WO1991006010A1 - Procedure de diagnostic - Google Patents

Procedure de diagnostic Download PDF

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Publication number
WO1991006010A1
WO1991006010A1 PCT/GB1990/001564 GB9001564W WO9106010A1 WO 1991006010 A1 WO1991006010 A1 WO 1991006010A1 GB 9001564 W GB9001564 W GB 9001564W WO 9106010 A1 WO9106010 A1 WO 9106010A1
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WO
WIPO (PCT)
Prior art keywords
mbp
binding protein
mannan binding
mannan
capture
Prior art date
Application number
PCT/GB1990/001564
Other languages
English (en)
Inventor
Malcolm Winston Turner
Steffen Thiel
Original Assignee
Institute Of Child Health
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB898922905A external-priority patent/GB8922905D0/en
Priority claimed from GB909002481A external-priority patent/GB9002481D0/en
Application filed by Institute Of Child Health filed Critical Institute Of Child Health
Publication of WO1991006010A1 publication Critical patent/WO1991006010A1/fr

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Classifications

    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention is concerned with mannan binding protein (MBP) which is important in the initiation of MBP
  • the invention relates to procedures for detecting the level of MBP in body fluids.
  • Opsonisation is the process by which foreign agents are coated in order that they may be cleared from the body by phagocytic cells.
  • the failure of serum to opsonise bakers' yeast (Saccharomyces Cerevisiae) for phagocytosis by normal polymorphonuclear leucocytes was first described by Miller et al., Lancet, 1968 (ii):60-63 in an infant with severe recurrent infections, diarrhoea and failure to thrive. This functional defect was subsequently reported in a series of paediatric patients with frequent unexplained infections (Soothill,
  • the present inventors have identified a deficiency in serum levels of MBP as being closely correlated with the opsonic defect and have identified the function of MBP in initiating complement binding to foreign agents having mannan-rich or N-acetylglucosamine-rich surfaces.
  • the wide- spread occurrence of these sugars in the cell walls of pathogenic Gram negative bacteria, mycobacteria and certain yeasts and viruses suggests that low levels of MBP may be linked to a susceptibility to a wide range of common
  • HIV human immuno-deficiency viruses
  • MBP is a macromolecular calcium dependent animal lectin which has been isolated from the serum and liver of many mammalian species including man, rat, cow and rabbit.
  • the human serum protein has a molecular mass of approximately 700,000 daltons based on Sepharose 6B gel filtration and appears to consist of some 18 subunits each of approximately 32,000 daltons as deduced from other physicochemical measurements and electron-microscopy. Structurally the protein resembles Clq and comprises three distinct domains: a cysteine-rich amino terminal domain, a collagen-like domain and the carbohydrate recognition domain (CRP) at the
  • the present invention provides a method for assessing the opsonic function of an individual comprising determining the concentration of MBP in a body fluid of the individual.
  • the body fluid may be any convenient fluid such as blood or a component thereof, for instance plasma, and is
  • MBP concentration in the body fluid may be achieved by any conventional method for quantitating specific proteins, preferably by the techniques which will be described below.
  • Typical serum concentrations of MBP in normal individuals are in the region of 150 ⁇ g l -1 whereas individuals having 30 ⁇ g l -1 or less particularly 20 ⁇ g l -1 or less are likely to have impaired opsonic
  • the invention provides a diagnostic process of measuring an individual's MBP levels which process comprises contacting a sample of a body fluid of the
  • an MBP-capture system comprising a solid support and an MBP-capture agent so as to bind MBP to the solid support via the MBP-capture agent and contacting the bound MBP with an MBP-detection system.
  • MBP-capture agent any material having a surface rich in either or both of these sugars may be used as the MBP-capture agent.
  • materials include mannans (for instance yeast zymosan as described below).
  • Other MBP-capture agents are the sugars N-acetyl-mannosamine, fucose and glucose.
  • the binding of MBP to saccharides such as the foregoing is a calcium-dependant interaction and when any of these saccharides is used as MBP capture agent the capture medium must contain calcium ions, preferably at a concentration of at least 5 mM.
  • MBP- capture agents which are calcium independent, are polyclonal
  • the solid support may be any conventional solid support such as multi-well titration plates and polystyrene or latex beads.
  • the solid support may be coated with MBP-capture agent by any conventional method.
  • the MBP-capture system comprises multi-well titration plates wherein the wells are coated with mannan or an anti-MBP antibody, for instance by the methods described in the Examples below.
  • the diagnostic process may be conducted by direct detection of the bound MBP or using a competition method in which the MBP in the sample competes for sites on the MBP- capture system with a predetermined amount of a labelled MBP.
  • the MBP-detection system comprises an MBP-recognition agent and means for detecting the binding of the MBP-recognition agent to MBP from the test sample.
  • MBP binding initiates classical pathway activation and C4 fixation which leads to the generation of C3b and which in turn forms a 1:1:1 complex with properdin and factor B.
  • C4, C3 properdin and factor B are all components of serum, when the diagnostic process is conducted using the individual's blood or serum, the MBP-capture system will necessarily become coated with C4b and with C3b, properdin and factor B.
  • the MBP-recognition agent may recognise MBP itself or one or more of C4b, C3b, properdin and/or factor B bound thereto.
  • Antibodies against MBP, C4b, C3b, properdin and factor B are all readily available and may be used as the MBP-recognition agent.
  • the MBP- recognition agent should be one which directly recognises MBP itself, for instance polyclonal or monoclonal anti-MBP antibodies mannan and saccharides such as N-acetyl
  • MBP-recognition agents which directly bind to MBP can also be used when Complement is present in the reaction medium.
  • MBP-capture and recognition agents rely on specific binding interactions with MBP and the available binding sites on MBP are limited in number, it is preferable that different agents are used as the MBP-capture and
  • the MBP-recognition system further comprises means for detecting the binding (directly or indirectly via C3b, properdin and/or factor B) to MBP of the MBP-recognition agent.
  • This may be achieved by any of the conventional labelling techniques.
  • the label may be linked to the MBP-recognition agent so that the binding of the MBP- recognition agent to MBP may be detected at once.
  • the label may be linked to a specific binding agent which itself recognises and specifically binds to the
  • MBP-recognition agent is a rabbit anti-MBP antibody and the labelled specific binding agent is a labelled anti-rabbit IgG antibody; the label is only attached to the MBP-recognition agent once the labelled specific binding agent has been incubated with the complex of MBP and MBP-recognition agent on the solid support.
  • the labels which may be used in accordance with the present invention are conventional labels such as a radio- labels, fluorescent labels or enzyme labels or labelling systems comprising a specific binding agent (which is linked to the MBP-recognition agent) and its specific binding partner, the latter being linked to a label such as an enzyme label.
  • a labelling system comprises biotin as the specific binding agent and avidin or,
  • streptavidin as the specific binding partner.
  • the diagnostic process of the invention may be any one of the diagnostic process of the invention.
  • the MBP-detection system comprises MBP linked to a label which is added to the reaction mixture and competes with the individual's MBP for sites on the MBP-capture system.
  • Any conventional label or labelling system as described above may be used.
  • Labels may be linked to other components of the MBP detection system by conventional techniques.
  • the presence of the label bound to the solid support may be detected by any conventional method. Quantitation of the label gives a measure of the MBP concentration in the sample of the
  • the process is conducted on diluted serum, preferably 5% serum, under physiological conditions of temperature and pH, preferably at from 20 to 37'C in buffer at pH from 6 to 8 more preferably at about pH7.
  • the process may be conducted according to any conventional immuno-assay protocol including appropriate washing steps and incubation steps as necessary.
  • calcium ions When calcium ions are needed in order for MBP to bind to the capture system, they may be present in the sample of body fluid but are preferably added or supplemented by inclusion of calcium ions in the dilution buffer, preferably at up to 50 mM calcium ions or even more.
  • the body fluid is diluted to 1 to 20%, preferably about 5% by volume using a dilution buffer containing a calcium salt such as calcium chloride.
  • calcium ions are needed in order for the MBP- recognition agent to bind to the captured MBP these may be present from an earlier step in the process or they may be added as part of the detection system.
  • the capture system comprises multi-well titration plates coated with mannan, the body fluid is diluted serum and the MBP-recognition system is a rabbit polyclonal anti- MBP antibody (as MBP-recognition agent) and horse radish peroxidase-labelled anti-rabbit IgG antibody.
  • the MBP-capture system comprises a multi-well titration plate coated with a polyclonal anti-MBP antibody, the body fluid is diluted serum and the MBP-recognition system comprises biotin-labelled anti-MBP antibody or biotin- labelled mannan (as MBP-recognition agent) and horse radish peroxidase-labelled streptavidin.
  • streptavidin (as MBP-detection system).
  • the MBP-capture system comprises polyclonal anti- MBP antibody-coated micro-titre plates, the body fluid is diluted serum and the MBP-recognition system comprises mannan (as MBP-recognition agent) linked to biotin and streptavidin- labelled with alkaline phosphatase.
  • the diagnostic process is conducted twice using alternative MBP-recognition systems one of which directly recognises MBP itself and the other recognises C3b, properdin or factor B and thereby, indirectly, MBP.
  • the diagnostic process may further comprise the step of assessing an individual's opsonic function by comparing the MBP concentrations in the body fluid of the individual with MBP levels of normal individuals and/or comparing the MBP concentration in samples of body fluid of the individual taken at different times during the course of an infective disease.
  • concentration of MBP in the body fluid will increase during the acute phase of the infection as do other well known acute phase proteins.
  • individuals with impaired opsonic In individuals with impaired opsonic
  • MBP levels remain approximately constant during the acute phase of the infection.
  • the invention provides a diagnostic test kit comprising an MBP-capture agent and one or more components of an MBP-detection system such as an MBP- recognition agent or labelled MBP.
  • the kit comprises an MBP-capture system comprising a solid support and, bound thereto, the MBP-capture agent.
  • the kit comprises the MBP-recognition system including the MBP-recognition agent and any necessary labels or labelled reagents as well as agents for detecting the labels.
  • the diagnostic kit may also contain buffers, controls and standards for use in conducting the diagnostic test process according to the invention.
  • MBP deficiency would be a particular risk factor in early infancy when the antibody repertoire is restricted and the levels of potentially compensating IgG opsonins are low. This may explain not only the association of the defect with otitis media in 1 year-old infants but also the apparent
  • Fig. 1 shows that addition of increasing amounts of MBP enriched material containing approximately 0.2 ⁇ g/ml MBP (prepared as described in Example 1, Materials and Methods) corrects the opsonic deficiency of serum LB as indicated by measuring the deposition of C4 (-O-), C3b+C3bi (-X-) and factor B (-+- ) on a mannan coated plate.
  • Fig. 2 shows the results of an antibody capture assay for serum MBP:
  • Fig. 3 shows the results of binding of various complement proteins to mannan coated ELISA plates in a
  • Fig. 5 shows MBP serum levels in 103 apparently healthy adults as measured by the ELISA technique of
  • Example 3 The invention is illustrated by the following Examples which are not intended to limit the scope of the invention in any way:
  • mannan binding protein from (i) a pool of 50ml serum obtained from 100 healthy donors, (ii) 8ml of serum from an individual showing high levels of C3b binding to yeast subsequently called HB serum), and (iii) 8ml of serum from an individual with low C3b binding activity (subsequently called LB serum).
  • SPS-PAGE and Western blotting Purified protein fractions were vigorously reduced by boiling in the presence of 40 mM DTT and duplicate samples were electrophoresed on a 10% reducing SPS-PAGE slab gel by the method of Laemlli (1). The gel was divided; half was silver stained by the method of Morrissey (2), the other half was electroblotted using the BioRad Transblot and probed with rabbit anti-MBP antibody followed by 125 I-labelled anti-rabbit Ig (Amersham UK Ltd).
  • anti-MBP antiserum An antiserum to human MBP was raised in a rabbit using MBP prepared from 6 litres of outdated human plasma. Briefly, the protein was bound to a mannan-Sepharose column, displaced with EDTA, rechromatographed on a similar, smaller column eluted with mannose and then successively fractionated by Superose 6-gel filtration and Mono-Q ion exchange chromatography.
  • Contaminating IgM was removed using an anti-human IgM
  • IgG was isolated from the antiserum by Na 2 SO 4 precipitation and passage of the redissolved
  • HB C3b binding to zymosan
  • LB serum known to give low binding in the same system
  • anti-factor B and anti-C4 (Serotec UK Ltd, Kidlington, Oxford).
  • binding coefficient (BC) 1-
  • Sera were diluted to 5% in imidazole buffer (40 mM imidazole/HCl pH 7.8 with 1.25 M NaCl and 50 mM CaCl 2 ) and 100 ⁇ l aliquots adddd in duplicate to the wells of the mannan coated plate which were then incubated at 37oC for two hours.
  • the plates were washed four times with PBS-T and then rabbit anti-human MBP, diluted to 1/500 in PBS-T, was added to all wells and incubated at 37oC for two hours.
  • the plates were washed four times with PBS-T and then incubated at 37o C with horse radish
  • MBP was also assayed using an antibody capture sandwich ELISA in which the rabbit anti-MBP serum was used as both the capture and detector antibody.
  • the antibody was biotinylated by the method of Guesdon et al (3) and the assay developed with streptavidin peroxidase.
  • a calibrated gravimetric standard serum was also included in the assay and permitted quantitation of MBP levels in ⁇ g l -1 .
  • mannan binding protein is a Ca ++ dependent lectin
  • EDTA displacement from a mannan-Sepharose affinity column was chosen as the major purification step.
  • Pre-fractionation of serum on Sephacryl S300 to give material of approximately 700 kPa reduced the protein load on the affinity column and further purification steps (IgM depletion and Mono-Q FPLC) gave preparations which were analysed by SPS-PAGE under reducing conditions.
  • a major subunit of approximately 32 kPa was present in material derived from a 50ml pool of serum (100 donors) and from 8ml of serum obtained from an
  • Electroblotting and probing with the anti-MBP antiserum revealed both the major 32 kPa and the minor 64 kPa bands in the material derived from a serum pool and the donor with high C3b binding (HB) but no evidence of these bands in material derived from the individual with low C3b binding (LB).
  • fragments is approx. 30-115% and that for C4 fragments 15-120%.
  • mannan coated plates were washed three times with phosphate buffered saline pH 7.3 (Oxoid UK Ltd) containing 0.5% (v/v) Tween-20 (PBS-T), once with PBS (without Tween-20) and once with VBS.
  • PBS-T phosphate buffered saline pH 7.3
  • the serum samples were diluted in Micronic tubes (Flow Laboratories) to 5% in VBS containing 5 mM CaCl 2 and 5 mM
  • a serum known to give high levels of C3b binding to zymosan (HB) and serum known to give low binding in the same system (LB) were included in every assay and used to
  • Binding coefficient (BC) 1-
  • Immulon micro-ELISA plates pre-coated with mannan as above The plates were then incubated at 37oC for 2 hours, washed four times with PBS-T and rabbit anti-human MBP diluted to 1/500 in PBS-T was added before a further incubation at 37oC for 2 hours.
  • the plates were washed four times with PBS-T and then incubated at 37oC with horseradish peroxidase-sheep anti-rabbit IgG conjugate (Serotech UK Ltd) at 1/500 in PBS- T.
  • the plates were further washed four times with PBS-T and colour developed as above. An MBP binding coefficient was then calculated as described for the binding of complement components.
  • MBP was also assayed using an antibody capture sandwich ELISA in which the rabbit anti-MBP was used as both the capture and detector antibody.
  • the antibody was biotinylated by the method of Guesdon et al. (3) and the assay developed with streptavidin peroxidase. An MSP binding coefficient was then calculated as above.
  • mannan binding protein MBP
  • the assay system used in the present study included a step involving the Ca +2 -dependent binding of mannan-biotin to antibody bound MBP prior to incubation with an avidin-linked- indicator enzyme, and subtraction of any background binding due to Ca +2 -independent binding.
  • Purified MBP (20 ⁇ g) was used to immunise a rabbit and the rabbit was given a booster injection of MBP (20 ⁇ g) after one month. Blood was taken from the rabbit two weeks after the booster injection and the IgG fraction was purified by sodium sulphate precipitation and ion-exchange chromatography and then stored at 4oC in the presence of sodium azide (0.1%, w/v).
  • Microtiter plates (Nunc-Immuno plate, GIBCO) were coated at room temperature with rabbit anti-human MBP IgG (100 ⁇ l per well of a 10 ⁇ g/ml solution) in buffer (sodium carbonate, 15mM, sodium hydrogen carbonate, 35 mM, pH 9.6) for 16 hours and then washed three times with tris-buffered saline, (10 mM tris, 150 mM sodium chloride, 0.05% sodium azide, 0.05% Tween 20, pH 7.4, TBS-NT). Unbound sites were blocked by addition of bovine serum albumin (BSA, 100 ⁇ l 1 mg/ml solution in TBS- TBS-NT) to the wells for 2-3 hours at room temperature followed by washing as before. The plates were stored at 4oC with tris buffered saline (10mM tris, 150mM sodium chloride,
  • Serum samples were diluted in TBS-NT containing 10mM ethylene diamine tetraacetic acid disodium salt (TBS-NTE); and the dilutions were incubated in the wells for 2 hours at room temperature.
  • TBS-NT 10mM ethylene diamine tetraacetic acid disodium salt
  • ExtrAvidin-alkaline phosphate conjugate (Sigma E2636), diluted 1 /10 4 in TBS-NT Ca +2 , was added followed by
  • the assay was standardised by using a preparation of purified MBP the concentration of which had been determined by amino acid analysis.

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  • Health & Medical Sciences (AREA)
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Abstract

Procédé d'évaluation de la fonction opsonique d'un individu incluant la détermination de la concentration en protéine de liaison mannan dans un fluide corporel humain.
PCT/GB1990/001564 1989-10-11 1990-10-11 Procedure de diagnostic WO1991006010A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB898922905A GB8922905D0 (en) 1989-10-11 1989-10-11 Diagnostic procedure
GB8922905.8 1989-10-11
GB9002481.1 1990-02-05
GB909002481A GB9002481D0 (en) 1990-02-05 1990-02-05 Diagnostic procedure

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WO1991006010A1 true WO1991006010A1 (fr) 1991-05-02

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0492530A1 (fr) * 1990-12-24 1992-07-01 E.R. SQUIBB & SONS, INC. Anticorps monoclonaux qui lient la protéine de liaison de mannose
EP1140171A1 (fr) * 1998-12-15 2001-10-10 The Brigham And Women's Hospital, Inc. Techniques et produits permettant de reguler l'activation du complement associee a la voie du complement a lectine
WO2003033522A1 (fr) * 2001-10-19 2003-04-24 Natimmune A/S Isolation des lectines
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
US8524453B2 (en) 2006-02-10 2013-09-03 The Brigham And Woman's Hospital, Inc. Lectin complement pathway assays and related compositions and methods

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001519A1 (fr) * 1987-08-20 1989-02-23 Children's Hospital Corporation Proteine humaine liant le mannose

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001519A1 (fr) * 1987-08-20 1989-02-23 Children's Hospital Corporation Proteine humaine liant le mannose

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Clinical and Experimental Immunology, Vol. 79, No. 2, February 1990, Blackwell Scientific Publications, M. SUPER et al.: "The Level of Mannan-Binding Protein Regulates the Binding of Complement-Derived Opsonins to Mannan and Zymosan at Low Serum Concentrations", pages 144-150 see the whole article *
The Journal of Experimental Medicine, Vol. 169, No. 5, 1 May 1989, M. KUHLMAN et al.: "The Human Mannose-Binding Protein Functions as an Opsonin", pages 1733-1745 see pages 1733-1735 cited in the application *
The Journal of Immunology, Vol. 144, No. 6, 15 March 1990, The American Association of Immunologists, J. LU et al.: "Binding of the Pentamer/Hexamer forms of Mannan-Binding Protein to Zymosan Activates the Proenzyme Clr2Cls2 Complex, of the Classical Pathway of Comlement, without involvement of Clq", pages 2287-2294 see the Abstract; page 2288, Column 2; page 2289, Column 2; page 2291, column 2 - page 2293, column 1 *
The Lancet, 25 November 1989, M. SUPER et al.: "Association of Low Levels of Mannan-Binding Protein with a Common Defect of Opsonisation", pages 1236-1239 see the whole article *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0492530A1 (fr) * 1990-12-24 1992-07-01 E.R. SQUIBB & SONS, INC. Anticorps monoclonaux qui lient la protéine de liaison de mannose
EP1140171A1 (fr) * 1998-12-15 2001-10-10 The Brigham And Women's Hospital, Inc. Techniques et produits permettant de reguler l'activation du complement associee a la voie du complement a lectine
EP1140171A4 (fr) * 1998-12-15 2002-03-13 Brigham & Womens Hospital Techniques et produits permettant de reguler l'activation du complement associee a la voie du complement a lectine
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
WO2003033522A1 (fr) * 2001-10-19 2003-04-24 Natimmune A/S Isolation des lectines
CN100447150C (zh) * 2001-10-19 2008-12-31 纳蒂芒公司 凝集素的分离方法
US8524453B2 (en) 2006-02-10 2013-09-03 The Brigham And Woman's Hospital, Inc. Lectin complement pathway assays and related compositions and methods

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