DD300382A7 - METHOD FOR ACTIVATING POLYSTYRENE-SOLUBLE CARBON SURFACES - Google Patents

Abstract

Method of activating polystyrene solid surfaces. The invention relates to Polystyrenoberflaechen, which are used after activation for the binding of Biomakromolekuelen, biological materials and low molecular weight compounds. Areas of application are the pharmaceutical industry, life sciences, biotechnology and medicine. The activation of the surface takes place according to the invention with organosilanes of the general formula {activation; Surface; polystyrene; Binding; biological macromolecules; biological materials; low molecular weight compounds; organosilane; Proof; immobilization}

Description

Field of application of the invention

The invention relates to a method for activating polystyrene solid-state surfaces for the subsequent binding of proteins, nucleic acids, low-molecular-weight ligands, microorganisms, cells and other biological materials. The process is used in the pharmaceutical industry, life sciences, biotechnology and medicine.

Characteristic of the known state of the art

Specific and sensitive immunological methods for the quantitative determination of antigens, antibodies, haptens, viruses, microorganisms etc. have gained outstanding importance in recent years in medical and life science research as well as in clinical practice. In particular, the methods referred to as immunoassays for the determination of antigens, antibodies, haptens, etc. have found widespread use because they combine the advantages of high specificity of immunological reactions with the extremely high sensitivity of the detection system (radioimmunoassay, enzyme immunoassay, fluoroimmunoassay, etc.) ,

Among the numerous variants described, the so-called solid-phase technique, in which one of the immunological reactants is bound to a solid phase and thus allows easy separation of the formed antigen-antibody complexes from the free, unbound reactants, has At present, almost exclusively the method of adsorptive biodimetation of proteins on solid plastic phases, preferably polystyrene, which is based on KJCatt and GWTregear (Science 15 [1967] 1570), is currently used (review: JEHerrmann: Methode in Enzyinol. 1981] 239) However, this process has some disadvantages:

- The loading density of plastic materials and thus the sensitivity of the immunoassay are limited by the respective plastic material, the available surface and the size of the protein.

- During the individual incubation and washing procedures in the immunoassay significant amounts of protein (68% in viral antigens: JE Herrmann, RM Hendry, MF Collins: J. Clin Microbiol 10 [1979] 210; 50% in antibodies: W. Schößler, G Wegner: Z. med., Labordiagn., 24 [1983] 177). Such desorptions, in turn, adversely affect the sensitivity and decisively determine the error of the method.

- The loaded with proteins plastic materials can be used only once after the immune reaction, so that very valuable and expensive to be prepared proteins are lost.

The use of these plastic materials as disposable containers represents a not inconsiderable economic factor for larger numbers of samples in clinical routine.

- The possibilities of standardization are limited in this method.

To circumvent these difficulties, various authors have described the use of chemically modified carriers for the covalent coupling of proteins in the immunoassay (for review: J. E. Herrmann: Methode in Enzymol 73 [1981] 239). Of such materials in particular cellulose (DD-WP 225795 EP 0134025), dextrans, agar and its derivatives [review: S. Fuchs, M. Seia, in: Handbook of Experimental Immunology (DM Weir, ed.) Blackwell Scientific Publications, Oxford 1978) in addition to polyacrylic acid derivatives or polyamides (RM Hendry, JE Herrmann: J. Immunol., Methods 35 [1980] 285) found as a matrix application. However, all of these materials have not been able to assert themselves to the desired extent, once, since the chemical modifications are often associated with oinem considerable effort, and on the other hand, since these materials nonspecifically bind proteins are thermally and chemically relatively unstable or microbially attacked easily ,

In the patents DD-WP 214659 and DD-WP 218189 the covalent bond to glass is described as a solid phase in the immunoassay. This method, which is characterized by a number of advantages and has also found application in practice (W. Schößler, F. Hiepe, T. Montag, HE Schmidt: Hiomed.Biochim.Acta 44 (1985) 1247), can only be relatively difficult to adapt to the commercial device technology, which allows half or fully automatic execution of enzyme immunoassays using 96-well microtiter plates.

On the other hand, there has been no lack of attempts to chemically modify polystyrene to covalently attach proteins and other biologically active compounds to such activated polystyrene surfaces. The process of activation of polystyrene solid surfaces with nitric acid and subsequent reduction of the introduced nitro groups to amino groups, now for the reaction with glutaraldehyde (review: GH Parsons: Methods in Enzymol 73 (19811 224) or for diazotization (H. Filippusson, WE Hornby: Biochem J. 120 [1970] 215), leads to a coloration of the polystyrene during the nitration, which significantly impairs the evaluation of the microtiter plates used in the enzyme immunoassay by vertical photometric.

Boehnisch (in: Prot., Biol., Fluids, Proc., 24th, Cofl., Bruges, 1976 [H. Peters, ed.], Pergamon Press, Oxford, 1976, page 743) described a method of activating polystyrene surfaces by glutaraldehyde. Unfortunately, this method did not lead to the desired results since no increase in sensitivity could be detected in the enzyme immunoassay in this manner (M. Steiner, JL Stratt, C'in Chem 24 [1978] 339, E. Nugel, B. Porstmann , T. Porstmann, R. Seifert, H. Schmechta: Z. med., Labor.-Diagn. 22 [1981] 202; own results).

U.S. Patent No. 4,001,583 discloses a method of covalent attachment of proteins by glutaraldehyde to polystyrene surfaces previously wetted with diphatic amines. However, the relatively weak binding, preferably hydrophobic interactions, between the polystyrene and the diphalic amines may be due to detergents used in the incubation and washing media of e.g. As immunoassays apply, are disturbed, so that it can come to not inconsiderable desorptions during the execution of the immunoassay. Therefore, almost exclusively the method of adsorptive binding to polystyrene mentioned at the outset has hitherto been used.

Object of the invention

The aim of the invention is to activate polystyrene surfaces cost with little technological effort.

Explanation of the essence of the invention

The invention is based on the object by chemical Oberflächenmodifizi srung to achieve a high binding capacity and binding capacity of polystyrene surfaces. According to the invention the object is achieved by a method in which Polystyrenfestkörperoberflächen with organosilanes of the general formula

(XRi) _n SiR ₄ _ _n (I)

where X is an amino, carbonyl, carboxy, isocyano, diazo, isothiocyano, nitroso, sulfhydryl or halocarbonyl group and R 'is an alkyl, alkylphenyl or phenyl group, while R is an alkyl, carbonyl, carboxy, isocyano, diazo, halocarbonyl; is an alkoxy, phenoxy or halogen group and n 'can have the value between 0 and 20 and η can have the value between 1 and 3. Common organosilanes can be represented by the following formula:

Y _n SiR ₄ -H (ID

where Y is an amino, carbonyl, carboxy or sulfhydryl group and R is an alkoxy, phenoxy-halogen group and η can take the value between 1-3. Commonly used and widely used organosilanes have the general composition

XCH ₂ CH ₂ CH ₂ -Si (OR) ₃ · (III)

where X is the reactive organic group corresponding to formula I and R is a methyl or ethyl group. The conversion is also carried out with at least two organosilanes of the formula I in a mixture or in succession. The preferred environment for the organosilanes is the liquid or the gaseous phase, wherein the activation can take place in aqueous systems, water-solvent mixtures or organic solvents. Subsequently, if appropriate, further treatment of the modified polystyrene solid surfaces with homo- or heterobifunctional reagents takes place. It has been known for some time that the hydroxyl groups on SiO ₂ surfaces, preferably glass, but also on other solid surfaces, are available as targets for organosilanes. Surprisingly, however, it has been found that organosilanes of suitable composition and concentration react with polystyrene solid surfaces which should not have any free OH groups. This binding can be done by strong secondary valence forces, but also conceivable covalent bonds with impurities and / or by-products of the polystyrene. Thus, the organofunctional group is available to the usual chemical reactions using amino, carbonyl, carboxy, isocyano, diazo, isothiocyano, sulfhydryl, nitroso or halocarbonyl groups, so that these compounds act as a bridge between the Solid-state surface and organic / biological compound act. The polystyrene solid surfaces activated in this way can now in the following directly via the corresponding functional groups or else by mediation of homo- or heterobifunctional reagents in a manner known per se with the proteins, nucleic acids, low molecular weight ligands, cells, microorganisms and other biologicals to be bound Materials are implemented.

This is done, for example, so that the polystyrene solid surfaces with mixtures of organosilanes, which are composed of aminopropyltriethoxysilane and glycidoxypropyltriethoxysilane, are activated and the now on the solid surface located amino and epoxy groups react with proteins directly or through the agency of glutaric dialdehyde. It is important that the reaction with the organosilanes, which are non-toxic and are produced industrially to a considerable extent, by simple Inkontaktbringon odor dipping, or can also be carried out in the gas phase. Of great importance here is that the reaction with organosilanes infl'jssiger phase with organic solvents such as acetone, toluene, dioxane, methanol, ethanol, etc., solvent mixtures such as methanol / ethanol, and in an aqueous medium or water-solvent mixtures, such as Ethanol / water and methanol / water, can be done so that the technological cost is low. The activation in gaseous phase should be particularly advantageous by using aerosols or by negative pressure.

A further advantage is that by choosing different organosilanes and optionally bifunctional coupling reagents virtually all possible reaction options can be realized because using the underlying and inventive principle polystyrene solid surfaces with a variety of functional groups (X in the formula I) can be erha th , In addition, due to the spacer effect of the organosilanes (R '= 0-20) and / or the bifunctional coupling reagents (such as glutaraldehyde), the bound biological materials almost without exception retain their biological activity is the use of mixtures of organosilanes, since so-called Silanhaftmittel alone hardly lead to an activation of polystyrene solid surfaces. In addition, such mixtures of organosilanes have different functional groups and thus biological materials can be bound via different binding mechanisms in high yield. Thus, mixtures of aminopropyltriethoxysilane and glycidoxypropyltriethoxysilane prove to be particularly suitable because the bonding of the ligands is realized via aldehyde and epoxy groups. The chemical modifications resulting from binding of organosilanes to polystyrene solid surfaces are very stable, so that so activated polystyrene surfaces can be stored in the dry or wet state for long periods without loss of activity and that bound biologically active ligands for a long time under extreme conditions remain stable. The unspecific bonds to polystyrene surfaces activated in this way are low. Organosilanes are characterized by good biocompatibility. Proteins such as antigens and antibodies, nucleic acids, low molecular weight ligands, microorganisms, cells and other biologically active materials are bound to the polystyrene solid surfaces activated in this way and for the separation and isolation of corresponding interaction partners or, above all, for the qualitative and quantitative detection of specific interaction partners in the Solid phase immunoassay used

 The invention has the following advantages:

- By using prefabricated moldings made of polystyrene, the targeted activation of only the surfaces is possible, to which the subsequent binding is to take place.

- The technological process of activation is simple and inexpensive.

- The active surfaces have a high binding capacity.

- The optical properties of the moldings are not changed after activation.

Ausführungsbelsplele

In the following the invention is explained with some examples, which do not claim to be exhaustive.

example 1

Polystyrene tubes (4 ml, diameter 9 mm, height 40 mm, VEB Polyplast Halberstadt, GDR) are brought into contact with a mixture of aminopropyltriethoxysilane and glycidoxypropyltriethoxysilane (adhesion promoter 6130, VEB Chemiewerk Nünchritz, DDR) by filling and incubated for 1 h at room temperature. After suction of the coupling agent and drying in vacuo, the tubes are washed three times with 0.1 M phosphate buffer, pH 6.8, and with 200 μΙ ¹²⁵ J-IgG (directed against the enzyme alkaline phosphatase) in a concentration of 10pg / ml (0.1 M phosphate buffer, pH 6.8) and incubated at 37 ⁰ C for ⁴ h. After aspirating the excess amount of protein and subsequent intensive washing three times with 2 ml of PBS buffer containing 0.05% Tween 20 and 0.22% NaN ₃ , the bound protein amount is determined by measuring the radioactivity with the introduction of the protein solution used as a standard. Thereafter, the polystyrene tubes are again washed ten times with the above PBS buffer and again the amount of bound protein determined by determining the radioactivity. The tubes thus loaded with ²⁶ J-IgG are then used several times in the enzyme immunoassay (see Example 4). After a period of four weeks, the amount of protein bound is again determined by determining the radioactivity. The results are summarized in Tab. 1.

Example 2

Polystyrene tubes (4mi, VEB Polyplast Halberstadt, DDR) are activated as described in Example 1 with coupling agent 6130 and then mixed with 3% glutaric dialdehyde and incubated at 37 ° C. for 2 h. After aspirating the excess reagent and then washing three times, the tubes are each mixed with 200 μl ²⁶ J-IgG (directed against the enzyme alkaline phosphatase) in the concentrations of 10 μg / ml and 50 μg / ml and incubated at 37 ° C. for 4 hours Suctioning off the excess amount of protein and subsequent washing determines the amount of bound protein by measuring the radioactivity as described in Example 1. Again, the bound protein is determined several times after further washing cycles and the tubes are used in the enzyme immunoassay. 1 shown.

Bet game 3

Polystyrene beads (6 mm diameter, Northumbrte England) are activated as described in Example 2 with coupling agent 6130 and glutaraldehyde and subsequently bound with ¹²⁵ I-IgG in the concentration of 1 μg / ml. The further treatment is also carried out as indicated in Example 2. These results are also shown in Tab. 1.

Table 1: Determination of the binding of ²⁶ J-IgG to polystyrene (all values represent ten-fold determinations, for comparability the amount of bound protein was based on equal area units, as reference the binding of ^'26 J-IgG by adsorption with a carbonate bicarbonate Buffer, pH 9.6)

protein binding I.Messung 2nd measurement 3rd measurement (dreiWasch- (another ten (after repeated cycles) Wash cycles) One. i. EIA) adsorptive: polystyrene tubes 10pg / ml 760ng / cm ² 700ng / cm ² 488ng / cm ² 50mg / ml 1 598ng / cm ² 1464ng / cm ² 1110iig / cm ² polystyrene beads lOpg / ml 1150ng / cm ² 1 026ng / cm ² 522ng / cm ² chemically activated according to Example 1: polystyrene tubes 10pg / ml 669ng / cm ² 598ng / cm ² 488ng / cm ² chemically activated according to Example 2: polystyrene tubes 10pg / ml 1 149ng / cm ² 1 023ng / cm ² 1 047ng / cm ² 50pg / ml 2283ng / cm ² 2047ng / cm ² 2047ng / cm ² chemically activated according to Example 3: polystyrene beads 10mg / ml 1 404ng / cm ² 1327ng / cm ² 1 327ng / cm ²

Example 4

The polystyrene carriers which are activated in accordance with Examples 1 to 3 and loaded with ^'26 J-IgG (directed against the enzyme alkaline phosphatase) are first of all three times as described in Examples 1 to 3 (1st measurement) and then a further 10 times (2. Measurement) with a PBS buffer, pH 7.4, containing 0.05% Tween 20 and 0.02% NaN ₃ , and then treated with 1 M ethanolamine for 2 h at 37 ° C. This will eventually block free reactive groups on the solid surface. After washing with the above buffer, the Polystyrenträger are added to the enzyme alkaline phosphatase (Boehringer-Mannheim, FRG) at a concentration of 1,5IU / mg og in PBS buffer and incubated at 4 ⁰ C 20h. After removal of the unbound excess enzyme by washing four times with PBS buffer indicated, the enzyme activity on the solid phase is determined by hydrolysis of 4-nitrophenyl phosphate in 1 M diethanolamine buffer, pH 9.8, and photometric determination of the reaction product at 405 nm after previous stop determined with 2N NaOH. The results are summarized in Tab. 2.

Table 2: Comparison of antibody reactivity to polystyrene by measuring enzyme activity (tenfold determinations)

Protein binding PS tubes PS tubes PS spheres

(Example 1) (Example 2) (8eisp.3)

enzyme activity

A _405n H ₁ 1,504 0,469 0,861

0.689

Example 5

Commercial polystyrene tubes (VEB Polyplast Halberstadt, GDR) are activated as described in Example 2 and mixed with 200 μM human IgG in a concentration of 37 pg / ml and incubated for 2 h at room temperature and then overnight at 4 ⁰ C. After removal of the excess protein by washing, as in Example 4, possibly free reactive groups with 1 m of ethanolamine (2h, 37 ° C) are blocked. Nnch washing again, the polystyrene tubes with a conjugate consisting of alkaline a rabbit anti-human IgG antibody and the enzyme phosphatase, incubated at 37 ⁰ C für4h. After repeated washing with specified PBS buffer, the enzyme activity is determined by photometric measurement as set forth in Example 4. Regardless, are polystyrene tubes in a known manner by adsorptive binding in a 0.1 m carbon? -Bicarbonate buffer, pH 9.6, loaded with human IgG in the same concentration and determined in parallel with the above conjugate under identical conditions. The results of the comparison are shown in Tab.

Table 3: Comparison of the immunological reactivity of human IgG as a function of the binding procedure (tenfold determinations)

adsorptive bond chemical bond

enzyme activity

A _40611n , 0253 1 ^ 050

Example 6

A commercial microtitration plate made of polystyrene (VEB Polyplast Halberstadt, DDR) with 96 wells is activated as described in Example 2 and then with 200μΙ human immunoglobulin G in a concentration of 20μg / ml in a 0.1 M phosphate buffer, pH 6.8, and incubated overnight at 4 ⁰ C. Subsequently, the wells now loaded with human IgG are incubated with a conjugate consisting of a rabbit anti-human fgG antibody and the enzyme alkaline phosphatase as mentioned in Example 5, and the enzyme activity as described in Example 4 is used of a vertical measuring photometer (MTF 10, VDE Berlin-Buch AdW of the GDR) determined, the enzyme reaction was stopped by 0.2m EDTA. Independently of this, a microtitration plate, analogously to Example 5, was loaded with human IgG in the same concentration and treated under otherwise identical conditions. The results are shown in Tab. 4. Nnch course of the immune response and completion of the Entzymimmunoassays the bound Immunreaktanden by incubating the microtitration plate with 3M KSCN (30min, 39 ° C) and then 1 M NaCl (30 min, 37 ⁰ C) split off, so that the loaded human IgG plate for a renewed use is available. This is then in turn mixed with specified conjugate and determines the enzyme activity. The results of a triple use of such a microtitration plate are also shown in Tab.4.

Table 4: Comparison of the immunoreactivity of human IgG with multiple use

Protein binding adsorptive binding * chemical bonding

I. Measurement 2. Measurement 3. Measurement

enzyme activity

Atosnm 0.280 1.173 0.878 0.890

Since significant protein levels are desorbed during adsorptive binding during the washing and incubation steps required in the enzyme immunoassay (see Table 1), this microliter plate was used only once.

Example 7

A commercial microtitration plate (VEB Polyplast Halberstadt, DDR) is activated as described in Example 2 and then measured in the dry state with a vertically measuring photometer (MTF, VED AdW of the DDR) against an unactivated microtitration plate. The measured absorbances were below 0.01 absorbance units in all cases.

Example 8

Polystyrene tubes (VEB Polyplast Halberstadt, DDR) are activated as described in Example 1 below 2 and subsequently to this purified human factor VIII at a concentration of 3.3 pg / ml in a 0.1 M phosphate buffer, pH 6.8 , bound. After removal of the unbound, excess factor VIII molecules by washing in a known manner, the enzyme immunoassay for the quantitative determination of factor VIII antigen (W. Schößler, M. Stepanauskas, C.Dittrich, H.Heine: Acta biol Med. germ. 41 [I982] 263, W. Schossler, M.Stepanauskas, C.Dittrich: Acta biol. med. germ., 41 [1932] 695) in the manner described. For this purpose, the tubes are mixed with an incubation mixture consisting of the plasma to be determined and an excess of rabbit anti-human factor VIII antibody and incubated at 37 ⁰ C for 6 h. After washing again, 200 μl of a conjugate consisting of a goat anti-rabbit IgG antibody and the enzyme alkaline phosphatase are added, and after several hours of incubation and subsequent washing, the enzyme activity is determined as described in Examples 4 and 6. After the enzyme immunoassay, the Polysty. Enröhrchen with 0.5 ml of 0.2 M HCI-glycine buffer, pH 2.8, for 30 minutes at room temperature, so that the bound immunoreactants are completely eliminated and the human factor VIII loaded tubes again in the enzyme immunoassay analog Example 6 can be used.

Example 9

Commercial polystyrene tubes are loaded with human factor VIII as described in Examples 1, 2 and 8 and subsequently used in the manner described for the determination of the Factor VIII antigen in the enzyme immunoassay. However, the detection of the bound antibody is carried out with a conjugate consisting of a sheep anti-rabbit IgG antibody and the enzyme horseradish peroxidase. After removal of unbound conjugate molecules by washing three times, the enzyme activity is monitored with 2,2'-azino-di- (3-ethyl) -benzthiazoline-sulfonate (6) (ABTS) (Boehringer-Mannheim, FRG) and H ₂ Oj and photometric Measurement determined at 418nm. After carrying out the enzyme immunoassay, the tubes were washed twice with 0.5 ml each of PBS buffer and subsequently treated with one of the following dissociation reagents for 1 h at room temperature: 1) PBS buffer containing 2 mM NaCl and 5% dioxane, 2) 1 mM KSCN, 3) 3m KSCN, 4) 4.5m MgCl ₂ , 5) 1m NaJ, 6) HCl-glycine buffer, pH 2.8, 7) HCI Glycine buffer, pH 2.2. By all these agents, the bound antigen /. Antibody complexes are cleaved, so that the human factor VIII loaded tubes can be reproducibly used in the enzyme immunoassay again.

Claims (3)

1. A process for activating polystyrene solid surfaces, characterized in that the polystyrene solid surfaces with organosilanes of the formula (XFU ₁ SiR ₄ -H (D. where X is an amino, carbonyl, carboxy, isocyano, diazo, isothiocyano, nitroso, sulfhydryl or halocarbonyl group, and R 'is an alkyl, alkylphenyl or phenyl group, while R is an alkoxy, phenoxy or halogen group, where η 'the value between 0-20 and η have the value between 1-3, or that the reaction with at least two organosilanes of the formula I in a mixture or successively in liquid or gaseous phase ErkJgt and that then optionally carried out a further treatment with homodor heterofunctional reagents. 2. The method according to claim!, Characterized in that the reaction of the polystyrene solid surfaces with compounds of formula I in organic solvents, solvent mixtures or mixtures of water and organic solvents. 3. The method according to claim 1, characterized in that the reaction of the polystyrene Festkörperoi 'in the gas phase takes place with an aerosol or at a pressure below atmospheric pressure.