WO1999039209A1 - Immunoassay and test kit for assaying fucosylised protein in a biological sample - Google Patents

Abstract

The invention relates to a sandwich immunoassay for determining the quantity of fucosylised protein (fuc-protein) in a biological sample and to corresponding reagent sets. A preferred version of the invention relates to an immunoassay for assaying fucosylised alpha -foetoprotein (AFP) which is significant in the early detection of hepatocellular carcinoma (HCC), a common form of cancer. Said immunoassay is characterized in that it uses a lectin selected from the group consisting of Ulex-europaeus agglutinin (UEA), Lotus-tetragonolobus agglutinin (LTA) and Anguilla-anguilla agglutinin (AAA).

Description

 Immunoassay and test kit for the determination of fucosylated protein in a biological sample

description

The invention relates to a sandwich immunoassay for determining the amount of fucosylated protein (Fuc protein) in a biological sample and associated reagent sets. In a preferred embodiment, the invention relates to an immunoassay for the determination of fucosylated α-fetoprotein (AFP), which is important in the early detection of hepatocellular carcinoma (HCC) as a common form of cancer.

The pathologically increased fucosylation of glycoproteins plays an important role in tumor diagnosis, since these glycosylation forums in the oligosaccharide side chain of

Glycoproteins are recognized as tumor-associated. For example, in Aoyagi, Q. et al. (1985) Biochim. Biophys. Acta ^ 3C_, 217-223 and Aoyagi, Y. et al. (1988) Cancer

_61, 769-774 or Wu, J.T. (1990) Ann. Clin. Lab. Be.

20, 98-105 described the fucosylation of the pathological AFP as specific for hepatocellular carcinoma. The determination of the amount of fucosylated protein can also be used when evaluating recombinant Glycoproteins produced, which are to be used as therapeutic agents, play a role, in particular in process control.

It is known to detect pathologically fucosylated proteins by means of affinity and immunoaffinity electrophoresis, mostly after blotting on antibody-coated membranes (Taketa K., J. Chromatogr. 569, 229-241, 1991). Affinity chromatography methods are also used. However, these methods are unsuitable for use in clinical practice.

To distinguish between fucosylated and non-fucosylated AFP, lectin-enzyme immunoassays with the lectins Concanavalin A (ConA), Lens-culinaris-Lectin A (LCA) and Phaseol us-vulgaris hemagglutinin E (PHA -E) (Yasufumi Suzuki et al., Ann. Clin. Biochem. 1990, 2T7, 121-128; Noriaki Kinoshita et al. (1989), Clinica Chi ica Acta 179, 143-152). However, it has been found that lectin enzyme immunoassays with the lectins mentioned do not meet the requirements regarding specificity and sensitivity that have to be made of a clinical test for the quantitative determination of fucosylated proteins. In the case of Lectine ConA and LCA, the results were also poorly reproducible. The object of the invention was therefore to provide a detection method for fucosylated proteins which is sufficiently sensitive, specific and reproducible and can be used in clinical practice without problems. The detection method should be easy to use and also suitable for routine examinations in the clinical area. In particular, it was the object of the invention to provide a detection method for fucosylated α-fetoprotein, a tumor marker in liver diagnosis.

The object of the invention is achieved as specified in the claims. The immunoassay according to the invention for determining the amount of fucosylated protein in a biological sample is characterized in that a) an immobilized anti-fuc protein antibody or an immobilized anti-fuc protein antibody fragment with the biological sample to form a fuc protein -Antibody complex is incubated, b) a lectin selected from the group Ulex-europaeus-agglutinin (UEA), lotus-tetragonolojbus-agglutinin (LTA) and Anguilla-anguilla-agglutinin (AAA) is added, which has appropriate labels to the amount to determine the fuc-protein-antibody complex and therefrom the amount of fucosylated protein in a manner known per se or b) an unlabeled lectin selected from the group mentioned is added and the detection of the fuc-protein-antibody complex by means of labeled anti-lectin antibodies against the lectins mentioned are carried out in a manner known per se. It has been shown that with the assay according to the invention and through the use of the selected lectins UEA, LTA and AAA a much better sensitivity and specificity is achieved than with the assays described in the literature, which use the marker lectins ConA, LCA and PHA-E.

In a preferred embodiment, UEA is used as lectin in the sandwich immunoassay according to the invention. Both polyclonal and monoclonal antibodies can be used as capture antibodies on the solid phase.

In one embodiment, the sandwich immunoassay is carried out in such a way that a lectin labeled with an acceptor selected from UEA, LTA and AAA is added to the immobilized anti-Fuc protein antibody and then with a labeled receptor that binds to the acceptor of the lectin , is incubated. In this case, the detection is carried out via the marker on the receptor. Haptens or low molecular weight ligands can be used as acceptors for labeling the lectin; biotin is preferably used. Accordingly, in a preferred embodiment, labeled avidin, streptavidin or their derivatives are used as the receptor. If haptens are used as acceptors, hapten-specific antibodies are used as receptors. According to the invention, enzymes, dyes, radioisotopes, metal colloids, chelators or spin markers can be used as receptor markers. An enzyme such as horseradish peroxidase (POD), alkaline phosphatase, β-galactosidase, urease or glucose oxidase is preferably used as the receptor marker and the amount of the fuc-protein-antibody complex is detected by a substrate reaction of this enzyme. In a particularly preferred embodiment, POD is used for labeling according to the invention and the substrate reaction is carried out by means of a chlorogenic substrate, for example using H0-tetramethylbenzidine. However, according to the invention it is also possible and familiar to the person skilled in the art that the substrate reactions for the most diverse enzymes can be detected by means of different chromogenic substrates, chemiluminescence or fluorescence.

In a particularly preferred embodiment, the immunoassay according to the invention is therefore carried out as an enzyme immunoassay. Alternatively, the receptor can also be used with a radioisotope (e.g. ^{1Z <:} 'J), with a fluorescence marker (e.g. FITC), a metal colloid (e.g. gold), a chelator (e.g. DTTA), with polynucleotides or with a spin marker (e.g. PROXYL) must be marked.

According to the invention, it is also possible for the lectin labeled with an acceptor and the receptor as the preformed complex for the antigen-antibody complex add so that a process step is omitted when carrying out the assay variant mentioned.

In a further embodiment of the invention, the immunoassay can also be carried out as a direct assay. In this case, the lectin according to the invention itself carries the markers mentioned. Here, too, the implementation as an enzyme immunoassay is preferred.

In a third variant, unlabeled lectin is used in the assay according to the invention and the detection is carried out using labeled anti-lectin antibodies. Both polyclonal and monoclonal antibodies against UEA, LTA or AAA or their fragments can be used. Here, too, the implementation as an enzyme immunoassay is preferred.

For the purposes of the invention, a biological sample is to be understood as any sample of a human or animal body fluid that can contain fucosylated proteins. For example, the blood can be plasma, serum, urine, tissue fluid, lymph, gastric juice, ascites or saliva. With the method according to the invention, fucosylated proteins such as, for example, .alpha.-fetoprotein, fucosylated, genetically engineered or pharmaceutical proteins or adhesion proteins (for example CD22) obtained from biological material can be determined. It has been shown that the assay according to the invention is particularly suitable for the differential diagnosis of liver diseases and that the determination of the amount of fucosylated α-fetoprotein (AFP) in the serum enables a reliable statement to be made about any hepatocellular carcinoma (HCC) present, since the Fucose-specific AFP content in HCC patients is significantly increased (see Fig. 1).

In a particularly preferred embodiment, the AFP assay according to the invention contains polyclonal antibodies or fragments thereof from mouse, rabbit or chicken as capture antibodies on the solid phase. Biotinylated UEA is added to the antigen-antibody complex and the detection of the Fuc-protein-antibody complex is carried out with an avidin, modified avidin or streptavidin-POD conjugate.

The technical solution according to the invention thus opens up the possibility of determining the total AFP content, for example by means of a conventional enzyme immunoassay, in the event of suspected liver tumor diseases and, in parallel, determining the content of fucosylated AFP (for example on a microtest plate). The structure of these two assays can, for example, be as shown below: Total AFP Assay:

1. Immobilized anti-AFP antibody or antibody fragment of species 1 (e.g. mouse, rabbit, chicken)

2. AFP standard dilution or diluted patient serum

3. Enzyme-labeled anti-AFP antibody or antibody fragment of species 2 (e.g. mouse, rabbit, chicken). Enzyme e.g. Horseradish peroxidase (POD) or alkaline phosphatase (AP)

4. Substrate reaction

Fucosylated AFP Assay:

1. Immobilized anti-AFP antibody or antibody fragment of species 1 (e.g. mouse, rabbit, chicken)

2. Diluted patient serum 3. Labeled lectin selected from UEA, LTA or AAA (e.g. biotinylated UEA)

4.Label-specific enzyme conjugate (e.g. avidin / streptavidin-POD conjugate or complex)

5. Substrate reaction

In addition to the determination method, the invention also relates to the associated reagent sets, as set out in the claims. Examples:

Example 1 a) Preparation of anti-human AFP-IgG-F (ab) ₂ ~ fragments

aa) Antiserum production

Rabbits are immunized with high purity umbilical cord blood human AFP. After coagulation, the antiserum is separated from the blood of the immunized animals by centrifugation.

ab) Isolation of the total IgG fraction

The serum is applied to an AFP-Sepharose. Unbound serum protein is washed out with TRIS-buffered saline (TBS). The anti-AFP-IgG is then eluted with a 0.2M glycine-HC1 buffer pH 2.5, neutralized with IM Tris-HCl pH 7.5, concentrated with Centricon 30 kD cartridges and against 50 mM acetate buffer pH 4.0; 0.5M NaCl, 0.02% NaN buffered.

ac) Antibody fragmentation using pepsin

An enzyme-IgG ratio of E: P = 1:50 is set and incubated for 4 hours at 37 ° C. The reaction is terminated with IM Tris-HCl pH 7.5. The anti-AFP-F (ab) 2 fragments are then isolated by immunoaffinity chromatography on an AFP column as described under ab), concentrated, dialyzed against TBS and stored at 4 ° C. 10

b) Immunoassay for total AFP determination

The wells of a microtest plate are coated with anti-human AFP-IgG (F (ab) 2 fragment, rabbit) by adsorption from a solution of 10 μg F (ab) 2 / ml in bicarbonate buffer pH 8.5-9 , 5 occupied. Non-specific protein adsorption is reduced by blocking the plastic surface with an inert protein solution (e.g. bovine serum albumin in phosphate-buffered physiological saline (PBS)). Either a standard dilution series of human AFP (concentration range 2 to 300 ng / ml) or a centrifuged, 1: 5 to 1:10 diluted sample of patient serum is placed in the wells coated in this way. Unbound material is washed out, then incubated with an appropriate dilution of anti-human AFP-IgG-POD conjugate. Unbound material is washed out, then the substrate reaction is started with H ^ O ^ tetramethylbenzidine and stopped after a defined time with sulfuric acid. The absorption is measured at 450 nm. The AFP content in the patient's serum is determined using the standard dilution series.

Example 2

Immunoassay according to the invention for Fuc-AFP determination

The wells of a microtest plate are coated with anti-human AFP-IgG (F (ab) 2 fragment, rabbit), as prepared in example la, by adsorption from a solution. 11

solution of 10 μg F (ab) 2 / ml in bicarbonate buffer pH 8.5-9.5. Non-specific protein adsorption is reduced by blocking the plastic surface with an inert protein solution (e.g. bovine serum albumin in phosphate-buffered physiological saline (PBS)). A centrifuged, 1: 5 to 1:10 diluted sample of patient serum is placed in the wells coated in this way. Unbound material is washed out, then biotinylated Ul ex-Europeus lectin (bio-UEA) is incubated with a suitable dilution. Unbound material is washed out, then acylavidin-biotinylated POD complex (AbP) is incubated with a suitable dilution. Unbound material is washed out, then the substrate reaction is started with H ^ 0-tetramethylbenzidine and stopped after a defined time with sulfuric acid. The absorption is measured at 450 nm.

Example 3

The results of Examples 1 and 2 for different subjects are shown (Fig. 2). It becomes clear that the total AFP content does not correlate with the content of UEA-reactive AFP, so that it is only with the test according to the invention that it can really be determined whether an increased amount of fucolyzed AFP is actually also present with increased AFP values is present. 12

Show it :

Fig. 1: Content of fucosylated AFP in patient serum samples control - healthy volunteers, SLE - systemic lupus erythromateus, AIH - autoimmune hepatitis, PBC - polynuclear cheap cirrhosis, HCC - hepatocellular carcinoma

Fig. 2: Comparison of the total and fucosylated AFP content in different HCC patient sera

Claims

13 Patent claims 1. Immunoassay for determining the amount of fucosylated protein (Fuc protein) in a biological sample, characterized in that a) an immobilized anti-Fuc protein antibody or an immobilized anti-Fuc protein antibody fragment with the biological sample Formation of a fuc-protein-antibody complex is incubated and b) a lectin selected from the group Ul ex europaeus agglutinin (UEA), lotus tetragonol obus agglutinin (LTA) and Anguilla anguilla agglutinin (AAA) is added, which has suitable markings in order to determine the amount of the fuc-protein-antibody complex and therefrom the amount of fucosylated protein in a manner known per se or b) an unlabeled lectin selected from the group Ulex-europaeus-agglutinin (UEA), lotus-tetragonol o-j us-agglutinin (LTA) and Anguilla-anguilla-agglutinin (AAA) are added and the detection of the Fuc-protein-antibody complex by means of labeled anti-lectin antibodies or their fragments against the lectins mentioned are carried out in a manner known per se. 14 2. Immunoassay according to claim 1, characterized in that b) a lectin labeled with an acceptor according to claim 1 is added, incubated with a labeled receptor, which binds to the acceptor of the lectin, and in a conventional manner by means of the marker on the receptor Amount of the Fuc-protein-antibody complex and from this the amount of fucosylated protein is determined. 3. Immunoassay according to claim 1, characterized in that the amount of fucosylated protein is determined directly by b) addition of a labeled lectin such that the amount of the Fuc-protein-antibody complex can be determined by means of the marker on the lectin. 4. Immunoassay according to one of claims 1 to 3, characterized in that the amount of fucosylated α-fetoprotein (AFP) is determined. 15 5. Immunoassay according to claim 2, characterized in that a lectin is used which is labeled with a hapten or a low molecular weight ligand as an acceptor. 6. Immunoassay according to claims 2 and 5, characterized in that the lectin is marked with biotin as the acceptor and avidin, streptavidin or their derivatives are used as receptors. 7. Immunoassay according to claims 2 and 5, characterized in that the lectin is marked with a hapten and a hapten-specific antibody is used as a receptor. 8. Immunoassay according to claims 2 and 5 to 7, characterized in that an enzyme, a dye, a radioisotope, a metal colloid, a chelator, as an receptor marker Nucleotide or a spin marker is used. 16 9. Immunoassay according to claim 3, characterized in that an enzyme, a dye, a radioisotope, a metal colloid, a chelator, a nucleotide or a spin marker is used as the lectin marker. 10. Immunoassay according to claim 1, characterized in that an enzyme, a dye, a radioisotope, a metal colloid, a chelator, a nucleotide or a spin marker is used as the marker of the anti-lectin antibody. 11. Test kit for determining the amount of fucosylated protein (Fuc protein) in a biological sample containing labeled or unlabeled lectin selected from the group [/ lex-europaeus-agglutinin (UEA), lotus-tet-ragonolobus-agglutinin (LTA) and Anguilla Anguilla Agglutinin (AAA) 12. Test kit according to claim 11, characterized in that it contains an anti-Fuc protein antibody or an anti-Fuc protein antibody fragment. 17 13. Test kit according to claim 11 or 12, characterized in that it contains a lectin labeled with an acceptor and a labeled receptor which binds to the acceptor of the lectin, which may also already be present as a preformed complex. 14. Test kit according to claim 13, characterized in that it contains biotinylated UEA as lectin labeled with an acceptor. 15. Test kit according to claim 11 or 12, characterized in that it contains unlabeled lectin and labeled anti-lectin antibodies against this lectin. 16. Test kit according to one of claims 11 to 15 for determining the amount of fucosylated α-fetoprotein (AFP).