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

Immunoassay and test kit for assaying fucosylised protein in a biological sample Download PDF

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Publication number
WO1999039209A1
WO1999039209A1 PCT/EP1999/000639 EP9900639W WO9939209A1 WO 1999039209 A1 WO1999039209 A1 WO 1999039209A1 EP 9900639 W EP9900639 W EP 9900639W WO 9939209 A1 WO9939209 A1 WO 9939209A1
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Prior art keywords
lectin
protein
fuc
agglutinin
labeled
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PCT/EP1999/000639
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German (de)
French (fr)
Inventor
Martin Holtzhauer
Sergej Ovodov
Alexander Knoll
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Biogenes Gmbh
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Priority to EP99904839A priority Critical patent/EP1053476A1/en
Priority to JP2000529612A priority patent/JP2002502037A/en
Publication of WO1999039209A1 publication Critical patent/WO1999039209A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins

Definitions

  • the invention relates to a sandwich immunoassay for determining the amount of fucosylated protein (Fuc protein) in a biological sample and associated reagent sets.
  • the invention relates to an immunoassay for the determination of fucosylated ⁇ -fetoprotein (AFP), which is important in the early detection of hepatocellular carcinoma (HCC) as a common form of cancer.
  • AFP fucosylated ⁇ -fetoprotein
  • 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
  • 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.
  • ConA Concanavalin A
  • LCA Lens-culinaris-Lectin A
  • PHA -E Phaseol us-vulgaris hemagglutinin E
  • 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.
  • 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 lectin selected from the group Ulex-europa
  • 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.
  • 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.
  • 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.
  • enzymes, dyes, radioisotopes, metal colloids, chelators or spin markers can be used as receptor markers.
  • An enzyme such as horseradish peroxidase (POD), alkaline phosphatase, ⁇ -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.
  • 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.
  • the substrate reactions for the most diverse enzymes can be detected by means of different chromogenic substrates, chemiluminescence or fluorescence.
  • the immunoassay according to the invention is therefore carried out as an enzyme immunoassay.
  • 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.
  • 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.
  • the immunoassay can also be carried out as a direct assay.
  • the lectin according to the invention itself carries the markers mentioned.
  • the implementation as an enzyme immunoassay is preferred.
  • unlabeled lectin is used in the assay according to the invention and the detection is carried out using labeled anti-lectin antibodies.
  • labeled anti-lectin antibodies Both polyclonal and monoclonal antibodies against UEA, LTA or AAA or their fragments can be used.
  • the implementation as an enzyme immunoassay is preferred.
  • a biological sample is to be understood as any sample of a human or animal body fluid that can contain fucosylated proteins.
  • the blood can be plasma, serum, urine, tissue fluid, lymph, gastric juice, ascites or saliva.
  • 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.
  • 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 ⁇ -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).
  • HCC hepatocellular carcinoma
  • 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:
  • Immobilized anti-AFP antibody or antibody fragment of species 1 e.g. mouse, rabbit, chicken
  • 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)
  • Immobilized anti-AFP antibody or antibody fragment of species 1 e.g. mouse, rabbit, chicken
  • lectin selected from UEA, LTA or AAA (e.g. biotinylated UEA)
  • the invention also relates to the associated reagent sets, as set out in the claims. Examples:
  • 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.
  • the serum is applied to an AFP-Sepharose. Unbound serum protein is washed out with TRIS-buffered saline (TBS).
  • TRIS-buffered saline TRIS-buffered saline
  • 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.
  • 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 ⁇ g 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)).
  • PBS phosphate-buffered physiological saline
  • 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.
  • 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.
  • F (ab) 2 fragment, rabbit anti-human AFP-IgG
  • 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

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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 α-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 und Testkit zur Bestimmung von fucosyliertem Protein in einer biologischen Probe Immunoassay and test kit for the determination of fucosylated protein in a biological sample
Beschreibungdescription
Die Erfindung betrifft einen Sandwich-Immunoassay zur Bestimmung der Menge an fucosyliertem Protein (Fuc- Protein) in einer biologischen Probe und dazugehörige Reagentiensätze. In einer bevorzugten Ausführungsform betrifft die Erfindung einen Immunoassay zur Bestimmung von fucosyliertem α-Fetoprotein (AFP) , das bei der Früherkennung des hepatocellulären Carcinoms (HCC) als einer häufigen Krebsform von Bedeutung ist.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.
Die pathologisch erhöhte Fucosylierung von Glycoproteinen spielt in der Tumordiagnostik eine wichtige Rolle, da zunehmend diese Glycosylierungs- for en in der Oligosaccharid-Seitenkette vonThe pathologically increased fucosylation of glycoproteins plays an important role in tumor diagnosis, since these glycosylation forums in the oligosaccharide side chain of
Glycoproteinen als tumorassoziiert erkannt werden. So wird z.B. in Aoyagi, Q. et al. (1985) Biochim. Biophys. Acta ^3C_, 217-223 und Aoyagi, Y. et al . (1988) CancerGlycoproteins 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 oder Wu, J.T. (1990) Ann. Clin. Lab. Sei._61, 769-774 or Wu, J.T. (1990) Ann. Clin. Lab. Be.
20, 98-105 die Fucosylierung des pathologischen AFP als spezifisch für ein hepatocelluläres Carcinom beschrieben. Die Bestimmung der Menge an fucosyliertem Protein kann daneben auch bei der Beurteilung von rekombinant hergestellten Glycoproteinen, die als Therapeutika Verwendung finden sollen, eine Rolle spielen, insbesondere bei der Prozeßkontrolle.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.
Es ist bekannt, pathologisch fucosylierte Proteine mittels Affinitäts- und Immunoaffinitäts-Elektrophorese, meist nach Blotting auf antikörperbeschichtete Membranen, nachzuweisen (Taketa K., J.Chromatogr . 569, 229- 241, 1991). Auch Affinitätschromatographie-Verfahren werden verwendet. Zur Anwendung in der klinischen Praxis sind diese Methoden allerdings ungeeignet.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.
Zur Unterscheidung von fucosyliertem und nicht-fucosy- liertem AFP werden in der Literatur auch Lectin-Enzym- i munoassays mit den Lectinen Concanavalin A (ConA) , Lens-culinaris-Lectin A (LCA) und Phaseol us-vulgaris- Haemagglutinin E (PHA-E) beschrieben (Yasufumi Suzuki et al., Ann. Clin. Biochem. 1990, 2T7, 121-128; Noriaki Kinoshita et al . (1989), Clinica Chi ica Acta 179, 143- 152) . Es hat sich allerdings herausgestellt, daß Lectin-Enzymimmunoassays mit den genannten Lectinen die Anforderungen bezüglich Spezifität und Empfindlichkeit, die an einen klinischen Test zur quantitativen Bestimmung von fucosylierten Proteinen gestellt werden müs- sen, nicht erfüllen. Für den Fall der Lectine ConA und LCA wurde zusätzlich auch eine schlechte Reproduzierbarkeit der Ergebnisse festgestellt. Aufgabe der Erfindung war es deshalb, eine Nachweismethode für fucosylierte Proteine bereitzustellen, die ausreichend sensitiv, spezifisch und reproduzierbar ist und in der klinischen Praxis ohne Probleme angewendet werden kann. Die Nachweismethode soll leicht handhabbar und auch für Routineuntersuchungen im klinischen Bereich geeignet sein. Es war insbesondere die Aufgabe der Erfindung, eine Nachweismethode für fucosyliertes α-Fetoprotein, einem Tumormarker in der Leberdiagno- stik, bereitzustellen.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.
Die Aufgabe der Erfindung wird wie in den Ansprüchen angegeben gelöst. Der erfindungsgemäße Immunoassay zur Bestimmung der Menge an fucosyliertem Protein in einer biologischen Probe ist dadurch gekennzeichnet, daß a) ein immobilisierter Anti-Fuc-Protein-Antikörper oder ein immobilisiertes Anti-Fuc-Protein-Antikörperfragment mit der biologischen Probe unter Ausbildung eines Fuc- Protein-Antiköperkomplexes inkubiert wird, b) ein Lectin ausgewählt aus der Gruppe Ulex-europaeus- Agglutinin (UEA) , Lotus-tetragonolojbus-Agglutinin (LTA) und Anguilla-anguilla-Agglutinin (AAA) zugesetzt wird, das geeignete Markierungen aufweist, um die Menge des Fuc-Protein-Antiköperkomplexes und daraus die Menge an fucosyliertem Protein in an sich bekannter Weise zu bestimmen oder b) ein unmarkiertes Lectin ausgewählt aus der genannten Gruppe zugesetzt wird und der Nachweis des Fuc-Protein-Antikörperkomplexes mittels markierter Anti-Lectin-Antikörper gegen die genannten Lectine in an sich bekannter Weise erfolgt. Es hat sich gezeigt, daß mit dem erfindungsgemäßen Assay und durch den Einsatz der ausgewählten Lectine UEA, LTA und AAA eine weit bessere Empfindlichkeit und Spezifität erreicht wird als mit den in der Literatur beschriebenen Assays, die die Markerlectine ConA, LCA und PHA-E nutzen.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 einer bevorzugten Ausführungsform wird im erfindungsgemäßen Sandwich-Immunoassay UEA als Lectin eingesetzt. Als Fängerantikörper an der festen Phase können sowohl polyklonale als auch monoklonale Antikörper Verwendung finden.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 einer Ausführungsform wird der Sandwich-Immunoassay so durchgeführt, daß dem immobilisierten Anti-Fuc- Protein-Antikörper ein mit einem Akzeptor markiertes Lectin ausgewählt aus UEA, LTA und AAA zugesetzt wird und danach mit einem markierten Rezeptor, der an den Akzeptor des Lectins bindet, inkubiert wird. Der Nachweis wird in diesem Fall über den Marker am Rezeptor geführt. Als Akzeptor zur Markierung des Lectins können Haptene oder niedermolekulare Liganden eingesetzt werden, vorzugsweise kommt Biotin zur Anwendung. Entsprechend finden in einer bevorzugten Ausführungsform markiertes Avidin, Streptavidin oder deren Derivate als Rezeptor Verwendung. Werden als Akzeptor Haptene eingesetzt, so finden als Rezeptor Hapten-spezifische Antikörper Anwendung. Als Rezeptormarker können erfindungsgemäß Enzyme, Farbstoffe, Radioisotope, Metallkolloide, Chelatoren oder Spinmarker eingesetzt werden. Vorzugsweise wird als Rezeptormarker ein Enzym wie beispielsweise Meerrettich-Peroxidase (POD) , alkalische Phosphatase, ß-Galaktosidase, Urease oder Glucoseoxidase eingesetzt und die Menge des Fuc-Protein-Antikörperkomplexes durch eine Substratreaktion dieses Enzyms nachgewiesen. In einer besonders bevorzugten Ausführungsform wird erfindungsgemäß POD zur Markierung verwendet und die Substratreaktion mittels eines chrorαogenen Substrats, z.B. mit H0-Tetramethylbenzidin, durchgeführt. Es ist erfindungsgemäß jedoch auch möglich und dem Fachmann geläufig, daß die Substratreaktionen für die verschiedensten Enzyme mittels unterschiedlicher chromogener Substrate, Chemolumineszenz oder Fluoreszenz nachgewiesen werden können.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.
Der erfindungsgemäße Immunoassay wird also in einer be- sonders bevorzugten Ausführungsform als Enzymimmuno- assay durchgeführt. Alternativ kann der Rezeptor jedoch auch mit einem Radioisotop (z.B. 1Z <:'J) , mit einem Fluor- eszenzmarker (z.B. FITC) , einem Metallkolloid (z.B. Gold), einem Chelator (z.B. DTTA) , mit Polynucleotiden oder mit einem Spinmarker (z.B. PROXYL) markiert sein.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.
Es ist erfindungsgemäß auch möglich, das mit einem Akzeptor markierte Lectin und den Rezeptor als präformierten Komplex dem Antigen-Antikörperkomplex zuzusetzten, so daß bei der Durchführung der genannten Assayvariante ein Verfahrensschritt entfällt.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 einer weiteren Ausführungsform der Erfindung kann der Immunoassay auch als direkter Assay durchgeführt werden. In diesem Fall trägt das erfindungsgemäße Lectin selbst die genannten Marker. Auch hier ist die Durchführung als Enzymimrαunoassay bevorzugt.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 einer dritten Variante wird im erfindungsgemäßen Assay unmarkiertes Lectin eingesetzt und der Nachweis über markierte Anti-Lectin-Antikörper geführt. Es können hierbei sowohl polyklonale als auch monoklonale Antikörper gegen UEA, LTA oder AAA oder deren Fragmente eingesetzt werden. Auch hier ist die Durchführung als Enzymimmunoassay bevorzugt.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.
Als biologische Probe ist im Sinne der Erfindung jede Probe einer Körperflüssigkeit von Mensch oder Tier zu verstehen, die fucosylierte Proteine enthalten kann. Beispielsweise kann das Blut, Plasma, Serum, Urin, Gewebeflüssigkeit, Lymphe, Magensaft, Ascites oder Speichel sein. Mit dem erfindungsgemäßen Verfahren können fucosylierte Proteine wie z.B. α-Fetoprotein, fucosylierte, gentechnisch erzeugte oder aus biologischem Material gewonnene Pharmaproteine oder Adhäsionsproteine (z.B. CD22) bestimmt werden. Es hat sich gezeigt, daß der erfindungsgemäße Assay insbesondere zur Differentialdiagnose von Lebererkrankungen geeignet ist und sich durch die Bestimmung der Menge an fucosyliertem α-Fetoprotein (AFP) im Serum eine zuverlässige Aussage über ein möglicherweise vorhandenes hepatocelluläres Carcinom (HCC) treffen läßt, da der Fucose-spezifische AFP-Gehalt bei HCC-Patienten signifikant erhöht ist (vgl. Abb. 1).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).
Der erfindungsgemäße AFP-Assay enthält in einer besonders bevorzugten Ausführungsform als Fängerantikörper an der festen Phase polyklonale Antikörper oder deren Fragmente aus Maus, Kaninchen oder Huhn. Biotinyliertes UEA wird dem Antigen- Antikörperkomplex zugesetzt und mit einem Avidin-, modifizierten Avidin- oder Streptavidin-POD-Konjugat die Detektion des Fuc-Protein-Antikörperkomplexes durchgeführt .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.
Die erfindungsgemäße technische Lösung eröffnet also die Möglichkeit, bei Verdacht auf Lebertumor- Erkrankungen den Gesamt-AFP-Gehalt z.B. mittels eines üblichen Enzym-Immunoassays zu bestimmen und parallel dazu den Gehalt an fucosyliertem AFP (z.B. auf einer Mikrotestplatte) festzustellen. Der Aufbau dieser beiden Assays kann beispeilsweise wie nachfolgend dargestellt aussehen: Assay Gesamt-AFP: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. Immobilisierter anti-AFP-Antikörper bzw. Antikörperfragmet der Spezies 1 (z.B. Maus, Kaninchen, Huhn)1. Immobilized anti-AFP antibody or antibody fragment of species 1 (e.g. mouse, rabbit, chicken)
2. AFP-Standard-Verdünnung bzw. verdünntes Patientenserum2. AFP standard dilution or diluted patient serum
3. Enzym-markierter anti-AFP-Antikörper bzw. Antikörperfragment der Spezies 2 (z.B. Maus, Kaninchen, Huhn). Enzym z.B. Meerrettichperoxidase (POD) oder Alkalische Phosphatase (AP)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. Substratreaktion4. Substrate reaction
Assay fucosyliertes-AFP:Fucosylated AFP Assay:
1. Immobilisierter anti-AFP-Antikörper bzw. Antikörperfragmet der Spezies 1 (z.B. Maus, Kaninchen, Huhn)1. Immobilized anti-AFP antibody or antibody fragment of species 1 (e.g. mouse, rabbit, chicken)
2. Verdünntes Patientenserum 3. Markiertes Lectin ausgewählt aus UEA, LTA oder AAA (z.B. biotinyliertes UEA)2. Diluted patient serum 3. Labeled lectin selected from UEA, LTA or AAA (e.g. biotinylated UEA)
4. Markierungsspezifisches Enzymkonjugat (z.B. Avidin/Streptavidin-POD-Konjugat bzw. -Komplex)4.Label-specific enzyme conjugate (e.g. avidin / streptavidin-POD conjugate or complex)
5. Substratreaktion5. Substrate reaction
Neben dem Bestimmungsverfahren hat die Erfindung auch die zugehörigen Reagentiensätze, wie sie in den Ansprüchen ausgewiesen sind, zum Gegenstand. Ausführungsbeispiele :In addition to the determination method, the invention also relates to the associated reagent sets, as set out in the claims. Examples:
Beispiel 1 a) Herstellung von Anti-human-AFP-IgG-F (ab) 2~FragmentenExample 1 a) Preparation of anti-human AFP-IgG-F (ab) 2 ~ fragments
aa) Antiserum-Gewinnungaa) Antiserum production
Kaninchen werden mit hochreinem humanen AFP aus Nabelschnurblut immunisiert. Aus dem Blut der immunisierten Tiere wird nach Koagulation das Antiserum durch Zentrifugation abgetrennt.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) Isolierung der Gesamt-IgG-Fraktionab) Isolation of the total IgG fraction
Das Serum wird auf eine AFP-Sepharose gegeben. Ungebundene Serumprotein werden mit TRIS-gepufferter physiologischer Kochsalzlösung (TBS) ausgewaschen. Anließend wird das Anti-AFP-IgG mit einem 0,2M Glycin- HC1 Puffer pH 2,5 eluiert, mit IM Tris-HCl pH 7,5 neutralisieret, mit Centricon-30-kD-Kartuschen konzentriert und gegen 50 mM Acetat-Puffer pH 4,0; 0,5M NaCl, 0,02% NaN umgepuffert.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) Antikörperfragmentierung mittels Pepsinac) Antibody fragmentation using pepsin
Es wird ein Enzym-IgG-Verhältnis von E : P=l : 50 eingestellt und 4h bei 37 °C inkubiert. Die Reaktion wird mit IM Tris-HCl pH 7,5 beendet. Anschließend werden die Anti-AFP-F (ab) 2-Fragmente durch Immunaffinitätschromatographie an einer AFP-Säule wie unter ab) beschrieben isoliert, konzentriert, gegen TBS dialysiert und bei 4° C gelagert. 10An 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 zur Gesamt-AFP-Bestimmungb) Immunoassay for total AFP determination
Die Vertiefungen einer Mikrotestplatte sind mit anti- human-AFP-IgG (F (ab) 2-Fragment, Kaninchen) durch Ad- sorption aus einer Lösung von lOμg F (ab) 2/ml in Bicar- bonatpuffer pH 8,5-9,5 belegt. Eine unspezifische Proteinadsorption ist durch Blockierung der Plastoberflache mit einer Inertprotein-Lösung (z.B. Rinderserumalbumin in Phosphat-gepufferter physiologischer Kochsalz- lösung (PBS)) reduziert. In die so beschichteten Vertiefungen wird entweder eine Standard-Verdünnungsreihe von humanem AFP (Konzentrationsbereich 2 bis 300 ng/ml) bzw. eine zentrifugierte, 1:5 bis 1:10 verdünnte Probe Patientenserum gegeben. Ungebundenes Material wird aus- gewaschen, dann wird mit einer geeigneten Verdünnung anti-human-AFP-IgG-POD-Konjugat inkubiert. Ungebundenes Material wird ausgewaschen, dann wird die Substratreaktion mit H^O^-Tetramethylbenzidin gestartet und nach einer definierten Zeit mit Schwefelsäure gestoppt. Die Absorption wird bei 450 nm gemessen. Der Gehalt an AFP im Patientenserum wird anhand der Standard-Verdünnungsreihe ermittelt.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.
Beispiel 2Example 2
Erfindungsgemäßer Immunoassay zur Fuc-AFP-BestimmungImmunoassay according to the invention for Fuc-AFP determination
Die Vertiefungen einer Mikrotestplatte sind mit anti- human-AFP-IgG (F (ab) 2-Fragment, Kaninchen), wie in Beispeil la hergestellt, durch Adsorption aus einer Lö- 11The 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
sung von lOμg F(ab)2/ml in Bicarbonatpuffer pH 8,5-9,5 belegt. Eine unspezifische Proteinadsorption ist durch Blockierung dr Plastoberfläche mit einer Inertprotein- Lösung (z.B. Rinderserumalbumin in Phosphat-gepufferter physiologischer Kochsalzlösung (PBS) ) reduziert. In die so beschichteten Vertiefungen wird eine zentrifugierte, 1:5 bis 1:10 verdünnte Probe Patientenserum gegeben. Ungebundenes Material wird ausgewaschen, dann wird mit einer geeigneten Verdünnung biotinyliertes Ul ex-euro- paeus-Lectin (bio-UEA) inkubiert. Ungebundenes Material wird ausgewaschen, dann wird mit einer geeigneten Verdünnung Acylavidin-biotinylierte POD-Komplex (AbP) inkubiert. Ungebundenes Material wird ausgewaschen, dann wird die Substratreaktion mit H^0-Tetramethylbenzidin gestartet und nach einer definierten Zeit mit Schwefelsäure gestoppt. Die Absorption wird bei 450 nm gemessen.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.
Beispiel 3Example 3
Es werden die Ergebnisse der Beispiele 1 und 2 für verschiedene Probanden dargestellt (Abb. 2) . Es wird deutlich, daß der Gesamt-AFP-Gehalt nicht mit dem Gehalt an UEA-reaktivem AFP korreliert, so daß erst mit dem er- findungsgemäßen Test wirklich festgestellt werden kann, ob bei erhöhten AFP-Werten tatsächlich auch eine erhöhte Menge an fucolysiertem AFP vorliegt. 12The 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
Es zeigen :Show it :
Abb. 1: Gehalt an fucosyliertem AFP in Patienten- Serumproben control - gesunde Probanden, SLE - systemischer Lupus erythromateus, AIH - autoimmune Hepatitis, PBC - polynucleare billäre Cir- rhose, HCC - hepatocelluläres CarcinomFig. 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
Abb. 2: Vergleich des Gehalts an Gesamt- und fucosyliertem AFP in verschiedenen HCC-Patienten- Seren Fig. 2: Comparison of the total and fucosylated AFP content in different HCC patient sera

Claims

13Patentansprüche 13 Patent claims
1. Immunoassay zur Bestimmung der Menge an fucosyliertem Protein (Fuc-Protein) in einer biologischen Probe, dadurch gekennzeichnet, daß a) ein immobilisierter Anti-Fuc-Protein-Antikörper oder ein immobilisiertes Anti-Fuc-Protein- Antikörperfragment mit der biologischen Probe unter Ausbildung eines Fuc-Protein-Antiköperkomplexes inkubiert wird und b) ein Lectin ausgewählt aus der Gruppe Ul ex- europaeus-Agglutinin (UEA) , Lotus-tetragonol obus- Agglutinin (LTA) und Anguilla-anguilla-Agglutinin (AAA) zugesetzt wird, das geeignete Markierungen aufweist, um die Menge des Fuc-Protein- Antiköperkomplexes und daraus die Menge an fucosyliertem Protein in an sich bekannter Weise zu bestimmen oder b) ein unmarkiertes Lectin ausgewählt aus Gruppe1. 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) und Anguilla-anguilla-Aggluti- nin (AAA) zugesetzt wird und der Nachweis des Fuc- Protein-Antikörperkomplexes mittels markierter Anti-Lectin-Antikörper oder deren Fragmente gegen die genannten Lectine in an sich bekannter Weise erfolgt . 14Ulex-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 nach Anspruch 1, dadurch gekennzeichnet, daß b) ein mit einem Akzeptor markiertes Lectin gemäß Anspruch 1 zugesetzt wird, mit einem markierten Rezeptor, der an den Akzeptor des Lectins bindet, inkubiert wird und in üblicher Weise mittels des Markers am Rezeptor die Menge des Fuc-Protein- Antikörperkomplexes und daraus die Menge an fucosyliertem Protein bestimmt wird.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 nach Anspruch 1, dadurch gekennzeichnet, daß die Menge an fucosyliertem Protein direkt bestimmt wird durch b) Zusatz eines derart markierten Lectins, daß die Bestimmung der Menge des Fuc- Protein-Antikörperkomplexes mittels des Markers am Lectin erfolgen kann.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 nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß4. Immunoassay according to one of claims 1 to 3, characterized in that
die Menge an fucosyliertem α-Fetoprotein (AFP) bestimmt wird. 15the amount of fucosylated α-fetoprotein (AFP) is determined. 15
5. Immunoassay nach Anspruch 2, dadurch gegekennzeichnet, daß ein Lectin eingesetzt wird, das mit einem Hapten oder einem niedermolekularen Liganden als Akzeptor markiert ist.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 nach den Ansprüchen 2 und 5, dadurch gekennzeichnet, daß das Lectin mit Biotin als Akzeptor markiert ist und als Rezeptor Avidin, Streptavidin oder deren Derivate eingesetzt werden.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 nach den Ansprüchen 2 und 5, dadurch gekennzeichnet, daß das Lectin mit einem Hapten markiert ist und als Rezeptor ein Hapten-spezifischer Antikörper eingesetzt wird.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 nach den Ansprüchen 2 und 5 bis 7, dadurch gekennzeichnet, daß als Rezeptormarker ein Enzym, ein Farbstoff, ein Radioisotop, ein Metallkolloid, ein Chelator, ein8. 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
Nucleotid oder ein Spinmarker eingesetzt wird. 16Nucleotide or a spin marker is used. 16
9. Immunoassay nach Anspruch 3, dadurch gekennzeichnet, daß als Lectinmarker ein Enzym, ein Farbstoff, ein Radioisotop, ein Metallkolloid, ein Chelator, ein Nucleotid oder ein Spinmarker eingesetzt wird.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 nach Anspruch 1, dadurch gekennzeichnet, daß als Marker der Anti-Lectin-Antikörper ein Enzym, ein Farbstoff, ein Radioisotop, ein Metallkolloid, ein Chelator, ein Nucleotid oder ein Spinmarker eingesetzt wird.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. Testkit zur Bestimmung der Menge an fucosyliertem Protein (Fuc-Protein) in einer biologischen Probe enthaltend markiertes oder unmarkiertes Lectin ausgewählt aus der Gruppe [/lex-europaeus-Agglutinin (UEA) , Lotus-tet-ragonolobus-Agglutinin (LTA) und11. 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
Λnguilla-anguilla-Agglutinin (AAA)Anguilla Anguilla Agglutinin (AAA)
12. Testkit nach Anspruch 11, dadurch gekennzeichnet, daß er einen anti-Fuc-Protein-Antikörper oder ein Anti- Fuc-Protein-Antikörperfragment beinhaltet . 1712. 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. Testkit nach Anspruch 11 oder 12, dadurch gekennzeichnet, daß er ein mit einem Akzeptor markiertes Lectin und einen markierten Rezeptor, der an den Akzeptor des Lectins bindet, beinhaltet, die gegebenenfalls auch bereits als präformierter Komplex vorliegen können.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. Testkit nach Anspruch 13, dadurch gekennzeichnet, daß er als mit einem Akzeptor markiertes Lectin biotinyliertes UEA beinhaltet.14. Test kit according to claim 13, characterized in that it contains biotinylated UEA as lectin labeled with an acceptor.
15. Testkit nach Anspruch 11 oder 12, dadurch gekennzeichnet, daß er unmarkiertes Lectin und markierte Anti-Lectin- Antikörper gegen dieses Lectin beinhaltet.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. Testkit nach einem der Ansprüche 11 bis 15 zur Bestimmung der Menge an fucosyliertem α-Fetoprotein (AFP) . 16. Test kit according to one of claims 11 to 15 for determining the amount of fucosylated α-fetoprotein (AFP).
PCT/EP1999/000639 1998-02-02 1999-02-01 Immunoassay and test kit for assaying fucosylised protein in a biological sample WO1999039209A1 (en)

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US7273925B1 (en) 1998-12-15 2007-09-25 Brigham And Women's Hospital, Inc. Methods and products for regulating lectin complement pathway associated complement activation
WO2001012212A1 (en) * 1999-08-13 2001-02-22 The Brigham And Women's Hospital, Inc. Inhibitors of the lectin complement pathway (lcp) and their use
US20210033627A1 (en) * 2005-05-05 2021-02-04 Drexel University Diagnosis of Liver Pathology Through Assessment of Protein Glycosylation
US8524453B2 (en) 2006-02-10 2013-09-03 The Brigham And Woman's Hospital, Inc. Lectin complement pathway assays and related compositions and methods
WO2008031288A1 (en) * 2006-09-13 2008-03-20 Beijing Hotgen Biotech Co., Ltd Pre-filled centrifugal column for detecting hepatocellular carcinoma-specific alpha-fetoprotein variant and test kit containing the column
CN107973855A (en) * 2016-10-24 2018-05-01 希森美康株式会社 Monoclonal antibody reacted with glycopeptide and application thereof

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