Antibodies

Recombinant antibodies within the ULTRA-DD project

Within work package 2 of the ULTRA-DD project one of the aims is to generate recombinant antibodies to selected target proteins to be used as research tools. A subset of these antibodies will be declared biological probes. These antibodies are targeting proteins present in the extracellular matrix and must be shown to have an effect on live cells in an assay format or to have a diagnostic use by detection of specific proteins in biological samples. The best antibody for each target protein will be made available through ModiQuest Research.

Antibody generation method:

A core aim of the SGC labs has always been the ability to produce high-quality proteins. Over the years, SGC labs have been able to produce thousands of expression clones and protocols for medically relevant proteins. From this gold mine we can now cherry pick the proteins of highest importance to our partner projects and use them as antigens for antibody generation. The SGC KI team is collaborating closely with the Drug Discovery and Development Platform at the Science for Life Laboratory in Stockholm. DDDP contributes with state-of-the-art combinatorial libraries of human antibody candidates where high-quality binders can be obtained through phage display technology. In contrast to traditionally used methods to generate antibodies that rely on immunizations of animals, phage display is animal-free and yields recombinant antibodies, which are completely renewable and can be converted to any desired format depending on downstream application.

Antibody validation method:

Selected antibody candidates are first validated for their binding against their antigen using various immune-based assays in 96-well format. Binders that pass this initial validation are then subjected to cell-based validation, testing whether they can bind their full-length target antigens produced endogenously in a human cell. This is done via immunoprecipitation – mass spectrometry, or IP-MS for short. The strength of this method is in its ability to detect not only the target protein that the antibody pulls down, but also its protein complex partners, and potential cross-reactivity targets. Compared to other traditional methods like Western blot where you see only a band on a gel, IP-MS also provides the identity of each protein in the immunoprecipitate.

Antibody validation criteria:

  • Binds its target antigen with an affinity of at least 20 nM
  • Can IP endogenous FL target protein from HEK293 cell lysate (or other relevant cell line)
  • Specific in IP-MS, or at least any cross-reactivity is known

Additional for biological probes:

  • Functional activity towards a target in the extracellular space measured in a cell-based assay
  • Diagnostic detection of proteins in biological samples

 

Protein Family Gene Gene ID UniProt ID Antigen domain Target Annotation Comment IP Modiquest Product
Bromodomain BRD4 23476 O60885 333-460 of 1362, Bromodomain 2 bromodomain-containing protein 4 isoform long   scFv, IgG MQR2.1201
Methyltransferases PRMT3 10196 O60678 211-531 of 531, HMT protein arginine methyltransferase 3 isoform 1   scFv, IgG MQR2.1601
WD40 repeat proteins WDR5 11091 P61964 1-334 of 334, Full-length WD repeat domain 5 [Homo sapiens]   scFv, IgG MQR2.1301
PWWP BRPF3 27154 Q9ULD4 1056-1195 of 1205, PWWP domain Bromodomain and PHD finger-containing protein 3   scFv, IgG MQR2.1401
PWWP BRD1 23774 O95696 925-1049 of 1058, PWWP domain Bromodomain-containing protein 1   scFv, IgG MQR2.1501
Bromodomain BRD4 23476 O60885 44-168 of 1362, bromodomain 1 bromodomain-containing protein 4 isoform long   scFv  
Bromodomain SMARCA4 6597 P51532 1451-1580 of 1647, Bromodomain SWI/SNF-related matrix-associated actin-dependent regulator of chromatin a4 isoform B Brings down complete SWI/SNF complex scFv  
Bromodomain SP140 11262 Q13342 688-862 of 867, Bromodomain Nuclear body protein SP140   scFv  
Genome integrity SPRTN 83932 Q9H040 1-216 of 489 SprT-like N-terminal domain   scFv  
Genome integrity CDAN1 146059 Q8IWY9 840-1202 of 1227 codanin-1   scFv  
Metabolism GALT 2592 P07902 1-379 of 379, Full-length galactose-1-phosphate uridylyltransferase   scFv  
Metabolism DHTKD1 55526 Q96HY7 45-919 of 919 dehydrogenase E1 and transketolase domain containing protein 1   scFv  
Transcription Factor IRF5 3663 Q13568,Q64GB1 M1-V120 (DBD) and E232-L434 of 498 interferon regulatory factor 5 Detected IRF5 in plasma serum scFv  
Chaperone SACS 26278 Q9NZJ4 1-339 (Ubl+sr1), 4441-4579 (HEPN) and 84-339 (sr1) of 4579 Sacsin, DnaJ homolog subfamily C member 29.   scFv  
tRNA synthetases SARS 6301 P49591 V2-A514 of 514, Alex' target seryl-tRNA synthetase   scFv  
tRNA synthetases WARS 7453 P23381 P2-Q471 of 471 tryptophanyl-tRNA synthetase   scFv  
tRNA synthetases HARS 3035 P12081 1-509 of 509, Catia's main target histidyl-tRNA synthetase   scFv  
tRNA synthetases KARS 3735 Q15046 A2-V597 of 597 lysyl-tRNA synthetase   scFv  
tRNA synthetases GARS 2617 P41250 55-739 of 739, Alex' target, Catia's target glycyl-tRNA synthetase   scFv  
tRNA synthetases NARS 4677 O43776 V2-P458 of 458 Catia's target asparaginyl-tRNA synthetase   scFv  
tRNA synthetases YARS2 51067 Q9Y2Z4 A31-L477 of 477 tyrosyl-tRNA synthetase 2, mitochondrial   scFv  
tRNA synthetases YARS 8565 P54577 G2–S528,G2–Y341 of 528 Catia's target tyrosyl-tRNA synthetase, cytoplasmic   scFv  
tRNA synthetases RARS 5917 P54136 D2-M660, T70-M660 of 660 arginyl-tRNA synthetase Brings down complete Multisynthetase complex scFv  
tRNA synthetases TARS 6897 P26639 F2-F723, N320-F723 3 of 723 Catia's target threonyl-tRNA synthetase   scFv  
Metabolism AASS 10157 Q9UDR5 455-926 of 926 aminoadipate-semialdehyde synthase TEP target scFv  
tRNA synthetases DARS 1615 P14868 P2-P501 of 501 aspartyl-tRNA synthetase   scFv  
tRNA synthetases AARS 16 P49588 D2-K455, D2-968 of 968 Catia's target alanyl-tRNA synthetase Antibodies with separate epitopes scFv  
tRNA synthetases MARS 4141 P56192 R2-L225 of 900, methionyl-tRNA synthetase Brings down LARS as well scFv  
tRNA synthetases LARS 51520 Q9P2J5 260-509 of 1176 leucyl-tRNA synthetase Brings down complete Multisynthetase complex scFv  
Myositis FHL1 2273 Q13642 323 aa (work on isoform 1, 280 aa) Four and a half LIM domains protein 1   scFv  
Interleukin IL8 3576 P10145 28-99 of 99 (1-20 signal peptide, CPR 21-99) interleukin-8   scFv  
SP type I MP TREM2 54209 Q9NZC2 19-131 of 230 Triggering receptor expressed on myeloid cells 2   scFv  
Metabolism ACACA 31 Q13085 1572 - 2338 of 2346 Acetyl-CoA carboxylase 1   scFv