Dry chemistry thin film immunoassay.

The authors report the development of a novel thin film multilayer immunoassay technology. The technology is applied to therapeutic drug monitoring and thyroid hormone testing in serum or plasma and can be extended to assays of other low molecular weight analytes. The assay detection range spans five orders of magnitude, from 1 x 10(-3) to 1 x 10(-8) M. The assay element comprises of multilayer coated chip, containing the active reagents in an agarose matrix and a plastic module serving both as a holder and a spreader. The assays are in the fluorescent competitive immunoassay format of the ligand displacement mode and are performed on an automated instrument with random access capability. The assays are fast and reliable and gave very good agreement when compared to reference methods.

Multilayer fluorescent immunoassay technique.

We describe a new multilayer immunoassay element for the determination of haptens in undiluted serum and plasma. Polysaccharide layers are coated onto a plastic base. The signal layer contains an immobilized antibody and a fluorescent-labeled hapten. A second layer, containing a pigment, acts as an optical screen. Sample spreading is achieved by a molded grid in contact with the upper layer of the immunoassay element. After sample is added to the element, endogenous analyte competes with the labeled hapten for the binding sites of the immobilized antibody; equilibrium is reached in 4-12 min. Because of the relative liquid-holding capacities of the layers and the grid, only a small amount of the free components remains in the signal layer. The signal is measured by front-surface fluorimetry. This technology has been applied to theophylline and thyroxin assays. Within- and between-run CVs range from 3% to 6%. Comparisons with fluorescent polarization immunoassays (Abbott TDx) showed excellent correlation (theophylline: r = 0.98, slope = 1.07, intercept = 0.3; thyroxin: r = 0.97, slope = 0.91, intercept = 0.8). The new method requires only one pipetting step (sample delivery) and is potentially applicable to a wide range of analytes.

Immunoelectron microscopy of amylase in the human parotid gland. Ultrastructural localization by use of both the protein A-gold and the biotin-avidin-gold technique.

Specimens of the human parotid gland were studied by immuno-electron microscopy for the presence of amylase. Both the protein A-gold technique and the biotin-avidin-gold technique were used on the same specimens. Different fixations were tried. Amylase was detected in the zymogen granules in high amounts. This enzyme could even be seen in glutaraldehyde fixed and routinely embedded material. The subcellular localization of this enzyme opens a new field of functional morphological studies and studies in special tumours including acinic cell carcinomas.

Amylase as an additional marker of salivary gland neoplasms. An immunoperoxidase study.

The presence of amylase in normal and neoplastic salivary gland tissue was investigated by immunoperoxidase techniques. Apart from normal and inflamed parotid glands, different kinds of tumours were studied with regard to amylase: acinic cell tumours, adenocarcinomas, adenoidcystic carcinomas, salivary duct carcinomas, mucoepidermoid tumours and squamous cell carcinomas. Amylase could be seen in acinic cell tumours, but not in other neoplasms. The results were discussed with respect to the diagnostic implications.

[A highly sensitive enzyme immunoassay for the determination of pancreatic lipase]. / Hochempfindlicher Enzymimmunoassay zur Bestimmung von Human-Pankreas-Lipase.

A highly sensitive and specific enzyme immunoassay for the determination of pancreatic lipase is described. The test follows the sandwich principle; total incubation time is 4.5 hours at 20--25 degrees C. Lipase concentrations between 3 and 300 micrograms/1 can be quantified (sample dilution 1 + 10); the lower detection limit is 0.3 micrograms/1 (sample dilution 1 + 1). Coefficients of variation from 2.9 to 6.5% for the in batch precision, and from 4.4 to 10.5% for the day-to day precision were found. A reference range from 7.7 to 56 micrograms/1 was calculated from the lipase concentrations in 369 serum samples from healthy adults (2.5 th--97.5 th percentile, median 23 micrograms/1). Very low concentrations were found in cord blood (median 2.8 micrograms/1). The lipase level remains quite constant between the ages of 3 and 50 years (median 15--20 micrograms/1); at the age of 70 years a median of 26 micrograms/1 is reached.

Enzyme immunoassay for the prostate-specific acid phosphatase (E. C. 3.1.3.2).

The clinical application of enzyme immunoassay for the determination of the prostate-specific acid phosphatase is reported. 227 sera were investigated in the diagnosis as well as in tumour monitoring and a good correlation with the clinical stage was found. In prostatic carcinomas 5 of 13 with stage T1, 11 or 12 patients with stage T2, 16 of 16 patients with T3 and 19 of 19 patients with stage T4 disease had values above 1 ng/ml. In prostatic adenomas (n = 69) in prostatitis (n = 40) and in other carcinomas of the urogenital tract (n = 28), renal carcinomas, carcinomas of the bladder and the penis) the values of the prostate-specific acid phosphatase measured by the enzyme immunoassay 131 of 137 were under 1 ng/ml. A comparison of random samples with the radioimmunoassay for this enzyme showed good correlation.

Coenzyme properties of NAD+ bound to different matrices through the amino group in the 6-position.

A method for the synthesis of N6-(2-aminoethyl)-NAD+ is given. The binding of this NAD+ derivative to different soluble and insoluble supports and the direct coupling of NAD+ to epoxyactivated Sepharose are described. Proofs are given that NAD+ is bound through the amino group in 6- position and the NAD+ derivative through the aliphatic amino group of the side chain. Non-enzymic reduction of the bound coenzyme to an almost quantitative extent is possible in all cases, but the enzymic reduction is largely influenced by the support. While N6-(2-aminoethyl)-NAD+ coupled to soluble dextran is nearly completely reducible by different dehydrogenases with a velocity of about 40% of that for free NAD+, the coenzyme bound to different insoluble matrices is very slowly reduced. Only 5% of the coenzyme derivative bound to BrCN-activated Sepharose are reducible, but 40% when it is bound through a spacer. From capacity determinations evidence is given that, even in this coenzyme gel, only those coenzyme molecules are useful in affinity chromatography which are on the surface of the gel grains; it is supposed that this may be due to the slow diffusion of an enzyme into the inner parts of an affinity gel.

[Coenzym properties of some Ade-1- and Ade-N6-substituted NAD derivatives (author's transl)]. / Coenzym-Eigenschaften einiger in 1- und N6-Stellung des Adeninringes substituierter NAD-Derivate

By reaction of NAD with different oxiranes or with aziridine, derivatives of the coenzyme are obtained with substituents in position 1 or on the amino group in position 6 of the adenine ring. While the Ade-1-substituted derivatives show high Km values with different dehydrogenases and are reduced only very slowly by these enzymes, the coenzyme derivatives substituted at the amino group behave very similarly to NAD. Correlations were found between coenzyme efficiency of the compounds and the lipophilic character of their substituents. The results can be interpreted from the structure of the active site of the dehydrogenases investigated.

Microcalorimetric methods for substrate determination in flow systems with immobilized enzymes.

The enthalpy of processes catalyzed by immobilized enzymes in the reaction cell of a LKB-flow calorimeter is used for determination of urea (0.5-5 mumol) and glucose (0.03-0.5 mumol). Accuracy is 2-5% and the time needed for one analysis is 20 min. A sensitive "enzyme thermistor" consisting of a flow through cell with an immobilized enzyme and two thermistors is described, which permits glucose determinations (0.05-1 mumol +/- 0.03 mumol) by means of temperature difference caused by reaction heat. Coupling of enzyme reactions for increasing reaction heat and consequently sensitivity in calorimetric determinations is demonstrated.