or has been bound to the antibody on the polymeric material. The antigen bound to the antibody can then optionally be reacted again with an antibody in solution. If in accordance with these Methods the antigen is bound directly to the polymeric material, the antigen can react with an antibody, which is directed against the antigen, or the antigen was reacted with such an antibody, which then in turn with E.g. an »antigen can be converted into solution.

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It is also well known that in the immunochemical analysis methods in question, preferably one of the Analysis components which are included in the analysis method and which are not directly bound to the polymer with an analytically detectable atom or an analytically detectable atom group, e.g. with a radioactive atom or a radioactive group, or e.g. with a fluorescent group or e.g. with an enzymatic active group is highlighted.

The literature discloses a large number of modifications of such analytical methods in which a labeled component, how a labeled antigen or a labeled antibody is used (see e.g. the references cited above). Examples of such modifications are:

a) the antibody bound to the polymer can with a

'Antigen in the sample and reacted with labeled antigen, or

b) the antibody bound to the polymer can be reacted with an antigen in the sample, so that the antigen is bound to the polymer-bound antibody, whereupon a labeled antibody is added, which is binds itself to the bound antigen, or

c) the polymer-bound antigen can with an antibody in of the sample are reacted so that the antibody binds itself to the antigen, whereupon a labeled Antigen is added which binds itself to the bound antibody, pder

d) the polymer-bound antigen can be reacted with an antibody in the sample, so that the antibody itself is bound to the antigen, whereupon a labeled antibody is added which is directed against the former Antibody, and binds itself to the same.

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The polymer-bound antibodies can also have a Antigen directly bound to the polymer can be bound, and the polymer bound antigens can also via antibodies that are bound directly to the polymer. The antibodies can be one or more Immunoglobulin classes belong to. A common feature of all of these analytical methods is that either the antigens or the antibodies are bound directly to the polymeric material.

According to the invention there is now a method for performing immunochemical analysis methods of the above Type provided, wherein a water-insoluble * but water-absorbent polymeric material is used, the contains hydrophilic groups, preferably hydroxyl groups and / or amide groups, with a Antibody (I) which is reacted or reacted with an antigen (A) against which the antibody (I) is directed has been, or an antigen (II) (preferably a polypeptide-containing antigen) which with an antibody (B), which is directed against the antigen (II), is converted or has been converted, is bound, wherein the polymeric material against antibodies and antigens included in the immunochemical analysis method, is immunochemically inert, which is characterized in that the polymeric material used has hydrophobic groups which are capable of the antibody (I) or the antigen (II) to bind by known so-called hydrophobic bond, and that the antibody (I) or the antigen (II) to the polymeric material is bound by a hydrophobic bond.

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By the term "water-insoluble" is meant that the polymeric material is insoluble in water at least within the temperature range of from ⁰ C to 30 C (preferably from 0 to I0 * ⁰ C). Polymeric material which is insoluble in water even at higher temperatures, for example temperatures up to 100 ° C., can also be used. Such polymeric material which has hydrophobic groups and which has no ionizable groups, as a result of which an ionic bond is avoided, can be used particularly advantageously.

As indicated above, the polymeric material having hydrophobic groups is used against antibodies and antigens, which are covered by the immunochemical analysis process, be immunochemically inert. Therefore, the polymeric material having hydrophobic groups does not become immunochemical Counterpart against the antibody or the antigen that is directly attached to the same by hydrophobic binding (also

referred to as a hydrophobic interaction), or against the other antigens or antibodies that are bound by the immunochemical analysis methods are included. Therefore, for example, the polymer containing hydrophobic groups Material no antigen or hapten against which the antibodies (I) or other antibodies which may be included in the immunochemical analysis method included.

The inventive method is _; compared to previously known methods in which polymer-bound antibodies or antigens are used, advantageous. The antibodies and antigens are bound under very mild conditions so that they are not chemically altered and their immunochemical properties are retained. The hydrophobic

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Bonds can still be made sufficiently strong ^ to prevent the bound immunochemical component be washed out under the conditions under which the analytical method is carried out. It was found that the hydrophobic binding of the "antibodies takes place at immunochemically inactive sites of the antibody, whereby the hydrophobically bound antibodies are attached to the polymer retain their ability to bind the antigen or hapten against which they are directed. The destruction more reactive Groups (which can occur with other methods when the antibodies or antigens are bound by covalent bonds are bound, and which can cause unspecific binding to the polymeric material when the analysis is carried out} can on the polymeric carrier material, if the binding of the antibodies or antigens to the same takes place according to the invention, do not occur. Preferably, the amount of directly bound antibody or antigen should be chosen so that that all binding sites on the polymeric material are saturated or blocked. In those cases where this is not possible should be, the remaining binding sites can be through a suitable substance, such as non-immunochemically active

■ y - globulin, albumin, etc. are blocked. For the hydrophobic binding of the antibodies or antigens to the carrier material, small quantities of antibodies or antigens in highly diluted solutions can be used and, if necessary, the binding process can be carried out over a long period of time. Thanks to the fact that no disruptive secondary reactions take place, the process is easily reproducible. As a result of the combination of the specific choice of the carrier polymer and the specific choice of the binding process, a surprisingly advantageous process is obtained by which

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immuno-chemical analyzes can be carried out at which have an immunochemical component to an insoluble one Carrier polymer is bound. It was also found that no interfering, non-specific binding to the solid Phase of non-polymer-bound immuno-chemical components, present during the analysis takes place or that such binding is only surprising to one takes place on a small scale. In the case of the previously known methods, such non-specific binding may not be possible make to perform the analysis, or such non-specific binding tends to reduce the accuracy of the Decrease analysis. In addition, the inventive Procedures are easily performed, thereby providing important practical advantages.

The water-absorbent polymeric material used in the present invention has a water absorption capacity which suitably at least 0.2 g of water per g of dry polymeric material and preferably at least 0.5 g of water per gram of dry polymeric material. The water absorption capacity can advantageously e.g. at least 1 g, like at least 2 gj at least 10 g or at least 20 g Water per gram of dry polymeric material. In general, a water absorption capacity which is less than 200 g, such as less than 100 g, e.g. less than 50 g of water per g of dry polymeric material is selected.

The water-insoluble but water-absorbing polymeric material having hydrophobic groups can advantageously contain water-absorbing polymeric or polymerized carbohydrates or sugar alcohols or derivatives thereof,

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wherein the polymers are preferably completely free or essentially free of ionizable groups. Examples of such materials include water-absorbent, water-insoluble polysaccharides _? such as agarose, cellulose or derivatives thereof.

A particular advantage is achieved when the water-insoluble, water-absorbing polymeric material is three-dimensional Network contains, which is water-insoluble but hydrophilic / swellable and which by bonds of a covalent nature is held together.

Such a three-dimensional network can advantageously by crosslinking, for example, polymers containing hydroxyl groups, such as polymer! or polymerized carbohydrates or sugar alcohols or polyvinyl alcohol, preferably polysaccharides, which are preferably not ionizable Contain groups, e.g. glucans such as dextran, starch, Cellulose, pollulan and postulan, galactans such as agarose, Mannans, arabinans, xylans etc. or derivatives thereof, be obtained by means of bridges with bonds of covalent Natua ?, the network being hydrophilic groups, such as hydroxyl groups having.

Such a virtually endless three-dimensional network can be achieved by reacting polymers containing hydroxyl groups, such as polymeric or polymerized carbohydrates or sugar alcohols, preferably polysaccharides or derivatives thereof, with an at least bifunctional crosslinking agent Funds are obtained.

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For the purpose of achieving networking bridges,
which are bound to the polymer chains, for example polysaccharide chains via ether bonds, can be the hydroxyl-containing polymer, for example a polysaccharide or polysaccharide derivative, in an alkaline aqueous solution with crosslinking agents, for example of the type

X A ₁ . Z. (I) uijLd χ. A ₂ . Z (II)

in which X, Y and Z each represent a halogen atom, preferably a chlorine atom or a bromine atom, and A .. and A "each represent a straight or branched aliphatically saturated hydrocarbon chain, which is replaced by a or flour? Hydroxyl groups is substituted and preferably 3 to 30 carbon atoms, for example 3 to 20 carbon atoms, such as 3 to 10 carbon atoms
contains and possibly by a or flour? Oxygen atoms is interrupted, means, or corresponding epoxy compounds which are obtainable from the compound I or the compound II by splitting off hydrogen halide.
Examples of bifunctional substances of the general
Formula XA ₁ .Z and corresponding epoxy compounds that
are obtainable from XA ^ .Z by splitting off hydrogen halide, include:

CH ₂ -CH. CH ₂ . 0. (CH ₂ ). 0. CH ₂ . CH - CH,

where η is an integer, e.g. from 2 to 4, and

CH ₂ - CH. CH ₂ .: 0. CH ₂ . CH ₂ . 0. CH ₂ . CH ₂ . 0. CH ₂ .CH -

and. _CH

\ ⁱ
CH «- CH. CH ₀ . 0. CH. CH ₅ . CH ₉ . 0. CH ₉ . CH - CH ₉

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and

- CH. C ^. O, CH ₂ . CH - CH ₂

CH ₉ - CH

CH ₉ · O. CH ₉ . CH (OH). CH ₀ . 0. CH ₅ . CH - CR, "Ζ * 2 2-2 ^ s * 2

or corresponding halohydrins and bifunctional glycerol derivatives of the formula X.CH ₃ .CH (OH) .CH ₃ .Z, for example dichlorohydrin and dibromohydrin, or corresponding epoxy compounds of the general formula

CH ₀ , - CH - CH ₀ - Z

e.g. epichlorohydrin and epibromohydrin, which are produced by cleavage are available from hydrogen halide. Another example of such a bifunctional compound is 1, 2 3, JJ-Diepoxybutane of the formula

CH ₉ - CH-CH - CH ₉

An example of a trifunctional crosslinking agent (which is an epoxy compound, corresponding to a compound the formula

I ·

X A ₂ . Z represents ₁ )

CH ₂ - CH. CH ₂ . 0. CH _{2 β} CH. CH ₂ . 0 _β Ci ₂ . CH -

. CH - *

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The hydroxyl group-containing polymer such as a polysaccharide or polysaccharide derivative is mixed with an amount of the at least bifunctional crosslinking agent reacted to form a water-insoluble gel, i.e. a practically infinite one to produce a three-dimensional network with the desired properties. Appropriate common amounts of different Hydroxyl group-containing polymers such as polysaccharides and polysaccharide derivatives, and crosslinking agents can be easily determined by one skilled in the art.

Crosslinking agents other than those mentioned above can also be used.

In addition to the formation of bridges, the crosslinking reaction can often lead to the introduction of singly bonded substituents (e.g. monoethers) from the crosslinking agent, i.e. only one reactive group in the at least bifunctional crosslinking agent has reacted with a hydroxyl group in a polymer chain, such as a polysaccharide chain ^, while the other reactive group or the other reactive groups in the crosslinking agent have reacted, for example, with water to form, for example, hydroxyl groups. Therefore, the polymeric product often also has mono-bonded substituents originating from the crosslinking agent, e.g. -0-CHp- CH (OH) -CH ₂ OH if the crosslinking agent is epichlorohydrin, and -0-CH-CH (OH) -CH ₂ -O- (CH ₂ ) ^ - O-CH ₂ -CH (Oh) -CH ₂ OH when the crosslinking agent is 1,4-butanediol diglycidether.

The mechanical properties of the water-absorbent polymeric material can be improved in that the polymeric material is represented by a three-dimensional network of a crosslinked polymer, e.g. crosslinked polyacrylamides or derivatives thereof, such as derivatives thereof that contain hydroxyl groups, e.g. by encircling

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the molecular chains of the first-mentioned polymeric material is supported by the network of the crosslinked polymer. In this Relationship can also include two different polymeric three-dimensional networks that are woven into one another, e.g. an agarose gel in which another crosslinked polymer, e.g. crosslinked polyacrylamide or derivatives thereof, is intertwined is to be considered. Two different polymeric three-dimensional networks can also be advantageous can be used, each network itself held together by bridges with bonds of a covalent nature becomes, and the two networks are joined together. An example of this type is a network of crosslinked polyacrylamide or derivatives thereof such as an N-methylol derivative thereof in a network of crosslinked agarose or crosslinked dextran, e.g. crosslinked by means of epichlorohydrin or diepoxides in the manner mentioned above and Way. In this way, one polymer can be crosslinked in the three-dimensional network of another polymer.

or

The polymeric gel product can be in shaped pieces in particulate form either by making the polymer in the form of large pieces (bulk polymerization) and then Divide these pieces, e.g. by grinding or by producing the polymer in the form of spherical particles Dispersion polymerization techniques can be obtained. In this latter case the reaction mixture will be in drop form dispersed in an inert liquid which is immiscible with the mixture, after which the pearl-shaped Gel particles that are formed in the droplets during the reaction are recovered. The particles preferably have spherical shape. The desired particle size can be obtained by fractionation, e.g. by sieving. The polymer product may also be in disk form or plate form, or in any other suitable form.

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A large number of such water-insoluble, but water-absorbent, hydrophilic are known in the art Polymers containing groups as well as those polymers which do not contain ionizable groups or which are im are essentially free of ionizable groups if they contain hydrophobic groups ^ for the inventive Methods are known to be useful. Some polymeric materials that contain hydrophobic groups make proteins by means of Can bind hydrophobic bonds are also known, e.g. from the Swedish patent 7304148-5.

The hydrophobic groups are preferably covalent to the matrix or the backbone of the polymer with bonds Bound to nature. The number of hydrophobic groups and the nature of such groups will depend on the number of hydrophilic groups and the nature of such groups adjusted to achieve the desired water-absorbing properties of the polymeric material to obtain. The number of hydrophobic groups is preferably smaller than the number of hydrophilic groups.

The hydrophobic groups are preferably arranged in branches on the backbone of the polymeric material, although such groups can optionally be incorporated into the framework. In the former case, the groups can contact directly the backbone of the polymeric material may be bound with carbon-carbon bonds, although the groups likewise to the framework via a bridging bond, e.g. an ether, ester or amide bridge, and advantageously be bound via a longer bridging bond, in which case the groups at a distance from the The basic structure of the polymeric material is arranged (spaced), which in terms of the hydrophobic bond (the hydrophobic Group) of polypeptides, e.g. polypeptides containing antibodies and antigens, is advantageous.

+) (i.e. only contains insignificant traces of such groups) 809815/0949

The hydrophobic groups advantageously include hydrocarbon chains of _s which are either straight-chain, branched-chain or a ring are closed in the form of saturated or unsaturated and preferably at least 4 carbon atoms, eg at least 6 carbon atoms, such as at least 8 carbon atoms. The number of carbon atoms may, for example, at most, 30, for example, at most, 20 carbon atoms, such as ^ highest if 15 carbon atoms betragend The hydrocarbon chains may optionally also hydrophobic _{substituents?} such as halogen atoms, preferably chlorine atoms or bromine atoms or alkoxy groups with preferably at most 6 carbon atoms, although such chains can also contain 1 or more hydroxyl groups or other hydrophilic groups as substituents, the number of groups being so low that the hydrophobic character of the substituted hydrocarbon group is not is lost, the carbon chains can optionally also be interrupted by one or more oxygen or sulfur atoms. Advantageously, hydrophilic groups are selected which contain at least 4, such as at least 6, for example at least 8 carbon atoms linked by single or double bonds, the atoms only bearing hydrogen atoms.

Examples of hydrophobic groups in the polymeric material correspond to the aforementioned groups of the general formula - (CHp) -CH-, where η is an integer. Preferably, η means at least 3, for example at least 5, such as at least J. For example, η can be at most 29, for example at most 19, such as at most 14. Higher values of η can also be selected. The alkyl group can advantageously comprise, for example, the octyl group or dodecyl group. Other examples of hydrophobic groups used by the

+) A large number of carbon atoms can also to be selected.

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present invention include branched alleyl groups, cycloalkyl groups, e.g. cyclohexyl, cycloalkylalkyl groups, the phenyl radical, phenylalkyl groups, the rings in the cyclic groups optionally with or a plurality of hydrophobic atoms or groups such as a halogen atom such as a chlorine atom or an alkylene group or an alkyl group, such as a methyl group or an ethyl group, or an alkoxy group may be substituted.

Examples of bridging bonds between the hydrophobic groups and the polymeric backbone are the following:

- 0. CH ₂ . CH (OH). CH ₂ . 0 - or

- 0. CH ₂ . CH (OH) .CH (OH). CHg .0 - or

- 0 .CHg. CH (OH) »CH ₂ . 0. CH ₂ . CH (OH). CHg . 0 - or ü

0. CH ₂ . CH (OH). CH ₂ . 0 0 -, _

0 . CHg . CH (OH)

where rv, an integer, for example an integer from 2 to 4, or any other bond, ¹ preferably contains no ionizable groups and contains, for example, at least 2 or 3 or more carbon atoms of such a length that the hydrophobic group is spaced from the polymer The basic structure is arranged so that the antibody or the preferably polypeptide-containing antigen is easily accessible for binding to the hydrophobic group by means of hydrophobic interaction.

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The coupling of a hydrophobic group R to the polymer of the type mentioned above, for example a polysaccharide or a crosslinked polysaccharide, can be achieved by means of substitution reactions which are customary with regard to such polymers. The coupling of the hydrophobic group R to the polymeric backbone via, for example, an ether bridge, ester bridge or amide bridge can be carried out with the aid of conventional esterification techniques, etherification techniques and amide-forming reactions. Therefore, the reactive derivatives of R can be reacted with, for example, hydroxyl groups in the polymer. An advantageous .. method is to prepare a compound RX ₁ , wherein X _{1 is} an epoxy-containing substituent, e.g.

-0-CH ₀ -CH - CH ₀ ,

² N / ²

react with e.g. hydroxyl groups in the polymer. It is also possible, e.g. one containing hydroxyl groups Polymer and a compound of the formula ROH with a bifunctional reagent of the above in connection with to implement the crosslinking of the polymer molecules mentioned type. If the substituent R is introduced, it is also possible a longer bond between the substituents and the Polymer chains are introduced.

The binding of antibodies or antigens to the hydrophobic groups on the polymer material is particularly simple and is effected under normal conditions for hydrophobic binding of proteins to hydrophobic groups. This bond can e.g. under mild conditions in the presence of an aqueous buffer solution at pH values which are suitable for the antibody or the antigen are not harmful, e.g. in the pH range of 5 to 9, e.g. about pH 7 can be achieved. if

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Not all hydrophobic groups can be used to bind antibodies or antigens, the excess can hydrophobic groups through e.g. a protein that does not affect the analytical method, e.g. serum albumin or immunoglobulins which do not contain antibodies against analytical components become saturated. In this way non-specific binding is avoided during the analytical process.

The following examples serve to further illustrate the present invention.

example 1

A gel in the form of a pearl containing 4 % agarose was prepared according to the method described by Hjerten in Biochem. Biophys. Acta 79 (1964), pages 393-398. The pearls on the order of 2 to 25 µm in size were collected by sieving. 60 ml of the beads obtained were transferred from water in 1,2-dichloroethane by suspending twice in 200 ml of ethanol and then four times in 200 ml of acetone. After the beads had been slurried twice with 200 ml of 1,2-dichloroethane, an alkylation process with octyl glycidyl ether (prepared according to the process described by V. UIbrieh in Coll. Czech. Chem. Commun. 29, 1964, page 1466) was carried out with boron trifluoride ether as the catalyst carried out:

1.2 ml of 48 Siigen BF XtJD (Pluka AG) were added, after which the mixture of gel and solvent was stirred for 5 minutes. Then 2.6 ml of octyl glycidyl ether were added added and the reaction continued for 40 minutes. The gel was made by repeated slurries, four times with acetone

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washed twice with ethanol and then with water. The gel obtained contained 9h% water and the NMR analysis showed that 1.5% of the hydroxyl groups in the agarose were replaced by the hydrophobic group Octy1-0-CH ₂ -CH (OH) -CH ₂ -O-,

Example 2

An alkylation of the agarose gel was carried out analogously to the process described in Example 1 with 2.6 g of dodecyl glycidyl ether (prepared by the same method as the octyl glycidyl ether). Following the same work-up procedure as described in Example 1, a gel was obtained which contained 95% water. The NMR analysis showed that 5 % of the hydroxyl groups in the agarose were replaced by the hydrophobic group dodecyl-O-CH ₂ -CH (OH) -CH ₂ -O-.

Example 5

Alkylation of the agarose gel was prepared as described analogously in Example 1 were carried out using 1.8 g Phenylglycidäther (Pluka AG). It was worked up in the same manner as described in Example 1 and the gel obtained contained 95 % water and 5 % of the hydroxyl groups were replaced by CgH ₅ -O-CH ₂ -CH (OH) -CH ₂ -O- .

Example 4

5 g of a dextran gel which was crosslinked with epichlorohydrin and which was substituted with hydroxypropyl groups (Sephadex LH-20, Pharmacia Fine Chemicals AB) and had a bead size of less than 25 μm were swollen in 100 ml of 1,2-dichloroethane. 1.2 ml of 48 # BP ₅₁ At ₃ O was added. After stirring for 5 minutes, 1.8 g of phenyl glycidyl ether was added. The reaction was allowed to proceed for 10 minutes at room temperature. The gel was

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then slurried three times with 500 ml of acetone and then ... three times with 500 ml of water. The gel beads obtained contained 65 % water. The NMR analysis showed that 10 % of the hydroxyl groups were substituted.

Example 5

8 g of agarose were dissolved in 200 ml of boiling water. 9.7 g of N-methylolacrylamide, 0.3 g of N, N-methylene-bisacrylamide, 0.2 g of potassium persulfate and 0.2 ml of N, N, N, N-tetramethylethylenediamine were added. The mixture was allowed to cool to 25 ° C. The resulting gel mixture of agarose and polymethylol acrylamide was ground and sieved through a metal net with a mesh size of HO μτη. After sieving, the gel was washed twice by slurrying in water.

The polymer product described above was prepared according to the in Example 1 described method in 1,2-dichloroethylene Convicted.

A suspension comprising 35 ml of sedimented gel and 35 ml of 1,2-dichloroethane was used for the further reaction with octyl glycidyl ether. After stirring for 5 minutes, 1 ml of octyl glycidyl ether was added. The reaction was carried out at 25 ° C. for Mo minutes. After careful washing with 1,2-dichloroethane and then with acetone, the substituted gel was transferred to water. The gel obtained contained 70 % water and 7.5 % of the hydroxyl groups were substituted by the hydrophobic groups.

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Example 6

An immunoglobulin G fraction was obtained from an anti-insulin antiserum (produced in guinea pigs) separated by precipitation with 8% sodium sulfate. The precipitation was in sodium phosphate buffer fer (0.05 m, pH 7 »containing also 0.5m NaCl) to the original serum volume. 50 μΐ of this anti-insulin immunoglabulin solution was dissolved with 3 ml of a gel suspension containing 1.5 ml of sedimented octyl agarose gel according to the procedure described in Example 1 was prepared, which three times with the phosphate buffer described above was washed. The reaction mixture was shaken continuously overnight at room temperature. The gel was then applied 8 times with 0.5 m

NaCl and then washed in a sodium phosphate buffer containing 0.3% human serum albumin. A suspension of the gel in the buffer was then produced with a gel content of 38 μl of sedimented gel per ml of suspension. The suspension was then identified as an immunosorbent in a radioimmunoassay performed essentially according to the technique described in the following literature:
L. Wide et al: Immunochemistry 4, p. 38I (1967), L. Wide, J. Porath: Biochem. Biophys. Acta I30 (1966), p. 257, was used.

Insulin and insulin ^{marked with V} J, which was used in this test, were taken from a commercially available insulin preparation (insulin kit, Phadebas Insulin Test from Pharmacia AB).

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100 μΐ ¹²⁵ I-insulin solution (0.3 μθί per ml), 100 μΐ different insulin dilutions (between 1 and 1000 microunits per ml) and 100 μΐ immunosorbent suspension were mixed in small plastic tubes as described above. The reaction mixture was left to stand at room temperature for 17 hours. The immunosorbents in the plastic

12 ¹ S tubes were then washed clean of unbound J-insulin by rinsing with 1 M NaCl and their radioactivity was measured.

For control purposes, tests were carried out with a gel in which normal immunoglobulin (from the blood of non-immunized guinea pigs) was absorbed instead of anti-insulin immunoglobulin, and with a gel which was not substituted. These tests resulted in an uptake of radioactivity, polygamy only reached 3 % of the activity that could be measured in the tests using specific anti-insulin immunoglobulin bound to substituted gel. The results obtained from the radioactivity measurements are shown in curve A of the accompanying drawing. The various concentrations of insulin (expressed in ^ U (micro units) per ml) were plotted on a logarithmic scale on the abscissa, while the ordinate represents the radioactivity as B./B, with B _{n being} the antibody

IO U

bound radioactivity (hence a measure of bound J-insulin) means in the absence of insulin, while B.

125

the amount of J-insulin bound at a given concentration of insulin indicates.

From curve A in the drawing it is clear that the Anti-insulin immunoglobulin preparation «produced according to

125 '

as described above, binds J-insulin and that the bound radioactivity with increasing concentration of insulin decreases. The requirements for a suitable

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Radioimmunosorbent system can therefore be considered fulfilled since the curve is suitable as a calibration curve for the determination of insulin in body fluids. So it is demonstrated that the immobilization of immunoglobulin by binding to an agarose gel that is substituted with octyl groups is a suitable method for performing radioimmunosorbent analyzes.

Example 7

The immunoglobulin 6 fraction obtained in Example 6 was separated from the anti-insulin antiserum and attached to a granulated gel prepared according to the method described in Example 5 in the same manner as in Example 6 described, bound. The final suspension, which was prepared for a radioimmunosorbent test, did not contain 38 / μl sedimented gel per ml suspension as described in Example 6, but only contained 2 μl per ml. A calibration curve for determining the insulin was prepared under the same conditions as they were described in Example 6, prepared. The results are shown in Figure B of the drawing in the same manner as the results of Example 6. From curve B in the drawing it can be seen that the immobilization of immunoglobulin by means of hydrophobic binding to a granulated agarose-polyacrylamide gel which is substituted with octyl groups is suitable for carrying out radioimmuno-analyzes.

The polymeric material with hydrophobic groups from Examples 2, 3 and 4 was prepared in the same way, as described in Example 6, used for binding immunochemical components in immunochemical analysis methods.

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Claims (1)

Patent claims : 1.NProcess for carrying out immunochemical analysis - methods using a water-insoluble but water-absorbent polymeric material that is hydrophilic Groups, preferably hydroxyl groups and / or amide groups contains, wherein the polymeric material an antibody (I), which with an antigen (A) against which the antibody (I) is directed, is converted or has been converted, or an antigen (II) which is reacted or has been reacted with an antibody (B) which is directed against the antigen (II), is bound, the polymeric material against antibodies and antigens, which are seen by the immunoehemis Analytical methods are included, immunochemically inert is, characterized in that a material is used as the polymeric ■ material which is hydrophobic Has groups that are capable of the antibody (I) or the antigen (II) by so-called hydrophobic binding to bind, and that the antibody (I) or the antigen (II) is bound to the polymeric material by hydrophobic bonding. 2. The method according to claim 1, characterized in that the polymeric material has a water absorption capacity of comprises at least 0.2 g, preferably at least 0.5 g of water per g of dry polymeric material. 3. The method according to claim 1 or 2, characterized in that the water-insoluble, but water-absorbing, polymer! at least one material containing hydrophilic groups 609815/0 9 49 water-absorbing, polymeric or polymerized carbohydrate or sugar alcohol or derivatives thereof contains as a polymeric backbone. H. The method according to claim 3> characterized in that the polymeric material also contains crosslinked polyacrylamide or derivatives thereof. 5. The method according to any one of the preceding claims, characterized in that the hydrophobic groups on the polymer Backbone with bonds of a covalent nature are bound. 6. The method according to any one of the preceding claims, characterized characterized in that the number of hydrophobic groups in the polymeric material is smaller than the number of hydrophilic groups. For: Pharmacia Diagnostics AB Uppsala / Sweden Dr H.3ftr.Beil Lawyer 60981 5/0949