11. Massenspektrometrie. II

10.4

145

Determination of Serum Insulin by Solid Phase Enzyme Immunoassay in Antibody Coated Tubes

G. Kleinhammer, H. Lenz, R. Linke, and W. Gruber

Boehringer Mannheim GmbH, Biochemica Werk Tutzing, Forschungszentrum, D-8132 Tutzing

Bestimmung yon Serum-Insulin im Solid-Phase Enzym-Immunoassay unter Verwendung Antik6rper-beschiehteter Polystyrolgliischen

Best. yon Insulin in Serum; Enzym-Immunoassay; antik6rperbeschichtete Glfischen

Using an Insulin-HRP-conjugate as tracer an enzyme immunoassay in antibody coated tubes for determina- tion of insulin was developed. The conjugate was synthesized according to N A K A N E with slight modi- fications and purified via ion-exchange and gel chro- matography. It could be used at least for 6 months when stored in the refr igerator .

Polystyrene tubes were coated with the y-globulin- fraction of guinea-pig anti-insulin anti-serum usually diluted.

The assay is performed by incubating first 100 gl of insulin standard solution or serum sample over night at 4~ in a total volume of 1.0 ml. Then, after washing the tubes with tap-water, I ml of a solution of insulin-HRP-conjugate is incubated in the tubes for 2 h at 4 ~ C. Finally, the conjugate- solution is aspirated, whereafter the HRP-activity bound to the tube wall is determined at pH 5.0 using 2,2'-azino-di- [3-ethyl-benzothiazoline-sulphonate (6)] (ABTS | as chro- mogen. The absorbance is read after 45 min at 405 nm.

The sensitivity of the assay, i.e., the amount of insulin displacing 10~o of the initially bound tracer was found to be 0 .5 -0 .75 pU of insulin per tube.

An inter-assay coefficient of variation (n = 10) of 11 ~o was obtained using a serum with about 80 p.U of insulin/ml.

Upon testing a panel of 18 human sera spiked with different amounts of insulin by enzyme- and radio- immunoassay in antibody coated tubes a coefficient of correlation between these two methods of r = 0.98 was found. The recovery of 60 and 150 pU of insulin/ ml was 113 and 114 ~ , respectively.

11. Massenspektrometrie. H 11.1

Determination of Cholesterol in Serum using Mass Fragmentography - a Reference Method in Clinical Chemistry

L. Siekmann, K. P. Hfiskes, and H. Breuer

Inst. f. Klin. Biochemie, Universitfit Bonn, Venusberg, D-5300 Bonn 1

Massenfragmentographische Bestimmung yon Cholesterin im Serum - eine kliniseh-chemische Referenzmethode

Best. von Cholesterin im Serum; Chromatographic, Gas/ Massenspektrometrie

Isotopic dilution-mass spectrometry (ID-MS) is widely accepted as a reliable analytical technique for the highly accurate determination of volatile compounds in biological material [1,2]. In the present investiga- tion, a mass fragmentography procedure (MF) for the quantitative determination of cholesterol in human serum was developed which makes use of the prin- cipIes of mass spectrometry and of isotopic dilution.

Cholesterol was measured in 100 serum samples by the use of this method; the analytical data are compared with those obtained (a) by the Lieber- mann-Burchard reaction [4], (b) by the enzymatic method according to R6schlau et al. [3] and (c) by a gas liquid chromatography (GLC) method.

0.5 pl of serum and 100 nCi = 1.8 nmol of [4-14C]cho- lesteryl oleate (55.5 mCi/mMol) are added to ethanolic KOH solution. Hydrolysis of the cholesteryl esters is performed at 55~ in 30 rain, and free cholesterol is extracted with 5 ml of cyclohexane. The organic solvent is evaporated and the residue reacted with N-methyl-N-trimethylsilyl trifluoroacetic amide (MSTFA) to form the TMS ether derivative of cho- lesterol. 5 pl of the reaction mixture are injected into the gas chromatography-mass spectrometer (GC-MS); the instru- ment is adjusted to record the m/e values 458 and 460. A mass fragmentogram obtained from the analysis of a serum sample is shown in Fig. 1. The intensities of the peaks of cholesterol (/458) and 14C-cholesterol (/46o) are determined