Direct IgG epitope mapping on bacterial AB toxins by cryo-EM

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

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SAT0031 Fine epitope mapping of immunodominant peptide epitopes by affinity matrix technology using yersinia-hsp60-specific synovial cd4+t-cells in reactive arthritis (rea) patients

10.1136/annrheumdis-2001.383 ◽

2001 ◽

Author(s):

A Thiel ◽

P Wu ◽

D Stauch ◽

M Rudwaleit ◽

A Radbruch ◽

...

Keyword(s):

T Cells ◽

Reactive Arthritis ◽

Cd4 T Cells ◽

Epitope Mapping ◽

Affinity Matrix ◽

Peptide Epitopes

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MONOCLONAL ANTIBODIES OF THE IgM AND IgG CLASS SPECIFIC FOR CROSSLINKED FIBRIN DEGRADATION PRODUCTS

10.1055/s-0038-1643651 ◽

1987 ◽

Author(s):

P J Gaffney ◽

L J Creighton ◽

A Curry ◽

B MacMahon ◽

R Thorpe

Keyword(s):

Monoclonal Antibodies ◽

Thrombolytic Therapy ◽

Epitope Mapping ◽

Degradation Products ◽

Subcutaneous Route ◽

Fibrin Degradation Products ◽

Conformational Epitopes ◽

Immunoglobulin Class Switching ◽

Unique Specificity ◽

D Dimer

Monoclonal antibodies (mabs) to crosslinked fibrin degradation products (XL-FDP) having the general formula D/Y[X]nY/D (known as X-oligomer) and D-D (known as D dimer) have been raised in balb/C mice by both a novel mtrasplenic and a conventional subcutaneous route of immunisation and by combinations of both these procedures. Mabs to X-oligomers (NIBn 52 and NIBn 123) obtained by an intrasplenic procedure have been demonstrated to crossreact only with X-oligomer in a 2-site ELISA procedure and not with D dimer or whole fibrinogen and have been shown to be of value m the examination of clinical material obtained from patients with various types of thrombosis and have also been useful in monitoring the efficacy of thrombolytic therapy. The X-oligomer mabs are immunoglobulins of the M class. It was demonstrated that their unique specificity for conformational epitopes on the large X-oligomer fragments does not reside in the IgM structure since alterative immunisation procedures have been used to generate mabs of the IgG class which have the same specificity. Using immunoglobulin class switching in culture rather than during immunisation was suggested by certain cell lines which produced both IgM and IgG specific for X-oligomer. This latter point needs rigorous validation.Immunoglobulin G type mabs to highly purified D dimer were raised by conventional subcutaneous immunisation of balb/C mice. One of these, NIBn-11, was found to crossreact with PVC-immobilised X-oligomer and D dimer but not with fibrinogen. However NIBn-11 did not bind to D dimer in a 2-site ELISA procedure while crossreactmg quite avidly with X-oligomer. This suggests that the D dimer epitope to which NIBn-11 is directed is expressed in some conformations and not m others and that these conformations are always expressed in the complex X-oligomer group of fragments. These mabs, whilst of value in measuring certain unique fibrin fragments m plasma, are useful in the epitope mapping of fibrinogen/fibrin and their plasmm-mediated

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