FabRICATOR for Antibody Fragmentation

Transcription

1 FabRICATOR for Antibody Fragmentation Fab and F(ab )2 antibody fragments are used in many applications or assays where the presence of the Fc region may cause problems with background staining (cells with Fc receptors) or complement binding (plasma). The divalency of F(ab )2 fragments also enables antigen crosslinking making it useful for rosetting, cellular aggregation, and precipitation assays. FabRICATOR Utilizing the unique specificity of a novel proteolytic enzyme, Genovis has developed a product to rapidly generate precise F(ab )2 fragments. FabRICATOR cleaves IgG at a singular defined site below the hinge region to yield F(ab )2 fragments in minutes. Why FabRICATOR? Rapid F(ab )2 fragments Complete cleavage of F(ab )2 fragments in minutes. Exquisite specificity Precise IgG cleavage ensures full-length fragments without subsequent degradation. Unrivaled yields and consistency Precision and efficiency results in unmatched yield and reaction to reaction consistency Lane 1 Molecular weight marker Mark 12 (Invitrogen) Lane 2 IgG fraction Lane 3 5 min digestion with FabRICATOR Lane 4 15 min digestion with FabRICATOR Lane 5 30 min digestion with FabRICATOR Lane 6 30 min digestion with FabRICATOR (reduced sample) Lane 7 After removal of Fc fragments Lane 8 After removal of FabRICATOR Lane 9 Molecular weight marker Mark 12 IgG species cleaved by FabRICATOR Human IgG Mouse IgG Rabbit IgG Rat IgG Monkey IgG Sheep IgG Goat IgG Bovine IgG Porcine IgG (IgG2a, IgG3) For Research Use Only. t for use in diagnostic or therapeutic procedures. QA0-FR1-020 FabRICATOR, cleaves 2mg IgG QA0-FR1-050 FabRICATOR, cleaves 5mg IgG Manufactured for QED Bioscience by Instructions for use on next page:

2 FabRICATOR for Antibody Fragmentation Instructions for product no. QA0-FR1-020 for cleavage of 2mg IgG QA0-FR1-050 for cleavage of 5mg IgG Product Description FabRICATOR is an enzyme used for preparation of F(ab )2. FabRICATOR is a digestive enzyme that cleaves IgG only at one specific site below the hinge region resulting in F(ab )2 and Fc-fragments. FabRICATOR is for Research Use Only and not for use in diagnostic or therapeutic procedures. Content and storage FabRICATOR is supplied as lyophilized powder from 50 mm sodium phosphate, 150 mm NaCl, ph 6.6, with no preservatives added. Contains sufficient material to produce F(ab )2 fragments from up to 2 mg IgG (QAO-FR1-020) or up to 5mg IgG (QA0-FR1-050). FabRICATOR is shipped on ice. It should be stored at -20 o C upon arrival. After reconstitution FabRICATOR is stable for 1 month at 2-8 o C. Unit Definition One unit cleaves 95% of 1ug IgG when incubated in 50mM sodium phosphate ph 6.6, 150mM NaCl at 37 o C for 30min. Quality Control FabRICATOR is tested to ensure lot-to-lot consistency. FabRICATOR is tested for absence of microbial contamination with blood agar plates, Sabaraud dextrose agar plates and fluid thioglycolate medium. Additional Materials Required 50mM sodium phosphate, 150mM NaCl, ph 6.6. Method Reconstitute QA0-FR1-020 in 30ul double distilled H 2 O and QA0-FR1-050 in 75ul double distilled H 2 O. This gives a concentration of 67 units/ul. To prevent microbial contamination, sodium azide can be added to the solution to a final concentration of % (w/v). 1mg IgG is digested by adding 1000 units (15uL) FabRICATOR and incubating for 30 minutes in 50mM sodium phosphate ph 6.6, 150mM NaCl at 37 o C. FabRICATOR contains a His-tag and can therefore easily be removed after digestion. The Fc fragments can be removed with Protein A or Protein G. To recover Fc fragments, simply apply the digested IgG to a Protein A or Protein G chromatography column. F(ab )2 fragments will come out in the column flow-through; Fc fragments can be eluted from the column as one would elute intact IgG. Best activity is obtained at ph 6.6 and 37 o C. It is possible to use a buffer with a higher ph and increasing the reaction time. Digestion can also be done at room temperature with prolonged incubation time. Additional Information: Cleavage of mouse IgG2a with FabRICATOR requires extended incubation time and higher concentration of FabRICATOR as compared with cleavage of human IgG. Using 5 units of FabRICATOR enzyme per ug mouse IgG2a with an incubation time of 2-3

3 hours is suggested, but the enduser might find it necessary to optimize these conditions for their sample of mouse IgG2a. It is possible to get F(ab )1 fragments from F(ab )2 fragments. After FabRICATOR cleavage, use 2-MEA (2-mercaptoethylamine) at a concentration of 50mM to break the disulfide bond in F(ab )2 fragments. FabRICATOR has been optimized for use at ph 6.6 and 37 o C. FabRICATOR is active at room temperature, but the reaction time should be increased by 15 min. Optimization studies are recommended before changing any other conditions. FabRICATOR is active in the presence of anticoagulants. FabRICATOR cleaves IgG in serum as well as purified IgG. Product References Ryan MH et al. Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid, Molecular Immunology 45: , Åkesson, P et al. IdeS, a highly specific immunoglobulin G (IgG)-cleaving enzyme from Streptococcus pyogenes, is inhibited by specific IgG antibodies generated during infection. Infection and Immunity 74: , Hess, J., L et al. Immunoglobulin clevage by the streptococcal cysteine proteinase IdeS can be detected using protein G capturing and mass spectrometry. J Microbiol Meth 70: , von Pawel-Rammingen, U et al. IdeS, a novel streptoccocal cysteine proteinase with unique specificity for immunoglobulin G. The EMBO Journal 21: , Vincents, B et al. Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine proteinase with strict specificity for IgG cleveage due to exosite binding. Biochemistry 43: , Wenig, K et al. Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG. Proceedings Natl Acad Sci USA 101: , Application te on next page:

4 FabRICATOR - perfect F(ab )2 fragments in minutes Sarah Fredriksson, Martin Kullman and Fredrik Olsson Genovis AB Introduction Fab and F(ab )2 antibody fragments are used in applications or assays where the presence of the Fc region may cause problems. If antibodies are used for staining of a specific target in tissues like spleen, lymph or in peripheral blood preparations, cells with Fc receptors (macrophages, monocytes, natural killer cells and B lymphocytes) can bind to the Fc region of the antibody and cause a background staining in areas that do not contain any antigen (1). Fab and F(ab )2 fragments are also desirable for staining or binding to cell preparations in the presence of plasma since they can not bind to complement, which would lyse the cells (2). The divalency of the F(ab )2 fragment enables it to cross-link antigens and hence makes it useful for rosetting, cellular aggregation and precipitation assays (3). F(ab )2 fragments are generally produced using pepsin. Here a novel method for antibody fragmentation using the enzyme FabRICATOR is introduced. This enzyme is a partially modified and his-tagged IdeS enzyme (4). FabRICATOR cleaves IgG in the hinge region leaving an intact F(ab )2 and two residual Fc fragments. Materials and Methods Generation of F(ab )2 fragments digestion of IgG 1mg of polyclonal human IgG (including all subclasses) was digested with 0.1nmol FabRICATOR for 30 minutes in 0.1M sodium phosphate buffer ph 6.6, 150mM NaCl at 37 C. Samples were withdrawn after 5,15 and 30 minutes for analysis on SDS Page ge lelectrophoresis. Purification of antibody fragments Particles carrying protein A and magnetic particles carrying chelated nickel ions (both particles are under development for the FabRICATOR kit) were added to the digested IgG in order to remove Fc fragments and FabRICATOR. The particles were added subsequently to make it possible to analyze each separation step separately. Both separation steps were carried out at room temperature for 30 minutes and then the particles were collected with a lab magnet. Sample from both separation procedures were collected for analysis on SDS PAGE gel electrophoresis. MW (kda) Lane 1 Molecular weight marker Mark 12 (Invitrogen) Lane 2 IgG fraction Lane 3 5 min digestion with FabRICATOR Lane 4 15 min digestion with FabRICATOR Lane 5 30 min digestion with FabRICATOR Lane 6 30 min digestion with FabRICATOR (reduced sample) Lane 7 After removal of Fc fragments Lane 8 After removal of FabRICATOR Lane 9 Molecular weight marker Mark 12

5 Analysis on SDS PAGE gel Cleavage products were analyzed by SDS PAGE in 4 to 12% gradient gels under both reducing and non-reducing conditions. Gels were stained using Simply Blue Safe Stain (Invitrogen) according to protocol recommended by the supplier. Results and Discussion All four subclasses are readily cleaved after 30 minutes of incubation with FabRICATOR and already after 5 minutes, the digestion is almost complete. The 150 kda IgG molecule is cleaved into one larger fragment of approximately 100 kda and one smaller fragment of 25 kda (the Fc region of the antibody). Upon reduction of the disulfide bridges, the 100 kda fragment is visible as two bands on the SDS Page gel, one 31 kda band and the light chain of 24 kda. After treatment with protein A and Ni chelating magnetic particles the Fc parts and FabRICATOR have been successfully removed from the reaction tube leaving the resulting pure F(ab )2 fragment in the supernatant. Conclusions FabRICATOR has unique proteolytic properties and hence serves as an excellent tool for antibody fragmentation. The yield of the reaction is high and the enzyme does not further digest the resulting products. In combination with magnetic particles it is possible to produce pure F(ab )2 fragments using FabRICATOR in less than 60 minutes with an easy and very user friendly process. References 1. Saito, K. et al., Decreased Fc gamma receptor III (CD16) expression on peripheral blood mononuclear cells in patients with Sjogren's syndrome. J. Rheumatol., 25, (1998). 2. De Reys, S., et al., Fc-independent crosslinking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation. Blood, 84, (1994). 3. de Saint Martin, J., Idiotypic and antiidiotypic determinants on lymphocytes during anti-rh immunization. Rev. Fr. Transfus. Immunohematol., 26, (1983). 4. von Pawel-Rammingen, U. Johansson, B.P., and Björck, L. IdeS, A novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO Journal 21, 7 ( ), 2002.