CA2055117A1

CA2055117A1 - Immunosensory transducer

Info

Publication number: CA2055117A1
Application number: CA002055117A
Authority: CA
Inventors: Richard Barner; Walter Huber; Josef Hubscher; Daniel Schlatter
Original assignee: Richard Barner; Walter Huber; Josef Hubscher; Daniel Schlatter; F. Hoffmann-La Roche Ag
Current assignee: F Hoffmann La Roche AG
Priority date: 1990-11-14
Filing date: 1991-11-07
Publication date: 1992-05-15
Also published as: EP0485874A2; EP0485874A3; EP0485874B1; JPH04268455A; DE59106527D1; JP3280404B2

Abstract

Ref. 4090/2 16 Abstract The immunosensory transducer for surface plasmoresonance has a gold carrier, a two-dimensional matrix in the form of a monomolecular organic intermediate layer on this surface and a ligand or antiligand layer immobilized on this. The transducer in accordance with the invention is considerably simpler to produce than known transducers having a three-dimensional matrix. For the production, a monomolecular layer of alkyl derivatives with sulphur-containing groups is applied to the gold surface by spontaneous chemisorption and subsequently a ligand or antiligand suitable for the desired immune detection is immobilized on this layer.

Description

2~5Sl~L7 The invention is concerned with an immunosensory transducer and with a method for its production.

The principle of an immunoassay is based on the selective 5 recognition of a ligand, e.g. an antigen (Ag) or a hapten (Hap), by an antiligand, e.g. an antibody (Ab). The immunoassays which are usually used in practice are so-called competitive assays or sandwich assays. Both procedures work with labelled ligands or antiligands. The nature of the so-called label differentiates o between a radioimmunonassay (RIA), enzyme immunoassay (EIA), a fluorescence immunoassay (FIA), etc.

Thus, for example, in the sandwich assay a first antibody against an antigen is immobilized on a solid phase (tube in the so-15 called "coated tube" technique; bead in the so-called "coated bead"
technique). Sample antigen and a labelled second antibody are incubated with each other and with the solid phase. In this way, binding sites on the surface, which are pre-formed by the immobilized first antibody, are filled by the complex between 20 antigen and second antibody. The determination of the concen-tration of sample antigen is subsequently effected via the determination of the label concentration at the surface, i.e. in the EIA procedure by incubation with the enzyme substrate, in the FIA procedure by measuring the fluorescence properties and in 2s the RIA procedure by measuring the radioactive radiation. All of the procedures innumerated above require time-consuming washing and incubation steps.

In the more recent literature there have accordingly been 3~ proposed to an increasing extent measurement techniques which would allow a detection of the Ag-Ab complex without the aid of a label (radioisotope, enzyme, fluorescing dye). The procedures are brought together under the term immunosensing. The immuno-sensors generally feature an immunologically-recognizing element Bu/3 .10.91 - lo- 2~5~1~7 of the dropwise addition the reaction mixture was stirred at room temp. for a further 2 h. Then, it was taken up with tert.butyl methyl ether and water, the ether phase was washed three times with water, dried over sodium sulphate and concentrated on a 5 rotary evaporator. The residue was taken up with toluene and the solution was filtered through silica gel. After removal of the toluene 11 -bromo- 1 -undecyltrimethylsilyl ether was obtained in almost quantitive yield. TLC (toluene) Rf = 0.8.

0.33 g (15 mmol) of metallic sodium in toluene was heated to 120 while stirring for fine distribution. After cooling to room temp. 4.85 g (15 mmol) of 11-bromo-undecyltrimethylsilyl ether were added dropwise while stirring and the mixture was again heated to 120, whereby sodium bromide gradually precipitated ts and the solution became blue in colour. After 1 h lithium tetrachlorocuprate was added as the coupling catalyst and the mixture was heated at reflux for a further 2 h. After cooling to room temp. the mixture was treated with 50 ml of tert.butyl methyl ether and traces of sodium were destroyed by the 20 dropwise addition of water. The organic phase was filtered on silica gel and concentrated on a rotary evaporator. (2.8 g = 77%) TLC (toluene) Rf = 0.75 TLC (toluene:EtOAc = 2:1) Rf = 0.9.

The 1,22-di-(trimethylsilyloxy)docosane obtained in the 2s preceding reaction was dissolved in 30 ml of tert.butyl methyl ether and the solution was treated with 20 ml of lN HCI while stirring. Crystalline 1,22-dihydroxydocosane separated rapidly.
After stirring for 1 h the precipitated material was separated and dried in a high vacuum. Yield almost quantitive (about 1.9 g) TLC
30 (toluene:EtOAc = 2:1) Rf = 0.25.

230 mg (0.67 mmol) of 1,22-dihydroxydocosane in 5 ml of pyridine were treated dropwise while stirring with 127 mg (0.67 mmol) of tosyl chloride in 1 ml of pyridine and the mixture 3s was subsequently stirred at room temp. for 2.5 h. Then, the reaction mixture was transferred into a separating funnel with 50 ml of toluene and shaken with 30 ml of water. After separating the aqueous phase the organic phase was washed with 3 2~5~
i) After the adsorption the proteins are present in random ~ orientation on the surface. Thereby, to some extent, the active domains (epitopes, antigen binding sites) of individual proteins are masked for the complex formation.
s ii) Proteins are denatured on such a surface by a strong hydrophobic interaction. Thereby, the selectivity of the surface is no longer guaranteed for only one analyte.

I o iii) An incomplete covering of the surface by adsorption results in accompanying proteins also adsorbing on the surface because of the adsorptive power of the surface. The tesc results are falsified considerably by this unspecific absorption not only in the case of an indirect detection, but also in the case of a direct I s detection.

While procedures for the suitable immobilization of antiligands on synthetic surfaces or on glass surfaces have become sufficiently known, for example from affinity chromatography, the 20 defined immobilization of protein molecules on a gold surface has only recently become known for the first time (see WO
90/05303). In this Patent Publication the necessity of a 3-dimensional matrix (preferably a polysaccharide matrix e.g.
consisting of dextran) is especially emphasized in order to achieve 2s a high sensitivity and an adequate dynamic range. The process relates to the immobilization of high-molecular polysaccharides via thioundecane derivatives, whereby the latter are responsible for the covalent anchoring of the polysaccharide to the gold surface. The production of a three-dimensional matrix is, 30 however, so expensive that the realization of a sensor which is disposable after being used once is put into question.

The object of the present invention is to provide an immunosensory transducer having high activity and selectivity 3 s with respect to the binding of analytes to a matrix of recognizing molecules (antibody, antigen).

4 Z~5~:~17 It has surprisingly now been found that a three-dimensional matrix is not necessary to achieve a high sensitivity and a sufficient dynamic range~ The relinquishment of such a three-dimensional, matrix simplifies the immobilization procedure s considerably.

The problem is solved in accordance with the invention by a transducer which comprises a gold carrier, a monomolecular organic intermediate layer applied to its surface and a ligand or o antiligand layer immobilized on this.

The method in accordance with the invention is characterized by applying a monomolecular layer of alkyl derivatives having sulphur-containing groups to a surface of a 15 metallic carrier by spontaneous chemisorption and subsequently immobilizing on this layer a ligand or antiligand suitable for the desired immune detection.

It has thus surprisingly been found that with this method, 20 proteins for example, can be immobilized on metal surfaces in a suitable manner. The surfaces which thereby result have a demonstrably higher activity with respect to the formation of the immune complex on the surface than the surfaces hitherto used in connection with the aforementioned measurement procedures 2s such as, for example, spontaneous adsorption or the application of a thin polymer layer on the surface by silanization or plasma polymerization and subsequent immobilization according to the known affinity chromatography procedure.

When proteins are to be immobilized on a surface in a defined and oriented manner, then this surface must be pre-prepared in such a manner that it has a bifunctionality; i.e. the surface must be provided with functional groups which prevent a spontaneous adsorption of the proteins and with functional groups 3 5 which give the covalent bonding with the groups provided therefor on the protein.

2C'5~117 According to a preferred embodiment of the method in accordance with the invention the gold surfaces are provided in a first step with a thin organic supplementary layer. The application of these organic supplementary layers is based on the s known spontaneous chemisorption of alkyl derivatives with sulphur-containing groups (thiols, sulphides, disulphides) from solutions of these compounds onto gold with the formation of monolayers [Langmuir 5, 723-727 (1989); Langmuir 4, 365-385 (1988); J.Phys.Chem 91, 6663 (1987)].
The chemisorption is effected in such a manner that the ~-containing group is anchored to the surface of the gold and the alkane chain extends approximately vertical to this surface. The layer thicknesses which can be produced by this procedure can be 5 varied in a range of about 1.3-3 nm by using alkane chains (Cl1-C20) of different lengths.

The formation of monolayers with these compounds is also effected when these compounds are provided with functional 20 groups, e.g. - COOH, -NH2, -OH etc. in the cl)-position to the S-containing group. With these reagents accordingly a gold surface is provided with a thin organic layer, and these organic supple-mentary layers are provided with a boundary surface (organic layer-surrounding medium) with densely packed functional 2s groups. Thus, the composition of the surface can be controlled with respect to the concentration of different functional groups via the concentration ratio of the corresponding reagents in the solution used for the adsorption. Surfaces for immobilization having the required bifunctionality can be produced by this 30 procedure in a surprisingly simple manner.

It has moreover been found that a spontaneous adsorption of proteins is suppressed surprisingly strongly when the S-containing reagents for the synthesis of the organic 3 s supplementary layers contain as the second functional group a hydroxyl, polyhydroxyl, an ethylene glycol, and oligoethylene glycol or a monosaccharide as a structural element.

- 6 - 2CC~ 7 Moreover, the hydroxyl groups present in these terminal groups can be used in a manner known per se, e.g. by cyanogen bromide activation, cyanogen chloride activation, tosyl chloride activation, epichlorohydrin activation or carbonyldiimidazole s activation (Meth. in Enzymology 135, 30-65 (1987)) for the covalent binding of proteins. Although to some extent very aggressive reagents must be used in these procedures, the once-formed monolayers surprisingly remain intact in these activation steps.
An alternative preferred embodiment of the method in accordance with the invention comprises bringing such hydroxyl-containing derivatives, which prevent the spontaneous adsorption of the proteins during an immobilization, on to the surface 15 together with derivatives which have special groups for the covalent binding of proteins. It is thereby possible to adjust the required bifunctionality of a surface to a freely chosen relative amount of the functional groups. This is not possible to a comparable extent in the direct activation of hydroxyl-rich 20 surfaces. The reagents which are available for the covalent binding via functional groups are divided into two groups. The first group contains derivatives with functional groups which can be used for the covalent binding only after an additional activation step. This activation is effected after the chemisorption 2s of the derivative on the gold surface. They are derivatives which contain, in addition to the S-containing structural elements, a carboxylic acid function (activation by ethyl chloroformate [J.Am.Chem.Soc. 74, 676-678 (1952)] or by reaction with carbodiimide, hydroxysuccinimide or thionyl chloride [Meth. in 30 Enzymology 135, 30-65 (1987)], an amino group (activation by reaction with succinic anhydride and subsequent activation of the carboxylic acid [ibid.]), an aromatic amino or nitro group (activation by conversion into the diazonium salt [ibid.]~ or a phenylazido group (photochemical activation [Biochemistry 16, 3s 5650-5654 (1977); Biochemistry 17, 1403-1408 (1978)]). Also here it has been found that the to some extent drastic methods for activation do not destroy these organic supplementary layers on the surface of the gold.

- 7 - Z~i5117 The second group comprises S-containing alkyl derivatives which do not require an activation step after the application to the surface of the gold. These reagents also arrange themselves on 5 the metal surface in the form of monolayers. They have functional groups which can react specifically with a group on the protein. Examples of these groups are maleimide (reaction with -SH groups of a Fab' fragment, p-benzoquinone, cyanuric chloride, epoxide, sulphonic acid derivatives, carboxylic acid azide (reaction o with NH2 function at the protein) or hydrazide (reaction with aldehyde function at oxidized carbohydrate residues). However, these compounds can only be produced preparatively wh~n the S-containing grouping is not thiol. The advantage of the compounds from the second group accordingly exists in that the surface of the 5 gold can be provided directly with a group which is reactive for the covalent protein immobilization.

The immobilization procedure on gold surfaces for molecules which can be used in immunodetection (e.g. Ab, Ag, Fab' 20 fragments, haptens) is based on the use of compounds of the general formula R I -(CH2)n-R2 2s wherein Rl is a group S-X and X = H or -(CH2)m-R2 whereby m and p can be = n and n = 1-20 or wherein Rl represents a cyclic disulphide group of the formula ~ ~
(CH2)q~S~S~CH2-CH~

with q = 1 or 2 and n has the above significance and R2 is a functional group of formula Y, 3s which, if desired, can be transformed into a group Z which can be coupled with protein or in which R2 directly has the above significance of Z whereby for R2 = Z Rl = SH (i.e. the compound is - 8 - ~ 7 produced as the thiolate and Y is transformed into Z after the production, or the compound contains Z and Rl = -S- or-S-S-).

Typical examples for Y are:
s -OH, -CHOHCEI2CHOHCH20H, -CHOHCH20H, -(OCH2CH2)mOH (m = 1-5), monosaccharide, 1 5 -COOH, -NH2, ~ ~H2 ~3 NO2 ~ N3 --O~ 3 -~'~ 3 -S ~ N3 Typical examples of Z are:

a) Activation of alcohols o o o-c~ ~ ~JY ~ s s--~o~

O'~ N~CI -O-CH~ ~5~

~O,S ~/I~r ' ~ >~ ~ ~

Cl-~3 N l=N

9 2C'~i5~ 7 b ) Activation of carboxylic acids O EIN--R' ~ ~S-S~.
-co-a CO~ NH2 -CO-N3 c) Activation of amines - N~ S S--~ ~;H_~'=C=O

Examples Example 1: 254 mg (0.5 mmol) of 22-tosyloxy-1-docosanol were heated at reflux for 2 h with 200 mg (1.75 mmol) of potassium thioacetate in 20 ml of ethanol. Then, 2 ml of lN
2s NaOH were added at room temp. and the mixture was stirred overnight. After acidification with lN HCI the reaction mixture was concentrated on a rotary evaporator and the residue was filtered on silica gel with toluene:EtOAc = 2:1, whereby 165 mg of 22-hydroxy-1-docosanethiol were obtained TLC (toluene:EtOAc =
30 2:1) RF value of product and educt are very similar, but the educt shows fluorescence quenching (aromatic).

The 22-tosyloxy-1-docosanol used as the starting material -~ was prepared as follows:
A solution of 25.1 g (0.1 mol) of 11-bromoundecanol in 60 ml of dry pyridine was treated dropwise while stirring with 20 g (0.12 mol) of bis-trimethylsilylacetamide. After completion - 10- 2C'~S~L7 of the dropwise addition the reaction mixture was stirred at room temp. for a further 2 h. Then, it was taken up with tert.butyl methyl ether and water, the ether phase was washed three times with water, dried over sodium sulphate and concentrated on a s rotary evaporator. The residue was taken up with toluene and the solution was filtered through silica gel. After removal of the toluene 11-bromo-1-undecyltrimethylsilyl ether was obtained in almost quantitive yield. TLC (toluene) Rf = 0.8.

0.33 g (15 mmol) of metallic sodium in toluene was heated to 120 while stirring for fine distribution. After cooling to room temp. 4.85 g (15 mmol) of ll-bromo-undecyltrimethylsilyl ether were added dropwise while stirring and the mixture was again heated to 120, whereby sodium bromide gradually precipitated s and the solution became blue in colour. After 1 h lithium tetrachlorocuprate was added as the coupling catalyst and the mixture was heated at reflux for a further 2 h. After cooling to room temp. the mixture was treated with 50 ml of tert.butyl methyl ether and traces of sodium were destroyed by the 20 dropwise addition of water. The organic phase was filtered on silica gel and concentrated on a rotary evaporator. (2.8 g = 77%) TLC (toluene) Rf = 0.75 TLC (toluene:EtOAc = 2:1) Rf = 0.9.

The 1,22-di-(trimethylsilyloxy)docosane obtained in the 2s preceding reaction was dissolved in 30 ml of tert.butyl methyl ether and the solution was treated with 20 ml of lN HCl while stirring. Crystalline 1,22-dihydroxydocosane separated rapidly.
After stirring for 1 h the precipitated material was separated and dried in a high vacuum. Yield almost quantitive (about 1.9 g) TLC
30 (toluene:EtOAc = 2:1) Rf = 0.25.

230 mg (0.67 mmol) of 1,22-dihydroxydocosane in 5 ml of pyridine were treated dropwise while stirring with 127 mg (0.67 mmol) of tosyl chloride in 1 ml of pyridine and the mixture 3s was subsequently stirred at room temp. for 2.5 h. Then, the reaction mixture was transferred into a separating funnel with 50 ml of toluene and shaken with 30 ml of water. After separating the aqueous phase the organic phase was washed with Z~-~5~17 2N sulphuric acid (removal of pyridine), subsequently with hydrogen carbonate solution and, after drying over sodium sulphate, evaporated on a rotary evaporator, whereby a white powder was obtained. Unreacted diol and traces of ditosylate 5 were removed by chromatography on silica gel with toluene:EtOAc = 2:1. 74 mg of pure 22-tosyloxy-1-docosanol were obtained.
TLC (toluene:EtOAc = 2:1) Rf = 0.55.

Example 2: 50 mg (0.1 mmol) of 22-tosyloxy-1-docosanol 0 were heated at reflux for 2 h together with 2.5 ml (0.05 mmol) of a 0.02M ethanolic solution of sodium sulphide. Then, the mixture was taken up with water/chloroform, the organic phase was separated and concentrated on a rotary evaporator. After chromatography of the residue on silica gel with toluene:EtOAc =
5 2:1 there were obtained 10 mg of pure di-(22-hydroxydocosyl)-sulphide.

Example 3: A solution of 230 mg (0.6 mmol) of 4-(3'-hydroxy-bromotridecanyl)-2,2-dimethyl-1,3-dioxolane and 20 140 mg (1.2 mmol) of potassium thioacetate in 4 ml of ethanol was heated at reflux for 2 h. Then, the mixture was concentrated on a rotary evaporator and the residue was chromatographed on silica gel with toluene:EtOAc = 2:1 and yielded 194 mg of 4-(3'-hydroxy- 13' -thioacetoxytridecanyl)-2,2-dimethyl - 1,3 -dioxolane.
2s After dissolving 4-(3'-hydroxy- 13'-thioacetoxytridecanyl)-2,2-dimethyl-1,3-dioxolane in 5 ml of methanol 0.5 ml of lN KOH
were added and the mixture was stirred at room temp. overnight (saponification of thioacetate), the solution was subsequently 30 stirred with 1.5 ml of ion exchang-resin Dowex 50W X8 (H+) and heated to 45 in order to cleave the acetonide (trans-acetatalization). After removing the methanol on a rotary evaporator the residue was chromatographed on silica gel. There were obtained 90 mg (51%) of 11,14,15-trihydroxy-1-35 pentadecanethiol. TLC (RP 8; 3% chloroform/ethanol) Rf = 0.4.

The 4-(3'-hydroxy-bromotridecanyl)-2,2-dimethyl-1,3-dioxolane used as the starting material was prepared as follows:

- 12- 2C'S~ .7 A solution of 2.1 g (10 mmol) of 4-bromoethyl-2,2-dimethyl-1,3-dioxolane in 25 ml of ether was converted into the Grignard reagent in the usual manner by reaction with magnesium. The ether solution obtained was cooled to 0 and treated dropwise 5 with a solution of 1 g (6.25 mmol) of 11-bromo-1-undecanal in 5 ml of ether while stirring. After stirring at room temp.
overnight sodium bicarbonate solution was added and the reaction product was extracted with ether. The organic phase was dried over sodium sulphate and concentrated on a rotary evaporator, o the residue was chromatographed on silica gel with toluene:EtOAc = 2:1 and 1.7 g (45%) of 4-(3'-hydroxy-bromotridecanyl)-2,2-dimethyl- 1,3-dioxolane were obtained.

Example 4: p-Azidobenzoyl chloride was prepared from 0.625 g 1 s (4 mmol) of the corresponding acid in thionyl chloride in analogy to the literature. The crude acid chloride obtained after concen-tration was added to a suspension of 0.994 g (2 mmol) of di(11-thioundecyl)disulphide disodium salt and the mixture was stirred at room temp. overnight. Then, the mixture was concentrated at 20 70 in a waterjet vacuum and the residue was taken up in toluene/conc. HCI, filtered over silica gel and eluted with toluene:EtOAc = 2:1. The eluate, which contained the pure fraction, was concentrated and gave 1.15 g (79%) of di(ll-(p-azido- benzoylmercapto)-undecyl)disulphide (Rf value = 0.1) 25 which was characterized by NMR and elementary analysis.

The di(11-thioundecyl)disulphide disodium salt used as the starting material was prepared as follows: A solution of 6.56 g (20 mmol) of 1,11-dibromoundecane in 200 ml of ethanol was 30 treated with 20.8 g (200 mmol) of potassium thioacetate. The mixture was refluxed for 16 h. The resulting solution was concentrated in a waterjet vacuum, subsequently taken up with toluene, filtered over silica gel and eluted with toluene. The eluate was concentrated and gave 6.32 g (99%) of l,11-diacetyl-3s mercaptoundecane as a yellowish oil which was characterized byMS.

- 13- 2~5~.7 3.185 g (10 mmol) of I,11 -diacetylmercaptoundecane were dissolved in 100 ml of ethanol (a solution initially resulted upon stirring and then crystallized after I h). The suspension was subsequently stirred at room temp. for 16 h. Then, it was 5 filtered, the residue was washed with ethanol and dried. There were obtained 0.88 g (33%) of 1,11-undecandithiol disodium salt of melting point = 61-62 having the correct elementary analysis.
The filtrate was treated with 200 ml of water and subsequently filtered. The residue was washed with water and dried and gave lo 1.36 g (54%) of di(11-thioundecyl)disulphide disodium salt of melting point = 292-294 which was characterized by MS and elementary analysis.

Example 5: 100mg of 11-phthalimildoundecyl thioacetate 5 were refluxed in 5 ml of hydrazine for 3 d. Then, the solution was concentrated and dissolved with boiling hydrochloric acid (conc.) and again conc~ntrated. The white crystalline crude product was taken up in methanol and chromatographed on silanized silica gel. The thus-obtained 11-aminoundecylthiol was 20 characterized as the hydrochloride by MS and NMR. Elementary analysis indicated that the product consisted of 19 mol% of the desired compound and 81 mol% of hydrazinium monohydro-chloride.

The 11-phthalimidoundecyl thioacetate used as the starting material was prepared as follows:

A solution of 12.56 g (50 mmol) of 11-bromoundecanol in 100 ml of DMF was treated with 18.52 g (100 mmol) of 30 potassium phthalimide and boiled at reflux for 161h. Then, the mixture was concentrated in a waterjet vacuum, chromato-graphed with toluene:EtOAc = 2:1 and gave 15.6g (98%) of 11-phthalimino-undecanol of melting point = 86-87, which was controlled by NMR. HPLC (30% MeCN; RP18 125-4) tR = 3.5 min.
A solution of 4.76 g (15 mmol) of 11-phthalimidoundecanol and 4.34 g (18 mmol) of tosyl chloride in 50 ml of pyridine was stirred at room temp. for 2 h. Then, the mixture was stirred with - 14- 2C`~i5~:17 saturated sodium bicarbonate solution for 1 h and subsequently extracted with methylene chloride. The organic phases were washed with water, then with lN hydrochloric acid and again with water. After drying over sodium sulphate and concentration 5 there were obtained 5.9 g (85%) of 1 l-phthalimido-undecyl tosylate, which was controlled by NMR and HPLC. TLC (toluene) Rf = 0.08.

3 g (6.36 mmol) of 11-phthalimidoundecanyl tosylate were o dissolved in 150 ml of ethanol and boiled with 1.45 g (12.7 mmol) of potassium thioacetate for 16 h. Then, the mixture was concentrated and subsequently taken up with toluene. The suspension was chromatographed on silica gel. The high vacuum-dried 1 l-pthalimidoundecyl thioacetate was 5 characterized by NMR. TLC (toluene) Rf = 0.14.

Example 6: Immobilization of IgG on the gold surface of a SPR sensor via aldehyde functions on sugar residues.

For this immobilization procedure, there are produced on the sugar residues of the IgG molecules by oxidation free aldehyde functions which then react with hydrazide functions on the gold surface.

2s For the production of a hydrazide-containing organic supplementary layer (30% hydrazide, 50% hydroxyl functions) on a gold surface suitable for the immobilization the gold surface is brought into contact for 12 h with a methanolic solution of mercaptoundecanoic acid (lx10-3 mol/l) and mercaptoundecanol 30 (lx10-3 mol/l). A monomolecular layer forms spontaneously during this treatment. The carboxylic acid functions present in this monomolecular layer are activated with methyl chloro-formate and transformed into acid hydrazide functions. The activation is effected by immersing the surface in a solution of 3s methyl chloroformate (5% v/v) in methylene chloride and pyridine (4% v/v) under an inert gas atmosphere for 1 h. The conversion of the activated carboxylic acid functions to hydrazides is effected by reacting the surface with a solution of hydrazine - 1 5 - 2C's~ ~.7 (about 1% v/v) in methylene chloride. This surface can then be reacted with at~tibodies which are modified for this immobilization procedure.

s The modification of the IgG molecules is effected in a manner known per se by treating the proteins with sodium periodate. For this purpose, the antibodies are dissolved in a solution of sodium periodate (Q.SM) in acetate buffer (pH 5.5;
O.lM) (20 mg/ml) and reacted for 20 minutes.
For the immobilization of the thus-modified antibodies, this reaction solution is brought into contact with the above hydrazide surface for 1 h.

Immobilization of Fab' fragments on the gold surface of a SPR sensor via the SH functions of free cysteine.

For the immobilization, the Fab' fragments are produced from Fab' fragments of antibodies in a manner known per se by 20 oxidation of the disulphide bridges. The Fab' fragments are obtained in a known manner by enzymatically cleaving the antibodies .

For the production of the supplementary layer on gold 2s surfaces suitable for the immobilization of the Fab' fragments, the gold surface is brought into contact for 48 hours with a methanolic solution of ll-aminoundecanamine (lx10-3 mol/l). In order to introduce SH-specific, reactive groups on the surface, the amino groups are reacted with N-succinimidyl-3-(2-pyridinyl)-30 dithiopropionate (Fluka) (lx10-3 mol/l in isobutanol). This surface can then be reacted with Fab' fragments of an antibody prepared for the immobilization.

Claims

1. An immunosensory transducer, characterized by a gold carrier, a monomolecular organic intermediate layer arranged on its surface and a ligand or antiligand layer immobilized on this.

2. An immunosensory transducer according to claim 1, characterized in that the intermediate layer consists of alkyl derivatives with sulphur-containing groups.

3. An immunosensory transducer according to claim 2, characterized in that the alkyl derivatives carry a second functional group in the .omega.-position to the sulphur-containing group.

4. An immunosensory transducer according to claim 2 and 3, characterized in that the second functional group is a hydroxyl or a hydroxyl-containing residue polyalcohol or ethylene glycol or a mononsaccharide.

5. An immunosensory transducer according to either of claims 2 and 3, characterized in that the second functional group contains a residue such as hydroxyl, carboxyl, aminophenyl, nitro-phenyl or phenylazido which can be activated for the protein immobilization.

6. An immunosensory transducer according to either of claims 2 and 3, characterized in that the second functional group is a residue such as cyanate, oxycarbonylimidazole, 2-oxyquinone, 2-oxymethylenequinone, -oxy(dichlorotriazine), 1-oxy-2,3-epoxypropane, (2-oxyethyl)-vinylsulphone, (2,2,2-trifluoroethyl)-sulphonyloxy, (p-tolyl)-sulphonyloxy, p-(oxybenzolyl)-nitrene, p-mercaptobenzoyl)-nitrene, p-(oxybenzyl)-diazonium chloride, pyridyldithiopropionic acid esters, mixed anhydride, isourea ester.
hydroxysuccinimide ester, acid chloride, acid hydrazide, acid azide, 2-acylaminoethyl pyridyl disulphide, maleimide, p-(aminobenzoyl)-nitrene, pyridyldithio-propionamide or an isocyanate and can be used without additional activation for the immobilization of proteins.

7. A method for the production of a transducer according to any one of the preceding claims, characterized by applying a monomolecular layer of alkyl derivatives with sulphur-containing groups to a surface of a gold carrier by spontaneous chemisorption and subsequently immobilizing on this layer a ligand or antiligand suitable for the desired immuno detection.

8. A method for the production of a transducer according to claim 7, characterized in that a monomolecular layer of alkyl derivatives with a sulphur-containing group and a second functional group is applied to the metallic (gold) surface of a transducer.

9. A method for the production of a transducer according to either of claims 7 and 8, characterized by applying to the gold surface of a transducer a monomolecular layer of a mixture of alkyl derivatives with a sulphur-containing group and different second functional groups according to any one of claims 3-6.