Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics

Although protein biomarkers have a great potential as biomarkers for diagnosis of diseases, they are seldom used in hospitals. There are many reasons for this, for instance, the difficulties to (i) find a biomarker for which the concentration in body fluids clearly differs between patients and healthy subjects, (ii) attain purification of the biomarker close to 100%, which is required for production of conventional protein antibodies as well as artificial gel antibodies for selective capture of a biomarker, (iii) design a standard curve for rapid and accurate determination of the concentration of the biomarker in the body fluid because of adsorption of the biomarker onto vials, pipettes, etc., (iv) determine accurately the sample volume delivered by a pipette, (v) avoid polymerization of the biomarker upon storage and to decide whether it is in the form not only of monomers, but also of dimers, trimers, etc., in the native state, (vi) determine the degree of possible glycosylation and amidation of the biomarker and (vii) decide whether glycosylation and amidation positively or negatively affects the possibility to use the protein as a biomarker. In this article, we discuss in quantitative terms the difficulties (iii–vii) and how to overcome them, which also may help to overcome the difficulty (ii), which in turn minimizes difficulty (i).     

Metallothionein isoforms from horse, rabbit and rat separated by capillary zone electrophoresis at low pH

A comparative study of MT isoforms in rat liver and in commercial Sigma MT preparations from rabbit liver and horse kidney was performed using capillary zone electrophoresis (CZE). Electropherograms revealed the co-migration of MT forms from these species. A special form, the a-form (not binding Cd), occurred in various MT samples in different amounts, depending on the method used for MT purification. In the rabbit liver electropherogram a main form appeared (the b-form), which might be a modified MT form. A band of unknown composition, running ahead of the rat liver MT-I and -II forms on polyacrylamide gels, not having Cd binding affinity, probably had its counterpart in a yet unidentified CZE peak. CZE electropherograms of purified MT samples may contain main peaks that do not represent genuine and functional MT isoforms. Results are also presented which indicate that at low pH the MT-II form is more unstable than MT-I.     

Analysis of decay data from neutron-rich nuclei

The β-decays of the neutron-rich nuclei ¹²Be and ²⁹Ne have been studied. The statistical correlations between the almost identical half-lives of ¹²Be and its daughter ¹²B are analysed for a large sample of ¹²Be decay data. Stringent mutual bounds are obtained on the parameter set, leading to a precise determination of the ¹²Be half-life of 21.50±0.04 ms. From a simultaneous detection of β-particles and neutrons from the decay of ²⁹Ne the neutron emission probability, P_n, is determined to 17±5%. No indication of two-neutron emission is seen from this nucleus. An upper limit of 2.2% (90% confidence level) is established for P_2n. Received: 3 May 2001 / Accepted: 6 August 2001     

Metallothioneins from horse kidney studied by separation with capillary zone electrophoresis below and above the isoelectric points

Capillary zone electrophoresis (CZE) was used to study metallothionein (MT) isoforms and non-MT components in the horse kidney MT preparation produced by Sigma. This technique was found to be well suited for studies of such forms. The non-MTs are heat resistant, they do not bind metal, but comigrate with MT in the various systems generally used for MT isolations. These forms may cause discrepancies between claimed MT content and sample weight as well as confusion in MT identification. The functional metal binding ability in the Sigma sample was measured, and the elution profile from an anion exchange column, commonly used for separation of tissue MTs, was determined in order to ascertain the MT-I isoform content. Optimum conditions for the separation of the MT isoforms have been studied in the polyacrylamide coated capillary at pHs below and above the isoelectric points (pIs) in various buffer systems. Evidence is put forward that MT forms oligomers or aggregates in the metal binding situation at pHs above pI. Our results may indicate that the oligomerized MT-IA form binds additional Cd atoms than the expected number of seven per monomer.     

Search for forbiddenβ-decays of the drip line nucleus12Be

Beta-coincidentγ-rays have been measured from implanted pure samples of¹²Be separated at the LISE3 spectrometer at GANIL. An intensity of 0.040(26) % can be estimated for the branching ratio of the isospin forbidden pure-Fermi transition to the 0⁺ excited state of¹²B and of 0.008(6)% of the transition to the 1^?1 excited state. Both are taken to represent upper limits. The half-life has been re-measured to be 26.1(2.4) ms.