Production and characterisation of monoclonal antibodies to the mid-region 37-67 sequence of parathyroid hormone-related protein.
The production and characterisation of monoclonal antibodies (MAb) to the mid-region sequence 37-67 of human parathyroid hormone-related protein (PTHRP) is described. In spite of the poor immunogenicity of this sub-fragment of PTHRP, a high percentage of specific hybrids were produced by boosting with conjugate and free peptide prior to cell fusion. Seven of the MAbs produced cross-reacted with PTHRP37-67, PTHRP1-86 and native forms of PTHRP. Inhibition studies with peptide sub-fragments of PTHRP37-67 indicated that the majority recognised the 45-59 region. In a RIA for PTHRP1-86, detection limits ranged from 0.17 to 0.9 ng PTHRP1-86/tube, and no cross-reaction was found with PTH1-84. Two MAbs 1D11 and 4B10 were shown to be of potential use in measuring PTHRP1-86 in a two-site immunoradiometric assay in combination with either a solid phase consisting of a MAb to PTHRP1-34, or iodinated affinity purified rabbit antibodies to PTHRP1-34. MAb 1D11 coupled to Sepharose was suitable for immunoextraction of PTHRP, and successfully localised PTHRP on immunoblots. Two additional MAbs were produced which recognised an epitope unique to PTHRP37-67 located in the 37-46 region of the peptide.
Wolfson Research Laboratories, Department of Clinical Chemistry, Queen Elizabeth Medical Centre, Birmingham, U.K.
W A
WA
Ratcliffe
S J
SJ
Bowden
J
J
Emly
S
S
Hughes
J G
JG
Ratcliffe
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D002846:Chromatography, Affinity; D003429:Cross Reactions; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D015151:Immunoblotting; D007132:Immunoglobulin Isotypes; D015592:Immunoradiometric Assay; D051379:Mice; D008807:Mice, Inbred BALB C; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000276:immunology; D011506:Proteins / Q000276:immunology*; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007132:Immunoglobulin Isotypes; C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins