SE462165B - MAKE SURE TO ADORABLE A COMPARATIVE PROTEIN COMPLEX, SPECIFIC SIGNIFICANT BY BIOSPECIFIC DETERMINATION METHODS - Google Patents

Description

462 165 at molar concentrations exceeding 1/5 of their solubility in water. In cases where the adsorption is promoted to be as complete as possible, the process enables a way to be able to quickly desorb this complex from the carrier in order to facilitate measurements with indicator systems where required and reuse of the carrier. This is desirable in many cases as the ligand can be very expensive and maximum utilization is therefore of great interest.

Reuse of the carrier also enables simplifications in the automation of diagnostic systems, where, for example, surfaces coated with a suitable polymer to which ligand is connected can either be used in a flow system with one measuring point or extended to several measuring points. For determination methods in biospecific affinity reactions, several reactants are used, one of which is labeled with at least one analytically indicative atom or atomic group, and is soluble in the aqueous solution in which the reaction takes place.

By biospecific affinity reactions, the reactants form a conjugate in which the labeled reactant is included and the analytically indicatable atom or group of atoms can be determined in the conjugate and / or in non-conjugate-bound, labeled reactant.

In such determination methods for biospecific affinity reactions, for example immunochemical reactions, a reactant which is labeled and soluble in the aqueous solution in which the reaction takes place is reacted with a reactant having a biospecific affinity, and optionally with a second reactant having a biospecific affinity. to the first and / or the second reactant may be included in the reaction chain, one having affinity for the support and another being labeled.

The course of the reaction can be described schematically: where P is P + Bi + A + Ax--) PBiA-I-PBiAX, the support, the polymer 10 15 20 25 30 35 462 165 B1; B2; ..Bi; ... Bn are affinity components for the polymer.

A is the unlabeled test substance, A? denotes labeled test substance, AB, AB1, AB¿, ABn .. is conjugate, as well as AXB, Axßl, A231, AXBn ..

Adsorbed is also P-Bi and free in the solution ABi + Bi.

Characteristic of the invention is that Bi and BiA and BiA ¥ are specifically adsorbed to the polymer in aqueous solution and that this adsorption is enhanced by chlorides, sulphates, phosphates or salts of polybasic organic acids. When applying the invention, one or more of these salts are added to a concentration suitably exceeding half or one third of the saturation concentration of the salt and so that the ratio Bi + Aßi / Afritt exceeds 1000.

The salt pushes the complex out of the solution so that the complex accumulates at the phase interface between solution and adsorbent. The ligand then penetrates into a surface region of the complex, where the dielectric constant is considered to be greatly reduced, and is in the range 2 - 15.

The assay system can be described generally as follows: Schematic image of immunoglobulin (Ig) T '\ / \ binding site' does not bind test substance test substance binding site ligand on carrier particle 10 15 20 25 30 35 462 165 A labeled test substance ¿ê§ unlabeled test substance Ig molecules and test substance molecules in admixture give, in addition to excess unbound substances, two kinds of immune complexes: labeled and unlabeled, respectively. When adsorbent particles are added, Ig molecules, free and bound, bind to the particle.

The higher the concentration of unlabeled substance, the less labeling of the particle is obtained.

Ig molecule that does not bind the test substance The particles are isolated and the labeling is measured. The course of the reaction when separating an antigen with antigen ß-Z-p. as a model is described schematically below.

With ß-Z-p. as a model, an analyte is obtained which is present in a high concentration in urine and serum, 1-10'7 M and thus also gives a high concentration of antibody concentration, total Y-globulin concentration 1-10 ' 5 M.

Portions of the structure of antibodies are almost identical to ß-2-u, which is therefore a very suitable antigen to illustrate the general principle and provide important information about the ability of the polymer to bind specific immune complexes and specific immunoglobulins. ß-2-u is commercially available from Pharmacia, Uppsala. ----- <: = antibody directed specifically to reactant A which in this case is antigen ß-2-p (Mv. 1150), antigen ß-2-ß labeled with 125 I or enzyme. --Å + - <: = non-specific antibodies, normally occurring antibodies in serum.

In solution there is at the same time _H_ <+ __ <+ ß-zu (in sample) + ß-2-u125I L whereby immune complexes are formed - {+ - <: + --- (f -ß-2-p125I + - «< -ß-2-u + ß-2-p or ß_2_u125I (unbound) 1. Addition of polymer with ligand which under specific conditions binds immunoglobulins, free or in complex + free proteins B-2-U-462 10 15 20 25 30 35 165 Washing gel with simultaneous separation of unbound antigen labeled with marker I (free from free proteins) Measurement of radioactivity Reactant A and labeled A 2 are a protein, but may also be another substance. eg a nucleic acid, which with at least one other reactant B which is a protein that binds A and forms a complex AB, which like B itself is adsorbed to a polymer P in an environment that contains a water-structuring salt (anti-chaotropic ) such as alkali, alkaline earth and ammonium salts of, for example, the anions chloride, sulphate, phosphate, citrate and of other water-soluble di- and tri-basic acids, preferably in a concentration of the rising half or third (about 20%) of the saturation concentration, after which the adsorbate and the adsorbent (the polymer to which AB has been adsorbed) are separated from the solution and the measurement is carried out.

In carrying out the assay method, a support is used to which only one of the reactants contained in the conjugate can be adsorbed or absorbed. The support used contains covalently bound ligands of structures XS, X-CH 2 S, X-CH 2 NR-, where X is an E-electron-containing isocyclic or heterocyclic ring system and / or ligands containing structures. SO 2 -CH 2 -CH 2 -Y where Y is S, N <or NR- and R is H or alkyl.

The support used contains either one or more sulfur atoms with or without a combination of one or more nitrogen atoms in such a combination as are found in, for example, mercaptopyridine, thiocyanate, ethyl sulfide, pyridyl sulfide, mercaptoethanol.

The antichaotropic salts in Hofmeister's series that promote the adsorption of the conjugate were used in combination with the above carriers and reactants at the concentrations that increase the specific adsorption of the conjugate.

The carrier may be a polymer which is soluble or insoluble in aqueous liquids, in the first case preferably having a molecular weight greater than that of the protein complex. For chromatographic purposes, the polymer must be particulate, but for diagnostic purposes either of the two forms can optionally be used, although we will only describe in this case insoluble, adsorbent particles for immunodiagnostic application in more detail. Both synthetic and natural polymers can be used and provided with different ligands, which, however, all contain at least one sulfur atom, alone or in combination with at least one nitrogen atom. The polymer may be crosslinked to a water-insoluble network which may be swellable in water.

Examples of such polymers are derivatives of agarose or other water-insoluble polysaccharides, silica gels or silicates, e.g. glass.

Examples of water-soluble polymers are derivatives of dextran, starch or other water-soluble polysaccharides.

Other examples of polymers are the native or modified, soluble or insoluble proteins or polypeptides which exhibit adsorption of conjugate or non-conjugate bound, labeled reactant A. There are many examples of polymeric substances containing sulfur or sulfur and nitrogen in various combinations. Such polymers are described, for example, in Febs Letters, vol. 185, No. 2, 11985. 462 165 10 15 20 25 30 35 Other examples are agarose substituted with groups - O - CH 2 - CH - CH 2, / to which are attached HS, HS I, HS - <@ N, N SCN and HS (CH 2) n - CH 3, where n is 1-5.

* Gel adsorbents with ligands containing structures -SO 2 -CH 2 -CH 2 -X, where X is S, NH or N, or other structures constituting heterocyclic or aromatic ring systems and gels with ligand structures -NH- in combinations with nitrile group are previously known through the Swedish patents 8402662-4, 8402663-2 and 8600641-8.

These adsorbents adsorb immunoglobulins at relatively low concentrations of water-structuring salts, after which the immunoglobulins are desorbed by lowering the salt concentrations and / or by adding to the medium a lower polarity organic solvent, e.g. glycerol, glycol or other lower alcohol. Although component B has previously been found to be adsorbed to P, adsorption does not necessarily have to take place after B has formed complexes with other high molecular weight reactants A ... An and this can be used for analytical and diagnostic purposes.

Characteristic of the invention is that conjugate A ... An can also be adsorbed in a similar manner to the same type of adsorbent and thus separated from the solution.

Those of the known methods which use a soluble polymeric support for one of the reactants have the advantage over other methods that separations of conjugate and non-conjugate-bound labeled reactant A? can be performed in a more accurate way. A disadvantage is that the bio-specific affinity reaction takes place more slowly when one of the participating reactants is bound to an insoluble carrier compared to if this reactant is free in solution. For the diagnosis of immunoglobulins or antigens, it is important that the analyzes require the least possible time. Complex formation takes place faster in solution than at a phase interface and is therefore carried out in solution and precipitation then takes place with a high ligand concentration on the solid phase. The invention therefore reduces the time for an assay to the time it takes for immunocomplex formation in solution since the adsorption of the immunocomplex to the solid phase is instantaneous. Immunoglobulin can also be diagnosed with anti-immunoglobulins.

Examples of reactant A and other reactants which exhibit biospecific affinity for each other are a) antigen and haptens and specific antibodies directed against them or modified and fragmented portions of antibodies, b) complement binding factors capable of binding to immunoglobulins, c) lectins which can bind to specific carbohydrate structures and specific antibodies directed against them, d) enzymes and enzyme inhibitors that can bind to these enzymes and specific antibodies directed against these reactants, 462 165 10 15 20 25 30 10 10 e) receptors and ligands thereto and specific antibodies bodies directed against these reactants, f) nucleic acid or nucleic acid derivatives thereof and specific reactants directed against them.

In order to be able to determine the content of the analytically indicable atom or atomic group in the conjugate or of labeled reactant AX that is not bound to the conjugate, the conjugate and the non-conjugate-bound labeled reactant AX are divided by the precipitation method or by chromatographic methods.

An example of a precipitation method is the so-called the double antibody method DASP (double antibody solid phase), according to which immunocomplexes are formed in a solution, after which conjugate and non-conjugate-bound, labeled reactant AX are separated by adsorbing the conjugate-bound reactant on the solid phase by antibodies directed against one of the components in the conjugate.

Examples of chromatographic methods are affinity chromatography! The partition is now often designed so that one of the reactants is bound to an insoluble polymer and to this is bound by a biospecific reaction other reactants, whereby the proportion of reactant A can be separated, which is not assumed to be provided with an indicator group or atom. , which is not biospecifically bound to the polymer. Examples of such systems are given in Radioimmunoassay Methods (Ed. K.E.

Kirkham and W.M. Hunter, Churchill Livingstone, London 1971, pp. 405 - 412). A large number of variants of determination methods in which a labeled reactant is used are reproduced in this publication.

The indicatable reactant is labeled with an analytical indicator group or atom, and the labeling of a reactant is now a well-established technique and takes place by coupling one of the constituent reactants to an analytically indicative atom or atomic group. by direct bonding or over a bridge. Common markers are radioactive atoms or atomic groups, fluorescent, luminescent or chromophoric groups, enzymatically active groups, enzyme inhibitor groups or coenzyme groups.

In the quantitative determination of one of the reactants, it is known to use varying known amounts of this reactant to establish standard curves which are then used to determine the amount of the reactant in a sample.

The invention is further explained by the following working examples and the accompanying drawings, in which Fig. 1 shows a standard curve for B-2-L1 with peroxidase as marker, Fig. 2 shows adsorption on agarose with different ligands, Fig. 3 shows adsorption of immunocomplexes and cursor as a function of time, and Fig. 4 shows the standard curve for B-2-u with l25I as cursor.

Example 1 Preparation of a standard curve for ß ~ 2-u to determine the concentration of 8-2-u in the samples.

Peroxidase was used as the marker. The antibodies were non-IS-purified (immunosorbent-purified) antibodies from immunization of B-2-β in sheep by precipitation of serum with 18% Na 2 SO 4 and desalting on Sephadex® G25.

The support consisted of epichlorohydrin-activated agarose (Sepharos®® 5B) to which mercaptopynun was coupled in the presence of 0.5 g NaBH 4 in 0.1 M Na-phosphate buffer, pH 7.5, by adding 462 165 10 15 20 25 30 35 Addition of 2 g of mercaptopyridine to 25 g of epichlorohydrin-activated gel and reaction at room temperature for 18 hours.

After completion of the reaction, it was washed alternately with distilled water, ethanol and finally with distilled water. 30 mg of dry suction agarose was swollen in 1 ml of 0.5 ml K 2 SO 4 - - 0.1 M Tris (trishydroxymethylaminomethane hydrochloride), pH 7.5. To Ellerman tubes with a volume of 3 ml was added 25 μl of anti-B-2-u solution diluted with 0.1 M Tris, pH 7.5 to a concentration of 1.8 x 10-6M with respect to total the concentration of immunoglobulin. Then 25 μl of 8-2-u solution was added to two series of tubes with decreasing concentration from 30 pg / ml to 0.0 pg / ml. After 5 minutes, 25 μl of 1 x 10-7M β-2-u-peroxidase conjugate was added and after 15 minutes of reaction time 500 μl of mercaptopyridine portion with a concentration of 30 mg gel / ml, corresponding to 1.8 mg carbohydrate / ml. After 10 minutes, the tubes were centrifuged, the supernatant was aspirated and 1000 μl of 0.5 M K 2 SO 4 - 0.1 M Tris was added. The tubes were centrifuged again, the supernatant was aspirated and 1 ml of 5-aminosalicylic acid solution (80 mg of 5-aminosalicylic acid in 100 μl of distilled water) was added. The solution was titrated to pH 6.0 and 20 μl of 3% H 2 O 2 was added to 10 μl of the solution. After 10 minutes reaction time, 100 μl of 5 M NaOH was added and the absorbance was measured in the supernatant at 449 nm. The standard curve above A 449 nm as a function of the B-2-p concentration is plotted in Figure 1.

Example 2 Effect of various ligands linked to cross-linked agarose I on the adsorption of immunocomplexes. a) Synthesis of 3- (2-pyridylsulfido) -2-hydroxypropylagarose (PyS). 40 ml of 4 M NaOH was mixed with 0.17 g of NaBH 4 and 50 g of washed and dry vacuum Sepharose 6B was added. Then first 4.25 ml of epichlorohydrin were added and after 30 minutes another 4 ml. During the following hour, 4 ml of epichlorohydrin were added 4 times at regular intervals each time. After allowing the mixture to react at room temperature for 18 hours, the reaction was stopped and the gel was washed with distilled water until neutral. 2 g of mercaptopyridine was dissolved in 100 ml of 0.1 M Na-phosphate buffer, pH 7.5, with the addition of 0.5 g of NaBH 4 and added to the gel after pH adjustment.

After reaction for 18 hours at room temperature, it was washed with ethanol and water. Analysis showed 810 μmol S and 800 μmol N per g of dried product. b) Synthesis of 3- (2-pyridyloxy) -2-hydroxypropyl agarose (PyO).

The epichlorohydrin activation of the gel was done in the same way as for a). Of 100 ml of 1 M NaOH, half was added with 0.5 - g NaBH4 and half with 5 g hydroxypyridine. The two solutions were mixed and 5 g of dry-sucked Sepharose 6 B was added and allowed to react at room temperature for 18 hours. Nitrogen analysis showed 1064 μmol N / g dried product. c) Synthesis of 3- (phenyloxy) -2-hydroxypropyl agarose (PhO).

The gel was epichlorohydrin activated in the same way as before. 0.25 g of NaBH 4 was charged to 25 ml of 1 M NaOH and 10 g of phenol was dissolved in likewise 25 ml of 1 M NaOH, the solutions were mixed together, 25 g of epichlorohydrin-activated gel were added and allowed to react for 18 hours at room temperature. The degree of substitution was determined by NMR to 670 nmol ligand / g dried product. d) adsorption of different ligands by different ligands at different gel concentrations. 25 μl of 1.8 x 10-6M anti-B ~ 2-u and 25 μl of 1 x 10-7 M ß-2-u-peroxidase conjugate were charged into Ellerman test tubes. After reaction for 30 minutes, varying concentrations of resp. gel to the tubes. The gel concentrations are indicated on the abscissa in Figure 2. After reaction for 30 minutes at room temperature, the polymers were washed thoroughly with 1 M 0.5 M K 2 SO 4 - 0.1 M Tris solution, pH 7.5, in 3 batches with intermediate 462 165 10 15 20 25 30 14 centrifugation in a table centrifuge at 3000 rpm and the supernatant is aspirated. Then 1 ml of 5-aminosalicylic acid solution (80 mg / 100 ml distilled water) was added and the substrate solution was adjusted to pH 6.0 with 1 M NaOH. To 1 ml of this substrate solution was added 20 μl of 3% H 2 O 2 and after 15 minutes the reaction was quenched by the addition of 100 μl of 5 M NaOH. After centrifugation of the gel, the absorbance of the supernatant was read in the different tubes. The curves are drawn in full in Figure 2. Corresponding dashed curves show the unspecified adsorption of marker to gel in the absence of antibody.

Example 3.

Desorption of adsorbed immunocomplex.

In the same manner as described in the previous example, anti-β-2-U and 3-2-U-peroxidase conjugates were charged into Ellerman tubes. The yellow mercaptopyridine-based cross-linked agarose was added and the resulting immunocomplex was adsorbed in the presence of 0.5 M K 2 SO 4 - 0.1 M Tris, pH 7.5. Two parallel test series were performed. In one series of experiments, unbound excess 5-2-β-peroxidase was washed with the vehicle with 0.5 M K 2 SO 4 - Tris, pH 7.5, and in the other series of experiments, 0.1 M Tris, pH 7.5 was washed. .

By measuring the amount of immunocomplex bound in the presence no.5 M K 2 SO 4 - 0.1 M Tris, pH 7.5, and comparing with when the corresponding immunocomplex was first adsorbed in the presence of 0.5 M K 2 SO 4 and then desorbed by washing of the 0.1 M Tris support, pH 75, it was possible to determine the desorbed amount of the adsorbed immune complex.

To 6 Ellerman tubes were added 25 μl of 1 x 10-6 M anti-B-2-u and 25 μl of B-2-u-peroxidase.

In tubes 1 and 2, the formed immunocomplex was adsorbed in the presence of 0.5 M K 2 SO 4 and washed with 0.5 M K 2 SO 4 - 0.1 M Tris, pH 7.5, after which the activity was measured. 462 165 In tubes 3 and 4, the support was washed with 0.1 M Tris, pH 7.5, and the enzyme activity was measured.

In tubes 5 and 6, instead of anti-B-2-U, the corresponding volume was added 0.1 M Tris, pH 7.5, and the support was washed with 0.5 M K 2 SO 4 - 0.1 M Tris, pH 7. 5.

Experimental results.

Absorbance at 449 nm 1.17 1.06 0.11 0.12 0.11 0.12 Tube No. ONUIàUJIUIJ Tubes 1 and 2 show the specific adsorption of the immunocomplex. Tubes 3 and 4 show the specific adsorption of immune complexes after desorption by eliminating salt. Tubes 5 and 6 show the undesired adsorption of marker to the polymer and at the same time the level of enzyme activity to be found in tubes 3 and 4 if the desorption of the immune complex is complete.

Example 4.

, The adsorption of the formed immunocomplex to 3- (2-pyridylsulfido) -2-hydroxypropylagarose as a function of time.

To a number of Ellerman tubes were added in each tube 25 μl of 1 x 10-7 M 5-2-U and 25 μl of 1 x 10-6 M anti-ß-2-U. Each tube was then filled with 500 μl of gel (30 mg of swollen, dry-sucked gel mixed with 1 ml of 0.5 M K 2 SO 4 - 0.1 M Tris, pH 7.5).

The reaction was stopped after 1, 7, 10, 15, 30, 60 and 120 minutes, respectively, by adding 1 ml of 0.5 M K 2 SO 4 - 0.1 M Tris, pH 7.5, to the respective tubes. The tubes were centrifuged and the supernatant was aspirated. The procedure was repeated twice. 462 165 10 15 20 25 30 16 The enzyme activity was read after 15 minutes of reaction with substrate in the same manner as described in Example 1. The enzyme reaction was stopped by adding 100 μl of 5 M'NaOH.

The test result is shown in Figure 3.

Example 5.

The carrier used was PyS according to Example 2, as a marker and as antibodies not IS-purified according to Example 1. 125 I 25 μl of anti-B-2-u solution diluted with 0.1 M Tris, pH 7.5, to 1.8 x 10 '6noglobulin, was charged into Ellerman tubes with a total volume of 3 ml.

To two series of tubes were added 25 μl of B-2-u in each tube at concentrations ranging from 31 g / ml to 0.48 g / ml.

After 5 minutes, 25 μl of B-2-u- labeled with 125 I, and with a concentration of 1.5 x 10-7 M, was added to each tube to each tube. After a total reaction time of 15 minutes, 500 μl of PyS gel with the concentration of 30 mg of swollen dry-sucking gel / / ml 0.5 M K 2 SO 4 - 0.1 M Tris, pH 7.5 was added. After 15 minutes, it was centrifuged and the supernatant was filtered off with suction. . 1 ml of 0.5 M K 2 SO 4 - - 0.1 M Tris, pH 7.5, was added, the tubes were centrifuged again and this was repeated twice. The activity in the Ms with respect to the total concentration of immuno-different tubes was determined in Y-counters.

Very good results have also been obtained with lectin, α2-macroglobulin, trypsin, chymotrypsin, enzyme complexes of α2-macroglobulin or soybean trypsin inhibitor and proteolytic enzymes etc. Even low molecular weight compounds with a molecular weight greater than 100 have been able to be diagnosed with good results and thus diagnostic tests have been performed on nortryptilin, whereby antibodies could be obtained by coupling to a carrier molecule whereby the hapten has become immune.

Claims (8)

10 15 20 25 30 35 f? 462 165 P a t e n t k r a v A method of adsorbing organic substances on a carrier, such as a polyhydric, organic polymer, silica gel, organic polyamine, polyamide or the like, with ligands containing at least one sulfur atom alone or in combination with at least one nitrogen atom, that the ligands contain one or more of the structures SX, S-CH2-X, RN-CH2-X, SO2-CH2-CH2-Y, where X is a W electron-containing isocyclic or heterocyclic ring system, Y is S, N <or NR and R is H or alkyl, and that the organic substances are composite protein complexes, in which one component is an immunoglobulin, an enzyme, an enzyme inhibitor, a lectin, a nucleic acid or a derivative thereof. 2. A method according to claim 1, characterized in that a hapten, such as a low molecular weight drug, hormone or the like, is bound to the immunoglobulin. 3. The method of claim 1, wherein the ligand contains any of mercaptopyridine, thiophenol, thiocyanate, ethyl sulfide, pyridyl sulfide or mercaptoethanol. 4. A method according to any one of the preceding claims, characterized in that the adsorption is enhanced by being carried out in a salt environment selected from salts which promote the adsorption at molar concentrations exceeding 1/5 of their solubility in water. 5. A method according to any one of the preceding claims, characterized in that the salt is antichaotropic and selected from alkali, alkaline earth or ammonium salts of hydrochloric acid, sulfuric acid, phosphoric acid or polyvalent organic acids. 6. A method according to any one of the preceding claims, characterized in that the carrier is a polyhydric polysaccharide, PVA, silica gel or derivative thereof. A method according to any one of the preceding claims, characterized in that the polymer is soluble in water to 0.1% or more. 8. A method according to any one of the preceding claims, characterized in that the polymer is present as a water-insoluble substance, such as in the form of a particle, wire or membrane.