Summary: We developed an enzyme immunoassay (ELISA) for quantitation of plasma cholinesterase substance concentrations in native plasma or serum samples. The ELISA assay is based on polyclonal (rabbit) antihuman cholinesterase and a highly specific monoclonal (mouse) antibody, with a commercially available peroxidase-conjungated (rabbit) antibody directed against mouse immunoglobulins as the signal carrier. The detected serum cholinesterase substance concentrations (mean: 4.51 mg/1, SD: 0.90 mg/1) in randomly selected serum samples from 33 healthy individuals were closely and linearly related to the corresponding catalytic activity concentrations.
The hydrophilic, salt-soluble (SS) form of acetylcholinesterase (AChE) from bovine brain caudate nucleus exists mainly as a tetramer sedimenting at 10 .3S
Monoclonal antibodies were raised against amphiphilic detergent-soluble (DS) acetylcholinesterase (AChE) from human brain caudate nucleus. Three mAb, 132-4 (IgG,), 132-5 (IgG,) and 132-6 (IgG,), specific for brain DS-AChE were selected and subcloned. These mAb reacted with native as well as heat-denatured and SDS-denatured DS-AChE, indicating that the epitopes to which mAb bound are continuous determinants. The mAb cross-reacted with DS-AChE from bovine and mouse brain and with brain DS-AChE from river trout (Sufmo rrurra f o m f u r i o ) and lake trout (SuZmo rrurru f o m u Zacusrris). No cross-reaction was detected with the following antigens : salt-soluble (SS) AChE from bovine brain, glycophospholipid-anchored AChE from human and bovine erythrocytes, DS-butyrylcholinesterase and SS-butyrylcholinesterase (BtChE) from the brains of human and bovine, DS-BtChE from chicken and BtChE from human serum. Deglycosylation of brain DSAChE with N-glycosidase F did not abolish the binding of mAb to DS-AChE. After reduction of brain DS-AChE by dithiothreitol, the mAb no longer reacted with the antigen, indicating that a disulfide bridge is important for the epitope. Monomerization of brain DS-AChE by trypsin and limited proteinase K treatment also abolished the binding of mAb to DS-ACE. Sucrose-densitygradient centrifugation showed that mAb reacted only with native tetrameric forms, but not with dimeric and monomeric forms. Western blot, after SDSPAGE under non-reducing conditions, showed that mAb reacted with those subunits carrying the hydrophobic anchor (i.e. tetramers, himers and heavy dimers) but not with those devoid of it (light dimers or monomers). Since mAb 132-4,132-5 and 132-6 recognized DS-ACE from fish up to mammalian brain in the evolutionary tree, it is concluded that the epitope to which these mAb bind, is conserved in nature.
Summary. A total of 111 amniotic fluid samples, clear or blood stained, with elevated levels of alpha‐fetoprotein and acetylcholinesterase was analysed by immunoassays specific for acetylcholinesterase and butyrylcholinesterase and the acetylcholinesterase/butyrylcholinesterase‐ratios determined. Samples from 40 pregnancies associated with anen‐cephaly, 47 pregnancies associated with open spina bifida or encephalocele and six pregnancies with fetal intrauterine death or miscarriage all had ratios of >0.14. All 11 pregnancies with fetal ventral wall defects had ratios <0.14 as had four pregnancies with normal outcome and elevated levels of alpha‐fetoprotein and acetylcholinesterase. Three fetuses with both open spina bifida and ventral wall defects were associated with ratios above 0.14. These results suggest that immu‐nochemical determination of acetylcholinesterase and butyrylcholinesterase can be used to distinguish pregnancies complicated by anencephaly, open spina bifida, encephalocele and miscarriage from those with ventral wall defects and samples with false positive elevated levels of alpha‐fetoprotein and acetylcholinesterase. The procedure is accurate and simple to carry out and well suited to routine use in a clinical chemistry laboratory.
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