Volume 36, Issue 6

Immunoassay is now the most widely used analytical technique in laboratory medicine embracing a vast repertoire of analytes and delivered through an increasingly diverse range of devices. This explosion of analytical techniques is complemented by an increasing knowledge base on the antigen antibody reaction that is being used to explore new and improved methodologies. Our knowledge of the molecular chemistry of the antigen antibody reaction coupled with the molecular biology of immunoglobulin expression has led to the development of antibody and antibody-like molecules with enhanced specificity, and reaction kinetics. These developments include novel fusion proteins, antibody mimics and antibodies that recognise antigen antibody complexes. The advances in the chemistry of the analytical systems are matched by the design of delivery vehicles: on the one hand the automated analyser that facilitates the analysis of large numbers of samples and on the other, the encapsulated, miniaturised device that enables an immunoassay to be performed at the point of care.

I deal with the process of standardization of steroid immunoassays on the basis of metrological traceability (SI units). First, I discuss the role theoretically attributed to the calibration of the measurement procedure. Since immunoassays show matrix sensitivity, I associate traceability of calibration with traceability of results pertaining to individual patient samples. From this, I stress that optimization of the immunoassays in terms of specificity and insensitivity to different kinds of interference is necessary before standardization, and that split-sample measurements should be included in the standardization process. Last but not least, I give an overview of the current status of standardization of steroid immunoassays.

Although affinity and kinetic measurements on their own provide useful information regarding the suitability of antibodies for various immunoassays, a thermodynamic analysis provices additional information that throws light on the molecular forces at work in the antigen-antibody interaction. It may then be possible to adjust assay conditions in order to favour either the association or dissociation of antigen-antibody complexes. A tenfold increase in binding affinity (K) corresponds to a free energy change of only 1.4 kcal/mol (5.8 kJ/mol) at 25 °C. This means that K values of 10 5 M −1 and 10 10 M −1 correspond to a free energy change (∆G) of 7.0 and 14.0 kcal/mol respectively. The entire range of affinity constants normally encountered in antigen-antibody interactions, therefore differs by no more than about 7 kcal/mol of free energy change, which is equivalent to only a few hydrogen bonds. In comparison, a single electrostatic interaction corresponds to about 4 kcal/mol of free energy change. A full description of the binding interaction requires an understanding of the change in hydration states of the reactants when the complex forms, and an assessment of the entropic andenthalpic effects of these changes. Contrary to earlier assumptions, it is now clear that antigen-antibody interactions are often accompanied by a large favourable enthalpy which more than compensates the unfavourable entropy.

Immunoassays are widely used for determination of the concentration of steroid hormones. However, obtaining specific anti-steroid monoclonal antibodies remains difficult. We used antibody engineering and phage display methods to change the specificity of an anti-cortisol monoclonal antibody towards estradiol. This work demonstrates that production of recombinant antibodies may be a valuable way of obtaining the high-specificity antibodies required for steroid immunoassays.

The presence of cardiac troponin I in the serum is now considered as one of the most specific biochemical markers of acute myocardial infarction. To improve the knowledge of the antigenic properties of cardiac Troponin I, a set of monoclonal antibodies and polyclonal antibodies against human cardiac troponin I has been tested with overlapping peptides covering the cardiac troponin I sequence. The results indicate that N-terminal and C-terminal cardiac troponin I regions were most often recognized by poly- and monoclonal antibodies. These observations are valuable for choosing the best combination of monoclonal antibodies to set up new immunoassays to detect serum cardiac troponin I earlier after myocardial damage, to understand better which forms are released, and finally to propose appropriate cardiac troponin I standards.

New approaches to obtain information about the residues of the antibody paratope involved in the interaction with antigen would be very useful to help perform rational mutagenesis of antibodies for improving sensitivity or selectivity in immunoassays. We have evaluated the possibility of dissecting the antibody paratope into large sets of short (12 residues) overlapping peptides to determine the contribution of each peptide to antigen binding. Our results show that the systematic analysis of the antigen-binding properties of heavy chain variable segment and light chain variable segment derived peptides of HyHEL-5, a model anti-lysozyme antibody, can be an experimental approach to the selection of paratope-derived peptides with antigen-binding activity. Detailed analysis of the contribution of each residue from each binding peptide permitted the identification of residues contributing to antigen binding. Of the 38 residues we identified, 22 corresponded to residues that had been shown by X-ray crystallography to be at the interface between HyHEL-5 and lysozyme. The peptide analysis we have developed is thus a way to map the subset of residues from the antibody paratope involved in antigen recognition. The same peptide approach was used to map the idiotope that an anti-idiotypic antibody recognized in the paratope of its cognate antibody.

Glycosylation is the most extensive of all post-translational modifications in proteins. It has important functions in their secretion, antigenicity and metabolic clearance through structural polymorphism. In recent years, advances in recombinant DNA technology allowed the production of recombinant therapeutic proteins, among which glycosylated proteins displayed differences compared to their native counterparts, including antigenic carbohydrates. In this review, we discuss the potential use of cloned glycosyltransferases in remodeling recombinant glycoprotein antigens as well as in synthesizing tumor-associated carbohydrate antigens.

Angiogenesis is a key component of the repair mechanisms triggered by tissue injury. Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis, as it acts directly and specifically on endothelial cells. VEGF produced locally in regenerating tissue may spill over into the systemic circulation, and measuring levels of circulating VEGF may allow monitoring of angiogenesis. To determine whether circulating VEGF is increased after severe injury, we measured concentrations of VEGF in serial serum samples of 23 mechanical burn patients, 55 patients with multiple trauma and 56 healthy normal controls, using a newly established ELISA assay. In burn patients, serum VEGF was increased on day 1 (369.4 ± 88.0 pg/ml) and on day 3 (452.0 ± 65.3 pg/ml), reached highest levels on day 14 (1809.5 ± 239.7 pg/ml) and was still elevated on day 21 post-burn (1339.8 ± 208.7 pg/ml) (mean ± SEM, p<0.01), when compared with healthy controls (82.2 ± 10.8 pg/ml (mean ± SEM)). Likewise, in trauma patients, serum VEGF showed a trend towards elevated values on the day of admission (186.9 ± 43.9 pg/ml) and on day 3 after injury (193.2 ± 62.1 pg/ml). Thereafter, serum VEGF increased further (day 7, 507.0 ± 114.7 pg/ml), peaked on day 14 (742.4 ± 151.8 pg/ml) and was still elevated on day 21 after injury (693.1 ± 218.6 pg/ml (mean ± SEM, p<0.01)). No significant correlation was observed between peak serum VEGF and initial severity of mechanical (Injury Severity Score) or burn injury (percentage of body surface burned). However, in both burn and trauma patients, the subgroup of patients with uncomplicated healing showed significantly higher increases of serum VEGF than the subgroup who developed severe complications during the post-traumatic course, such as sepsis, adult respiratory distress syndrome or multiple organ failure (p<0.05). Thus, markedly enhanced levels of serum VEGF are present one to three weeks after trauma or burn injury. Further, occurrence of severe complications during the post-traumatic period is associated with lesser increases of serum VEGF.

To identify pre-β-high density lipoproteins, a rapid two-dimensional separation by electrophoresis (1 hour 30 minutes) was performed on an automated Phast System™. This procedure used commercially available polyacrylamide gradient gels (4–15 %) and allows sensitive and reproducible results. Pre-β-1- and pre-β-2-high density lipoproteins were clearly identified by this method. In addition, our procedure was successfully applied to diagnosis of a patient with familial lecithin: cholesterol acyltransferase deficiency, characterized by the absence of α-high density lipoproteins.

This report represents the continuation of our studies on the effects of gemfibrozil therapy on high density lipoprotein cholesterol levels. Previously, we reported that despite an impressive mean increase in high density lipoprotein cholesterol (20 %), the response to 12 weeks of gemfibrozil therapy was highly variable. Accordingly, out of the 27 subjects studied, five actually had lower high density lipoprotein cholesterol at the conclusion of therapy compared to baseline values. The changes observed in plasma lipids, combined with correlational relationships suggest that the conversion of triglyceride rich lipoprotein components into high density lipoprotein may be impaired in those subjects that respond poorly or negatively to gemfibrozil therapy.

The aim of this study was to establish reference intervals for cerum cystatin C and serum creatinine in adults. Blood samples were collected from 270 healthy blood donors (135 men and 135 women between 20 and 65 years old with 15 men and 15 women in each five-year-interval). Serum cystatin C was analyzed using an automated particle-enhanced immunoassay (DAKO Cystatin C PET kit) on the Cobas Mira S analyzer. Serum creatinine was analyzed using the Vitros Creatinine Slide, an enzymatic method on the Vitros 950 chemistry analyzer. The calculated reference intervals for serum cystatin C were 0.62–1.15 mg/l in women (median 0.84 mg/l, range 0.56–1.29 mg/l) and 0.51–1.25 mg/l in men (median 0.87 mg/l, range 0.42–1.39 mg/l). The Mann-Whithey U-test revealed no gender-related difference for cystatin C (p = 0.48). A common reference interval in women and men was calculated to be 0.54–1.21 mg/l (median 0.85 mg/l, range 0.42–1.39 mg/l). The non-parametric reference interval for serum creatinine was 57–95 μmol/l in women (median 72 μmol/l, range 44–105 μmol/l) and 69–111 μmol/l in men (median 89μmol/l, range 58–123 μmol/l).

Several international standards and corresponding interpretation documents for quality management systems have been published. Although these standards are found useful to some extent, they are considered to be insufficient in several areas important for medical laboratories particularly in the pre- and post-examinational phases. The normative document for accreditation of laboratories (ISO/IEC Guide 25) is presently being revised and a document for medical laboratories (ISO/TC 212, CD 15189) is at draft stage. Both aim to include aspects of total quality management. The concept of total quality management is rather vague. Generally, its goal has been defined as "business excellence". This term, however, needs some explanation if applied to medical laboratories. Therefore, a project group of the European Confederation of Laboratory Medicine (ECLM) has developed a model for total quality management, which is based on a comprehensive management concept issued by the European Foundation for Quality Management. In the case of a medical laboratory, the term "business excellence" should be replaced by "good medical laboratory services". The proposed model could serve as a basis for future developments of total quality management standards in laboratory medicine. The goal of the "journey" should be clarified before it starts. To the best of our knowledge, this is the first attempt to develop a model of a good medical laboratory.

Cytokines and soluble adhesion molecules play an increasing role in laboratory medicine especially due to the increasing knowledge of the function of the immune system and the availability of cytokine assays on analyzers working in the "random-access" mode. This meeting report gives an insight of different applications for the measurements of these immunomediators. Examples from surgery, pediatrics, transplantation medicine and immunopharmacology show that these parameters could provide early and useful information for diagnosis, prognosis, therapeutic decision making and drug actions.