DE4037809A1 - Staphylococcal neutral phosphatase and 70 KD protein - isolated by immuno:affinity purificn., ion exchange chromatography or affinity chromatography and used to stimulate lymphocytes and bind immunoglobulins - Google Patents

Staphylococcal neutral phosphatase and 70 KD protein - isolated by immuno:affinity purificn., ion exchange chromatography or affinity chromatography and used to stimulate lymphocytes and bind immunoglobulins

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DE4037809A1
DE4037809A1 DE19904037809 DE4037809A DE4037809A1 DE 4037809 A1 DE4037809 A1 DE 4037809A1 DE 19904037809 DE19904037809 DE 19904037809 DE 4037809 A DE4037809 A DE 4037809A DE 4037809 A1 DE4037809 A1 DE 4037809A1
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phosphatase
immunoglobulin
matrix
affinity
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Yehia Mohammed Yousif
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

A process is claimed for the use of purified active immunoglobulin bound staphylococcal neutral phosphatase and 70 KD protein. The protein is prepd. by (a) immuno affinity purifcn. using antiphosphatase and anti 70 KD protein coupled antibodies on an activated matrix; (b) ion exchange chromatography using cation exchange matrix; or (c) affinity chromatography using immunoglobulin coupled on a solid phase matrix. Stimulation of terminal blood lymphocytes and immunoglobulin synthesis is fulfilled. Sepn. of immunoglobulins, F(ab)'2 fragments and the substrate analogue of phosphatase from solns. is effected by using immobilised phosphatases and 70 KD protein in activated solid phase matrix. USE/ADVANTAGE - Gram positive staphylococcal bacteria are nitrogenic factors for B-lymphocytes. Protein A is not the stimulating factor. In this invention, the purified B-cell stimulation factors, immunoglobulins and F(ab)'2 fragments binding factors are used. The purifcn. process separates phosphatase and 70KD protein from the whole protein soln. Pure phosphatase and 70KD protein can be used as a stimulation factor for B-lymphocytes for immunoglobulin synthesis, both with and without IL-2. Immobilised phosphatase and 70KD protein on an activated solid matrix can be used in the sepn. of immunoglobulin and substrate. The immobilised phosphatase and 70KD protein can bind polyclonal IgG, IgM, IgA and kappa myeloma immunoglobulin both in humans and in rats.

Description

Die Erfindung betrifft die Anwendungen von staphylokokkal neutrale Phosphatase und 70KD Protein nach dem Oberbegriff des Anspruchs 1 bis 2.The invention relates to the applications of staphylococcal neutral phosphatase and 70KD protein according to the generic term of Claims 1 to 2.

Pokeweed Mitogen, IL-2 und gram positive staphylokokkal Bakterien sind bekannt als Mitogenen Faktoren für B-Lymphozyten: Protein A war durch B-Zellen oberflächliche Immunoglobulinbindungs-Mechanismus der mögliche Stimulierungsfaktor. Es ist der Fachwelt bekannt, daß von keiner anderen Seite, Immunoglobulinbindungs-Proteine als Protein A von Staphylokokken vorhanden sind. Protein A bindet hauptsächlich nur Fc Teil von IgG und nicht IgM, IgA oder F(ab)′2 Teil.Pokeweed Mitogen, IL-2 and gram positive staphylococcal Bacteria are known as mitogenic factors for B lymphocytes: Protein A was superficial by B cells Immunoglobulin binding mechanism the possible stimulation factor. It is known to experts that no other Page, Immunoglobulin binding proteins as Protein A from Staphylococci are present. Protein A mainly binds only Fc part of IgG and not IgM, IgA or F (ab) ′ 2 part.

Hochgereinigtes Protein A und rekombinante Protein A hat keinen stimulierenden Effekt auf B-Zellen, gleichzeitig werden intakte Staphylokokkal-Zellen stimmuliert Igs synthetisieren. Nicht alle Immunoglobulinbindungs-Aktivitäten von Staphylokokken kann man durch Protein A allein erklären.Highly purified Protein A and recombinant Protein A have none stimulating effect on B cells, at the same time intact staphylococcal cells stimulate Igs to synthesize. Not all staphylococcal immunoglobulin binding activities can be explained by protein A alone.

Der Erfindung liegt die Aufgabe zugrunde, die gereinigten B-Zellenstimulierungsfaktoren, Immunoglobulinen und F(ab)′2 Teile Bindungsfaktoren, anzuwenden. Die Lösung der Aufgabe wird durch ein Reinigungs-Verfahren erreicht, dessen Zielsetzung die Trennung von Phosphatase und 70KD Protein aus der gesamten Protein-Lösung ist.The invention has for its object the cleaned B cell stimulation factors, immunoglobulins and F (ab) ′ 2 Parts of binding factors to apply. The problem is solved by a cleaning process achieved, the objective of which is the separation of phosphatase and 70KD protein is from the entire protein solution.

Bakterielleswachstum-Medien und -Kulturen:
Tryptic Soy Broth (TSB), Brain Heart infusion Medium und chemically defined medium (CDM) bei 37c für 18-24 Stunden mit rütteln.Bacterial growth media and cultures:
Shake Tryptic Soy Broth (TSB), Brain Heart infusion Medium and chemically defined medium (CDM) at 37c for 18-24 hours.

Es gibt 3 Wege um die Proteine zu erhalten. Diese Wege sind wie folgt unter A-C beschrieben:There are 3 ways to get the proteins. These ways are like described below under A-C:

A) Ionenaustauschkromatography-VerfahrenA) Ion exchange chromatography procedure

Die Kultur wird bei 3,000 rpm für 30 min zentrifugiert. Die Bodensatz-Zellen werden mit 1M KCl, Tris-Puffer pH 8,5 für eine Stunde inkubiert. Der Überstand wird bei 45,000 rpm für 15 Stunden ultrazentrifugiert und gegen 0,2M NaCl, PBS-Puffer dialysiert. Die Lösung (40 ml) wird auf dem Kationenaustauscher-Matrix (z. B. Mono S, Dowex resin) aufgetragen. Lineargradient bis 1M NaCl, PBS-Puffer wird durchgeführt. Phosphatase wird bei 25-30% Endpuffer eluiert und 70KD Protein bei 30-35% Endpuffer eluiert.
Die Ausbeute:
- 3-4 mg reine Phosphatase/40 ml Gesamt-Protein-Waschlösungs-Extrakt nach dem Ultrazentrifugieren (GPEU).
- 3,5-5 mg reine 70KD Protein/40 ml (GPEU). The culture is centrifuged at 3,000 rpm for 30 minutes. The sediment cells are incubated with 1M KCl, Tris buffer pH 8.5 for one hour. The supernatant is ultracentrifuged at 45,000 rpm for 15 hours and dialyzed against 0.2M NaCl, PBS buffer. The solution (40 ml) is applied to the cation exchange matrix (e.g. Mono S, Dowex resin). Linear gradient up to 1M NaCl, PBS buffer is carried out. Phosphatase is eluted in 25-30% final buffer and 70KD protein in 30-35% final buffer.
The yield:
- 3-4 mg pure phosphatase / 40 ml total protein wash solution extract after ultracentrifugation (GPEU).
- 3.5-5 mg of pure 70KD protein / 40 ml (GPEU).

B) Immunoaffinitäts-Verfahren zur Reinigung von Phosphatase oder 70KD ProteineB) Immunoaffinity method for the purification of phosphatase or 70KD proteins

Monoklonale Antikörper (Anti-Phosphatase oder Anti-70KD-Protein) wird mit dem aktivierten solide Phase Matrix (z. B. CN Br-aktivierte Sepharose 4B, 10 mg Protein/ml Matrix) in 0,5M Na inkubiert. Nach dem Waschen werden 10 Volumen von 10 mM Äthanolamine pH 7,4 mit dem Matrix für 4 Stunden bei Raumtemperatur inkubiert. (GPEU) wird gegen PBS, 0,1M NaCl dialysiert und mit dem Matrix nach dem Waschen für 4 Stunden bei 4c inkubiert. Das Matrix wird in eine Säule abgefüllt und gewaschen und mit einen Elierungs-Puffer (z. B. 100 mM Glycin pH 5) eluiert. Die entsprechenden Fraktionen werden gesammelt und neutralisiert.
Die Ausbeute:
- 5-9 mg reine Phosphatase/40 ml (GPEU).
- 6-10 mg reine 70 KD Protein/40 ml (GPEU).Monoclonal antibodies (anti-phosphatase or anti-70KD protein) are incubated with the activated solid phase matrix (e.g. CN Br-activated Sepharose 4B, 10 mg protein / ml matrix) in 0.5M Na. After washing, 10 volumes of 10 mM ethanolamine pH 7.4 are incubated with the matrix for 4 hours at room temperature. (GPEU) is dialyzed against PBS, 0.1M NaCl and incubated with the matrix after washing for 4 hours at 4c. The matrix is filled into a column and washed and eluted with an elution buffer (e.g. 100 mM glycine pH 5). The appropriate fractions are collected and neutralized.
The yield:
- 5-9 mg pure phosphatase / 40 ml (GPEU).
- 6-10 mg of pure 70 KD protein / 40 ml (GPEU).

C) Affinitätskromatography-Verfahren (IgG-Solide Phase Matrix) für Abtrennung von Phosphatase und 70KD ProteinC) Affinity Chromatography (IgG Solid Phase Matrix) for separation of phosphatase and 70KD protein

5 ml IgG-Matrix (z. B. IgG-Protein A-Sepharose 6 oder IgG-Sepharose 6) wird mit Probenpuffer (0,2M NaCl, PBS) gewaschen. 7 ml (GPEU) nach Dialysierung gegen Probenpuffer wird mit dem Matrix für 3-12 Stunden bei Raumtemperatur inkubiert. Nach dem Waschen wird das Matrix in eine Säule abgefüllt. 100 mM Glycin pH 5 wird wird dabei als Eluierungspuffer benutzt. Entsprechende Fraktionen werden gesammelt und neutralisiert.
Die Ausbeute:
- 2,5-5 mg Phosphatase-70KD Protein/40 ml (GPEU).5 ml IgG matrix (e.g. IgG protein A-Sepharose 6 or IgG-Sepharose 6) is washed with sample buffer (0.2M NaCl, PBS). 7 ml (GPEU) after dialyzing against sample buffer is incubated with the matrix for 3-12 hours at room temperature. After washing, the matrix is filled into a column. 100 mM glycine pH 5 is used as the elution buffer. Corresponding fractions are collected and neutralized.
The yield:
- 2.5-5 mg phosphatase-70KD protein / 40 ml (GPEU).

Die biologische Eigenschaften von staphylokokkal Phosphatase:The biological properties of staphylococcal Phosphatase:

Molekular-Gewicht:
- SDS-PAGE (12% Gel) zeigt 2 Banden (31,32 KD).Molecular weight:
- SDS-PAGE (12% gel) shows 2 bands (31.32 KD).

Isoelektrische Punkte (pI):
- Flach Gel Methode (Ampholiten 3-10 oder 9-11) zeigt keine Banden im Gel → pI<10.Isoelectric points (pI):
- Flat gel method (ampholites 3-10 or 9-11) shows no bands in the gel → pI <10.

Substrats-Aktivität:
- Maximal enzymatische Aktivität liegt bei pH 7,4 mit P-N-Nitrophenol phosphte Substrat.Substrate activity:
- Maximum enzymatic activity is pH 7.4 with PN-nitrophenol phosphate substrate.

Die biologische Eigenschaften von staphylokokkal 70 KD Protein:The biological properties of staphylococcal 70 KD protein:

Molekular-Gewicht:
- SDS-PAGE (12% Gel) zeigt 1 Band (70 KD).Molecular weight:
- SDS-PAGE (12% gel) shows 1 band (70 KD).

Isoelektrische Punkte (pI):
- Flach Gel Methode (Ampholiten 3-10 oder 9-11) zeigt keine Banden im Gel → pI<10. Isoelectric points (pI):
- Flat gel method (ampholites 3-10 or 9-11) shows no bands in the gel → pI <10.

I) Anwendung von reiner Phosphatase und 70KD Protein als Stimulierungs-Faktoren für B-Lymphozyten zur ImmunoglobulinsyntheseI) Use of pure phosphatase and 70KD Protein as stimulation factors for B lymphocytes for immunoglobulin synthesis

Reine Phosphatase und/oder 70KD Protein aktivieren die terminalen B-Lymphozyten, diese produzieren Immunoglobulin in der Kultur mit und ohne IL-2.Pure phosphatase and / or 70KD protein activate the terminal B lymphocytes, these produce immunoglobulin in the culture with and without IL-2.

Terminalen Blutlymphozyten (PBL) wurden von 15 ml defibriniertes Venenblut mit Ficoll-Hypaque-Trennlösung durch Gradientzentrifugation abgetrennt. Die vorhandenen (PBL) wurden nach dem Waschen in einer Konzentration von 10 Mio Zellen/ml in RPMI 1640 Medium + 10% fötalem Kälberserum in Falcon-Röhrchen über 6 oder 9 Tage bei 37c in einer angefeuchteten Atmosphäre von 5% CO₂+95% Luft kultiviert.Terminal blood lymphocytes (PBL) were defibrinated by 15 ml Vein blood with Ficoll-Hypaque separation solution by gradient centrifugation separated. The existing (PBL) were after the Wash in a concentration of 10 million cells / ml in RPMI 1640 Medium + 10% fetal calf serum in Falcon tubes over 6 or 9 days at 37c in a humidified atmosphere of 5% CO₂ + 95% Air cultivated.

Folgende Ansätze wurden durchgeführt:The following approaches were carried out:

  • 1. 6 und 9 Tage mit 2 µg/ml oder 20 µg/ml Phosphatase.1. 6 and 9 days with 2 µg / ml or 20 µg / ml phosphatase.
  • 2. 6 und 9 Tage mit 2 µg/ml oder 20 µg/ml 70KD Protein.2. 6 and 9 days with 2 µg / ml or 20 µg / ml 70KD protein.
  • 3. Pokeweed Mitogen, IL-2 und Staphylokokkus aureus intakt Zellen (Cowan I) wurden als Kontrolle verwendet.
    IgG, IgM und IgA wurden in den zellfreien Kulturüberständen mit Hilfe eines Peroxidase-ELISA-Sytems gemessen.    3. Pokeweed Mitogen, IL-2 and Staphylococcus aureus intact cells (Cowan I) were used as controls.
    IgG, IgM and IgA were measured in the cell-free culture supernatants using a peroxidase ELISA system.

Vorteilhaft wirkt sich die Phosphatase und 70KD Protein auf die B-Zellen als stimulierender Faktor aus. Gute Ergebnisse sind auch in geringer Konzentration (2 µg/ml) mit oder ohne IL-2 zu erzielen. Dieser Aktivierungs-Effekt war höher als der Effekt von intakten bakteriellen Zellen. Dies zeigt, daß die Phosphatase und 70KD Protein die Stimulatoren für B-Zellen sind. The phosphatase and 70KD protein have an advantageous effect on the B cells as a stimulating factor. Good results are even in low concentrations (2 µg / ml) with or without IL-2 achieve. This activation effect was higher than the effect of intact bacterial cells. This shows that the phosphatase and 70KD protein are the stimulators for B cells.  

II. Abtrennung von Immunoglobulin und Substrat von Phosphatase aus Lösungen durch Anwendung von immobilisierte Phosphatase und 70KD Proteine zu aktiviertem solidem MatrixII. Separation of immunoglobulin and substrate from Phosphatase from solutions using immobilized phosphatase and 70KD proteins too activated solid matrix

Aktiviertes solides Matrix (z. B. CN Br-aktivierten Sepharose 4B) wird mit Phosphatase und/oder 70KD Protein gekoppelt. Lyophilisiere Phosphatase oder 70KD Protein wird in Bindungs-Puffer (0,5M Na Phosphat pH 7,4) gelöst und mit dem entsprechenden Matrix (5-10 mg Protein/ml Matrix) bei 4c für 3-12 Stunden inkubiert. Nach dem Waschen 10 Volumen von 100 mM Äthanolamine pH 7,4 wird zugesetzt und bei Raumtemperatur für 4 Stunden inkubiert. Nach dem Waschen in PBS wird die entsprechende Probe dazu gegeben und bei Raumtemperatur für 4-12 Stunden inkubiert. Eine Säule wird mit Matrix abgefüllt und gewaschen. Das gebundene Material wird mit 100 mM Glycin pH 5 eluiert und dann neutralisiert. Das Matrix wird mit neutralem Tris maleate, 0,1% NaN₃ Puffer gewaschen und kann bei 4c gelagert werden.
Die Ausbeute:
- 0,1 mg immobilisierte Phosphatase oder 70KD Protein kann 500 µg Polyklonal IgG, IgM, IgA oder F(ab)′2 Teilen binden.Activated solid matrix (e.g. CN Br-activated Sepharose 4B) is coupled with phosphatase and / or 70KD protein. Lyophilized phosphatase or 70KD protein is dissolved in binding buffer (0.5M Na phosphate pH 7.4) and incubated with the appropriate matrix (5-10 mg protein / ml matrix) at 4c for 3-12 hours. After washing 10 volumes of 100 mM ethanolamine pH 7.4 is added and incubated at room temperature for 4 hours. After washing in PBS, the corresponding sample is added and incubated at room temperature for 4-12 hours. A column is filled with matrix and washed. The bound material is eluted with 100 mM glycine pH 5 and then neutralized. The matrix is washed with neutral Tris maleate, 0.1% NaN₃ buffer and can be stored at 4c.
The yield:
- 0.1 mg immobilized phosphatase or 70KD protein can bind 500 µg polyclonal IgG, IgM, IgA or F (ab) ′ 2 parts.

Der Vorteil besteht darin, daß die immobilisierte Phosphatase oder 70KD Protein von ihren biologischen Eigenschaften so angelegt ist, daß sie sowohl Polyklonal IgG, IgM, IgA und Kappa myeloma Immunoglobulin bindet. Diese Affinität besteht auch für Human und Rat Igs insbesondere Kappa oder Polyklonal Igs. Phosphatse enzymatische Aktivität kann man auch für eine Substrat-Analog-Abtrennung benutzen.The advantage is that the immobilized phosphatase or 70KD protein from their biological properties like that is designed to be both polyclonal IgG, IgM, IgA and Kappa myeloma immunoglobulin binds. This affinity is there also for Human and Rat Igs especially kappa or polyclonal Igs. Phosphatse enzymatic activity can also be used for a Use substrate-analog separation.

Claims (3)

Verfahren zur Anwendung von gereinigten aktiven Immunoglobulinen-Bindung-Staphylokokkal:
  • - Neutral Phosphatase.
  • - 70KD Protein.
Methods of using purified active immunoglobulin binding staphylococcal:
  • - Neutral phosphatase.
  • - 70KD protein.
Die Herstellung von Proteinen ist durch:
  • - Immunoaffinität-Reinigungs-Verfahren durch Anwendung von Anti-Phosphatase und Anti-70KD-Protein gekoppelte Antikörper zur aktiviertem Matrix,
  • - Ionenaustauschkromatography-Verfahren durch Anwendung von Kationenaustauscher-Matrix,
  • - Affinitätskromatography-Verfahren durch Anwendung von Immunoglobulin gekoppelt zur soliden Phase Matrix,
The production of proteins is through:
  • - immunoaffinity purification method using anti-phosphatase and anti-70KD protein-coupled antibodies to the activated matrix,
  • - ion exchange chromatography method using cation exchange matrix,
  • Affinity chromatography method using immunoglobulin coupled to the solid phase matrix,
dadurch gekennzeichnet, daß man
  • 1. eine Stimmulierung von terminalen Blutlymphozyten und Immunoglobulinsynthese erfüllt.
  • 2. Abtrennung von Immunoglobulinen, F(ab)′2 Teilen und die Substrat-Analoge von Phosphatase aus Lösungen durch Anwendung von immobilisierten Phosphatase und 70KD Protein zur aktiviertem soliden Phase Matrix.
 characterized in that one
  • 1. fulfilled a stimulation of terminal blood lymphocytes and immunoglobulin synthesis.
  • 2. Separation of immunoglobulins, F (ab) ′ 2 parts and the substrate analogues of phosphatase from solutions by using immobilized phosphatase and 70KD protein for the activated solid phase matrix.
DE19904037809 1990-11-28 1990-11-28 Staphylococcal neutral phosphatase and 70 KD protein - isolated by immuno:affinity purificn., ion exchange chromatography or affinity chromatography and used to stimulate lymphocytes and bind immunoglobulins Withdrawn DE4037809A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001019863A1 (en) * 1999-09-13 2001-03-22 Microbiological Research Authority Preparation of highly pure toxin fragments

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001019863A1 (en) * 1999-09-13 2001-03-22 Microbiological Research Authority Preparation of highly pure toxin fragments
US7193066B1 (en) 1999-09-13 2007-03-20 The Health Protection Agency Preparation of highly pure toxin fragments

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