Determination of proinsulin intermediates by means of an immunoradiometric method using polyclonal antibodies.
In our endeavour to develop a method for proinsulin determination, an immunoradiometric method was developed. Thereby guinea-pig antibodies to bovine insulin which were purified with an immunoadsorbent, were, in surplus, coupled to polyethylene tubes. These tubes were used to extract proinsulin and insulin from the sample and standard that or was to be determined. The proinsulin adsorbed onto the wall of the tube was distinguished from insulin by incubating, in a second step, a rabbit antibody to human C-peptide with the tubes. In order to render the proinsulin anti-C-peptide complex measurable, a donkey antibody to rabbit IgG was used, which had been purified via an immunoadsorbent and which was labelled with iodine-125. Since proinsulin extracted from human pancreata was available next to biosynthetic human proinsulin, it was striking to note that these substances were very differently recorded by the determination method applied. Thus biosynthetic human proinsulin dissolved in gelatin buffer could not be measured at all. After mild tryptic cleavage of the biosynthetic human proinsulin, a clear increase of the immunoreactivity was seen in this method. Therefore the claim could be made that partially cleaved proinsulin molecules with a retained C-peptide structure had come into existence. This could be verified by application of the proinsulin cleavage products 65/A1 and 32/33, which exhibited a behaviour very similar to that of pancreatic proinsulin in this method. In this way we could demonstrate that the originally planned immunoradiometric determination method for proinsulin, recorded partially hydrolized intermediates of the proinsulin, which represent a large part of the proinsulin immunoreactivity in the serum.
Zilker
T R
TR
II. Med. Klinik der Techn., Universität München, Germany.
Rainbow
S J
SJ
Hales
C N
CN
Saldern
S v
Sv
Gray
I P
IP
D000906:Antibodies / Q000276:immunology; D002021:Buffers; D002096:C-Peptide / Q000276:immunology; D005780:Gelatin; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007106:Immune Sera / Q000276:immunology; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods; D007074:Immunoglobulin G / Q000276:immunology; D007163:Immunosorbent Techniques; D007328:Insulin / Q000276:immunology; D007457:Iodine Radioisotopes; D010179:Pancreas / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D011095:Polyethylenes; D011384:Proinsulin / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
D000906:Antibodies; D002021:Buffers; D002096:C-Peptide; D007106:Immune Sera; D007074:Immunoglobulin G; D007328:Insulin; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011095:Polyethylenes; D005780:Gelatin; D011384:Proinsulin; D014357:Trypsin