How to use PS_380 antibody array of ArrayIt/BSI? BSI s monoclonal AB-s recognise accessible epitops on native plasma proteins

Transcription

1 How to use PS_380 antibody array of ArrayIt/BSI? BSI s monoclonal AB-s recognise accessible epitops on native plasma proteins

2 BSI s monoclonal antibodies......are made against mixtures of native proteins therefore BSI s antibodies are able to recognize accessible epitopes on these native polypeptides; Proteins in their native configuration, harbor numerous potential posttranslational modifications and carry the imprints of their also numerous interactions with other (macro)molecules including other proteins. Our goal is to cover all of the natural epitopes with at least one antibody. These antibodies are primarily planned to be used in experiments where no preconception is set toward antibody specificity and so these antibodies can serve as discovery tools capable of targeting high- as well as low-abundant analytes in complex, non-fractionated proteomes. The resolution of such proteome profiling efforts correlate directly with the number of antibodies included and specificity becomes important if new discoveries have been made. Therefore customer should do the profiling experiment first then characterize the selected target him/herself, or buy this service from BSI.

3 Protein ID of BSI s monoclonal antibodies... is not planned to be communicated, although a customer might have access to our continuously developing database for a price (final policy on this is currently being worked out); The protein ID of the antibodies at BSI is determined with either multiple immune-affinity chromatography or immunoprecipitation using antigenic mixtures where the representation of low abundance proteins is gradually (step after step) is increasing and the components of the immune-precipitates are detected by methods with increasing sensitivity. The protein ID of approximately one third of the antibodies on PS380 have been solved. From previous experiences at BSI we know that routinely (at the first two experimental levels) approximately 40% of the ID-s can be solved; in the rest of the cases the antigen is most likely low abundant protein, the identification of which requires more specific effort. In case the client is interested in the specificity of an antibody, this antibody can be placed in the frontline for ID-determination or the client can buy a limited amount of antibody for her/his own investigations.

4 Protein ID of BSI s monoclonal antibodies... Our protein IDs are experimental evidences that have not been fully validated so they only serve as guides for the end user, who will have to perform new experiments and characterize the cognate protein he/she identifies in a certain disease condition. We have plenty of data ( as the literature) to support the notion that pathological proteins may be different from what we ID-d. We also have data that demonstrates protein protein interactions in the blood, thus once you capture one protein by a mab you may pull down many others, then the ID interpretation (based on our current knowledge) is not necesserily straightforward. On the next few slides some examples of the characteristics of PS_380 antibodies are shown

5 Some examples of the antibodies on the PS_380 array recognizing different abundance antigenes in human plasma Recognized protein Complement component BSI AB-library Hemopexin C1q Ceruloplasmin C1r Fibronectin a-1-b-glycoprotein BP C5 Afamin C6 Vitamin D-binding protein C7 C8 Prothrombin C9 Serum amyloid P-component Factor H S-protein

6 BSI s monoclonal antibody library contains several members recognizing human complement component containing complexes. In most of the cases these complexes contain more than one complement component, and sometimes other plasma proteins as well. AB C1q C1r C1s C2 BP C5 C6 C7 C8 C9 Factor H Factor I S-protein Other proteins in the precipitated complex C1q A B C D E, not definrd Afamin C9 Afamin/Albumin FactorH BP S-protein F BP BP S-protein Factor H, not definrd C9 BP, not definrd G C7, not definrd FactorH BP, not definrd C6 FactorH C6 BP S protein C9 BP, not definrd Factor H Factor H RP

7 The epitopes recognized by the PS_380 antibodies on the same proteins (Complement component 3 in this case) are very likely different from each other as it is shown here by the phage display results; A NHKWLPLGLQFP C QIDMN_AWFVLPP E FGLDWNTYLDSL NHKWLPLGLQFP _TSL--PWFIIP EYS FGLDWNTYLDSL NHKWLPLGLQFP SP--PWFVMPGTQA FGRDWNTYLDSL NHKWLPLGLQFP SP--PWFVMPGTQA FGRERNTYLDSL SVKSLPLGLHLT SP--PWFVMPGTQA FGLDWNTYLDSL EHRTLPMGLTEL SP--PWFVMPGTQA FGLDRNTYLDSL TVKWLPMFPPPS SP--PWFVMPGTQA FGLDWNTYLDSL AFKPMPMGLPPL _DKH--PWFITPSDW- FGLDWNTYLDSL LDKVLPLGLEPR DT--PWFIMPQLHP HYPYQHPDYIPD SP--PWFVMPGTQA HLEPDHPWFVMP F No epitope defined yet B -DYLKVHLNLRPP D --ADPESRWIQRLS-- G YSFQQRLWELKP-- -DYLKVHLNLRPP --SLPALYWQIDEP-- YSFQQRLWELKP-- -DYLKVHLNLRPP --SLPALYWQIDEP-- YSFQQRLWELKP-- RDYITQHYLLGP- --SLPALYWQIDEP-- YSFQQRLWELKP-- RDYITQHYLLGP- --SLPALYWQIDEP-- YSFQQRLWELKP-- RDYITQHYLLGP- --SLPALYWQIDEP-- -G-PRPAYPSLPEY -DYLKVHLNLRPP --SLPALYWQIDEP-- YSFQQRLWELKP-- -DYLKVHLNLRPP --ESC--LICGSDPHT YSFQQRLWELKP-- -DYWKVHLNLRPP --ESC--LICGSDPHT YSFQQRLWELKP-- RDYITQHYLLGP- TRHLP--FILPTGP-- YSFQQRLWELKP-- RDYITQHYLLGP- TRHLP--FILPTGP-- YSFQQRLWELKP-- RDYITQHYLLGP- APALP--DLLPTGR-- YSFQQRLWELKP-- YSTIP--FLLPTGR--