A new method of measuring C-reactive protein, with a low limit of detection, suitable for risk assessment of coronary heart disease.

A new indication has been proposed for C-reactive protein (CRP) as a prognostic risk marker of coronary heart disease (CHD). The new indication calls for accurate (true and precise) measurement of CRP within the conventional reference range (< 5 mg/L). The existing turbidimetric and nephelometric methods do not cover the required measuring range and thus time-consuming and labour-intensive enzyme immunoassays have been used for the clinical studies focusing on CHD risk. We developed a new method based on microparticle enhanced turbidimetry, which attained the required limit of detection, while keeping the upper measuring limit comparable to the existing turbidimetric and nephelometric methods. The superior characteristics of the new method were realised by mixing two types of microparticle reagents differing in microparticle size and reactivity of coated antibody. The analytical detection limit of the method was 0.28 mg/L with use of only 2.5 microliters serum. The method showed good precision at 2 to 3 mg/L, the critical concentration range for CHD risk assessment. Other performance data including dilution linearity, method comparison, and interference study also met the requirements for the practical use in the clinical laboratories. Sera from 354 apparently healthy blood donors were measured in a reference range study. The reference range estimated after log-transformation was 0.16 mg/L to 7.57 mg/L CRP, with a total range of 0.09 mg/L to 21.0 mg/L. The distribution of CRP concentrations in this population was comparable to other results that established the use of CRP as a risk marker of CHD in a prospective study.

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.

Carbohydrate-enzyme conjugates for competitive EIA.

Enzyme glycoconjugates prepared from oligosaccharides or polysaccharides are efficient and durable reagents for direct, competitive enzyme immunoassays based on solid phase antibody. This assay format facilitates simple measurement of association constants and relative inhibitory power of oligosaccharides, while also providing a sensitive quantitative assay of bacterial polysaccharide antigens. Enzyme-saccharide conjugates were prepared by established glycoconjugate methodologies, and protocols for their optimal use were established by investigating the effects of solid phase antibody concentration on the kinetic equilibrium of binding for different antigen-enzyme conjugate concentrations, in the presence and absence of inhibitor. Four discrete monoclonal antibody-carbohydrate antigen systems were studied and it is demonstrated for each, that the relative inhibitory power of oligosaccharides may be readily measured in a convenient assay format, and that association constants can be extrapolated from the same assay data.

[Determination of fructosamine in chronic kidney diseases (dialysis-dependent patients)]. / Die Fructosaminbestimmung bei chronischen Nierenerkrankungen (dialysepflichtige Patienten).

The serum fructosamine normal range was confirmed. Correction to protein or albumin did not significantly affect the results. Therefore, correction of fructosamine values from patients with normal protein and albumin values would not improve the clinical significance of fructosamine. Fructosamine concentrations of heparin plasma from non-diabetics also fell within the serum fructosamine normal range. The fructosamine concentration from non-diabetic dialysis patients was significantly higher and more widely distributed than that of the reference collective despite normal blood glucose concentration. Relating fructosamine to protein had no substantial effect, whereas the differences were even increased when fructosamine was related to albumin. On the present stage of knowledge it might be considered to establish a reference interval for dialysis patients. It appears that the fructosamine estimation may then be successfully applied also to dialysis patients. Although dialysis resulted in hemoconcentration, the fructosamine concentration remained virtually unchanged. Referencing both values before and after dialysis to protein or albumin improved the correlation, but substantial differences were introduced as well. However, none of several parameters measured in parallel interfered to a degree which might explain such differences. In order to find a reasonable explanation for these findings further experiments are necessary.