Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2.
A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type 1 diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M13) is an IgG1/kappa and in an immunofluorescence test M13 at 1 microg/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound (35)S-labelled IA-2 (26% at 100 microg/mL) and (125)I-labelled IA-2 (34% at 100 microg/mL) in an immunoprecipitation assay and reacted well with IA-2 in western blotting analysis. Amino acids 777-808 in the PTP domain of IA-2 were found to be important for M13 binding in an analysis using modified (35)S-labelled IA-2 proteins. M13 V region genes were from VH1-3, D3-22, JH4b, VKI DPK8/Vd+ and JK3 genes and showed a high replacement/silent mutation ratio for both the heavy (11.0) and the light (6.0) chain genes. Mouse monoclonal antibodies (mMAbs) reactive with at least three different epitopes within IA-2 aa 604-686 corresponding to the juxtamembrane domain were also obtained. F(ab')(2) or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(ab')(2) caused inhibition in only 6/22 sera. M13 is representative of some patient serum IA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2.
Ananieva-Jordanova
R
R
FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, CF14 5DU, UK.
Evans
M
M
Nakamatsu
T
T
Premawardhana
L D K E
LD
Sanders
J
J
Powell
M
M
Chen
S
S
McGrath
V
V
Belton
C
C
Arnold
C
C
Baker
S
S
Betterle
C
C
Zanchetta
R
R
Smith
B Rees
BR
Furmaniak
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D001323:Autoantibodies / Q000737:chemistry / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology*; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D018604:Epitope Mapping; D006801:Humans; D006825:Hybridomas; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000276:immunology*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; C096322:ICA512 autoantibody; D008565:Membrane Proteins; C024211:islet cell antibody; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516863:Ptprn protein, mouse; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8