Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges for 14 proteins in serum based on the standardization against the IFCC/BCR/CAP Reference Material (CRM 470). International Federation of Clinical Chemistry. Community Bureau of Reference of the Commission of the European Communities. College of American Pathologists.

The release in 1993 of a new reference material for serum proteins, CRM 470/RPPHS 5 has given rise to a great improvement in the between-laboratory variability of serum protein measurements worldwide. Conversion to the new reference material results in significant changes in reference values for some proteins. The establishment of new reference ranges will take a considerable time, and in the interim several professional societies and diagnostic companies have agreed to use consensus reference ranges based on studies already undertaken.

A specific immunoassay for the detection of flunitrazepam.

The development of an immunochemical procedure for the determination of flunitrazepam in whole blood is described. Flunitrazepam was derivatized in position 3 of the benzodiazepine ring to a hapten which was coupled to a carrier protein. To obtain antibodies, rabbits were immunized with these immunogens and the collected antisera were tested in a heterogeneous, competitive RIA. The antibodies showed a very specific reaction with flunitrazepam and hardly any cross-reactivity with related 1,4-benzodiazepines. Because of its high specificity the antiserum has the advantage of a definite determination of low levels of flunitrazepam without the risk of false-negative results obtained by using the commercially available group-specific test systems. The drug was extracted from whole blood in a simple batch process with a polystyrene suspension and the extracts were measured by RIA. The advantages of an immunochemical system, such as short analysis time and simple sample preparation, and the exactness of a drug-specific method are combined in this procedure, which allowed the specific and very sensitive determination of flunitrazepam in the low therapeutic range.

[Immunochemical determination of saccharin]. / Immunchemische Bestimmung von Saccharin.

Saccharin is coupled to bovine serum albumin by spacers via the five membered as well as the six membered ring. Rabbits treated with these immunogenic conjugates give precipitating antibodies which are used for coating polystyrol tubes and microtiter plates. Saccharin is determined down to 0.1 mg/l, the detector reaction being performed by a saccharin horseradish peroxidase conjugate and hydrogen peroxide/1,2-diaminobenzen as substratum.

New international reference preparation for proteins in human serum (RPPHS).

Quality-control surveys in recent years, in various parts of the world, have shown poor between-laboratory agreement for measurements of plasma proteins. Despite the existence of international reference materials distributed by the World Health Organization, standards produced by diagnostics manufacturers and professional organizations differ significantly in their ascribed values. The reasons for this are complex but include poor availability of the primary materials, confusion about their use, and the fact that their turbidity on reconstitution precludes their use in modern optical immunoassays. This unfortunate situation led to an important initiative to produce sufficient quantities of a widely available, optically clear secondary reference material for plasma proteins that could be used worldwide by manufacturers, professional organizations, and laboratories. Here we present an overview on how the laboratory community, including manufacturers, clinical laboratories, professional societies, and regulators, has reached what we consider is a successful conclusion to a difficult problem.

Properties of the two isoenzymes of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum.

Methyl-coenzyme M reductase (MCR) catalyses the methane-forming step in the energy metabolism of methanogenic Archaea. It brings about the reduction of methyl-coenzyme M (CH3-S-CoM) by 7-mercaptoheptanoylthreonine phosphate (H-S-HTP). Methanobacterium thermoautotrophicum contains two isoenzymes of MCR, designated MCR I and MCR II, which are expressed differentially under different conditions of growth. These two isoenzymes have been separated, purified and their catalytic and spectroscopic properties determined. Initial-velocity measurements of the two-substrate reaction showed that the kinetic mechanism for both isoenzymes involved ternary-complex formation. Double reciprocal plots of initial rates versus the concentration of either one of the two substrates at different constant concentrations of the other substrate were linear and intersected on the abcissa to the left of the 1/v axis. The two purified isoenzymes differed in their Km values for H-S-HTP and for CH3-S-CoM and in Vmax. MCR I displayed a Km for H-S-HTP of 0.1-0.3 mM, a Km for CH3-S-CoM of 0.6-0.8 mM and a Vmax of about 6 mumol.min-1 x mg-1 (most active preparation). MCR II showed a Km for H-S-HTP of 0.4-0.6 mM, a Km for CH3-S-CoM of 1.3-1.5 mM and a Vmax of about 21 mumol.min-1 x mg-1 (most active preparation). The pH optimum of MCR I was 7.0-7.5 and that of MCR II 7.5-8.0. Both isoenzymes exhibited very similar temperature activity optima and EPR properties. The location of MCR I and of MCR II within the cell, determined via immunogold labeling, was found to be essentially identical. The possible basis for the existence of MCR isoenzymes in M. thermoautotrophicum is discussed.

Modern aspects and requirements for the standardization of immunoassays for human plasma proteins.

Some fundamental problems are summarized for an optimal standardization of protein determination in immunoassays. The relationship between a measurement signal in an immunoassay and the protein value followed by that is complicated and is explained on the basis of two immunochemical postulates and of two nonimmunochemical influences for which four postulates are derived.

Differential expression of the two methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme-specific antisera.

Methanobacterium thermoautotrophicum contains two isoenzymes of methyl-coenzyme M reductase (MCR), MCR I and MCR II, which catalyze the methane-forming step and which together represent more than 10% of the cellular protein. We describe here the preparation of isoenzyme-specific antisera against the two MCR isoenzymes and their use in the quantitative immunochemical determination of the two isoenzymes in the methanogen. The relative and absolute cellular concentration of the two proteins is shown to be strongly affected by growth conditions such as the temperature, pH, and substrate concentration. Conditions were found yielding cells which contained essentially only MCR I or MCR II. Using antisera against MCR I and MCR II, MCR from other methanogens were immunochemically compared. Evidence is presented that Methanobacterium wolfei also contains two isoenzymes of MCR.

Measurement signal versus value in immunoassays for human plasmaproteins.

Signal size proportionality to a measurement value will be obtained in immunoassays only if the following four--theoretical--postulates are fulfilled: The immunochemical reaction behaviour (number and affinity of the epitopes) of the protein analyzed is identical and uniform for both the reference preparations (Standard/Calibrator/Control) and for the analyte in the specimens (uniform physicochemical properties of the proteins). The immunochemical reaction behaviour of the antibody/ies containing reagent used in the immunoassay is identical and uniform from batch to batch with respect to the reactive antibody population. The immunochemical method is so well standardized with the consequence that the size of the measurement signal--caused only by the antigen antibody reaction product--will not be falsified (by matrix- and other interfering influences, or by the method principle). The values for the results of plasma protein determination are declared by arbitrary units, e.g. international units (IU). As a rule conversion factors to g/L (SI units) are not constant and depend on very many effects. It is very difficult to fulfill all the four theoretical postulates. Therefore the practical consideration of the medical requirements should be taken into account and should be the guideline for approachable solutions.

[Problems in standardization of the immunochemical detection of plasma proteins. I. International reference standards for plasma proteins. II. Immunochemical relation of measurement signals and values]. / Standardisierungsfragen zur immunchemischen Bestimmung von Plasmaproteinen. Teil I: Internationale Referenz-Präparationen für Plasmaproteine. Teil II: Die immunchemische Relation von Messsignal und Messwert für Human-Plasmaproteine.

When the results of plasmaprotein determination--dependent on the reagents, methods and other factors--are expressed in international units (IU) a better comparison of the results between different labs is possible. By this means and using the corresponding reference values and reference intervals the reliability of the interpretation of laboratory data will increase. The situation regarding the WHO IRP's for plasma proteins (approx. 20 parameters) and the relation from IU to mg in the reagents of the Behringwerke AG are reported. The overlability of WHO IRP's has not allowed yet to achieve an optimal standardization of the immunochemical methods for measurement of plasma proteins. This depends especially on the complexity of the antigen-antibody reaction. The relationship between the measured signal and the hence obtained value will be discussed in details in the second part of this paper. Hereby especially the reciprocal interdependence between physicochemical properties of proteins and the deriving immunochemical behaviour as well as the dependence of the results from the quality of the antibodies-containing reagents are also considered. Possible solutions for a better control system of the plasma protein determination by using the so called master calibrators as internal support for validation will be presented. Furthermore, general immunochemical postulates and rules for optimization of the standardization of plasma protein determination will be proposed and discussed.

Unusually high concentrations of Zn alpha 2-glycoprotein and the lack of alpha 2HS-glycoprotein in human ejaculates.

Ejaculates of 132 nonvasectomized and 129 vasectomized men were assayed for their Zn alpha 2- and alpha 2HS-glycoprotein contents by radial immunodiffusion. The respective mean concentrations of Zn alpha 2-glycoprotein were 319 and 309 mg/L, respectively, but these fluids were devoid of alpha 2HS-glycoprotein. In comparison, the concentration of these two proteins in serum of normal men were 62 and 617 mg/L for Zn alpha 2- and alpha 2HS-glycoprotein, respectively. Because albumin in the ejaculates is 100-fold less concentrated than in serum, the fivefold increase in the concentration of Zn alpha 2-glycoprotein of the ejaculates represents about a 500-fold enrichment of this protein in the ejaculates over the concentration expected by comparison with albumin.

Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin).

A binding protein for plasminogen activator inhibitor 1 (PAI-1-BP) was isolated from human plasma by a four-step procedure. 1) The 7 S globulin fraction of plasma was isolated by gel filtration on Sephacryl S-300. 2) Human endothelial cell-type plasminogen activator inhibitor (PAI-1), pretreated with 12 M urea, was added to this fraction (22 micrograms of PAI-1/ml of plasma), and a PAI-1 antigen peak with apparent mass 450 kDa (representing 65% of PAI-1 antigen and 85% of PAI activity) was isolated by gel filtration of this mixture. 3) The PAI-1.PAI-1-BP complex was further purified by immunoadsorption on an immobilized murine monoclonal antibody directed against PAI-1 (MA-7D4) and by elution with 4 M KSCN. 4) The complex was then dissociated by addition of excess human tissue-type plasminogen activator (t-PA), and t-PA and PAI-1 antigen (t-PA.PAI-1 complexes and free t-PA and PAI-1) were removed by immunoadsorption on monoclonal antibodies directed against t-PA (MA-62E8) and against PAI-1 (MA-7D4 and MA-12A4). Sodium dodecyl sulfate-gel electrophoresis of the purified material under nonreducing conditions revealed two bands with apparent mass approximately equal to 150 kDa and two bands with mass 74 and 68 kDa. Reduced sodium dodecyl sulfate-gel electrophoresis displayed two main bands with apparent masses of 73 and 64 kDa. The PAI-1-BP reacts with urea-treated, but not with inactive PAI-1. t-PA dissociates the complex between PAI-1 and PAI-1-BP. PAI-1 in complex with PAI-1-BP is 2-3-fold more stable at 37 degrees C than purified PAI-1, suggesting that PAI-1-BP may stabilize PAI-1 in blood. The concentration of PAI-1-BP in plasma determined by titration with PAI-1 is approximately 130 mg/liter. The isolated PAI-1-BP was shown to be identical to S protein (vitronectin) both by cross-reactivity with monospecific rabbit antisera and by NH2-terminal amino acid sequence analysis. The gel filtration behavior, mobility on sodium dodecyl sulfate-gel electrophoresis, and concentration in plasma suggest that PAI-1-BP is a multimer (presumably a dimer) of S protein accounting for approximately 35% of the S protein in plasma.

[Crystallized human plasma proteins. I. Albumin, transthyretin, retinol-binding protein, ceruloplasmin and beta 2-glycoprotein I]. / Kristallisierte Human-Plasmaproteine. I. Albumin, Transthyretin, Retinol-bindendes Protein, Coeruloplasmin und beta 2-Glykoprotein I.

The presently known possibilities and conditions for achieving the crystallization of Albumin, Transthyretin, Retinol-binding Protein, Ceruloplasmin and beta 2-Glycoprotein I are described with respect to our own experiences. For isolating some proteins the crystallization gives rise to a real purification step. Sometimes degrees of purity near to 98 till 99% will be reached. Furthermore crystalline proteins are very important for the determination of the three-dimensional structure by aid of the X-ray diffraction analysis. For this purpose large single crystals are needed with minimum requirements of 0.4 mm length for all the three dimensions. Additionally the crystals must have a high degree of order on the molecular level and should allow recording of the diffraction pattern out to 3 A resolution. From the five proteins described here the three dimensional molecular structure of Transthyretin and Retinol-binding Protein could be elucidated by using the method of the X-ray diffraction analysis.

A radioimmunoassay to measure piretanide in human serum and urine.

This paper describes a specific radioimmunological method for determining the diuretic agent piretanide (4 phenoxy-3-(1-pyrrolidinyl)-5-sulfamoyl benzoic acid) in human serum, plasma and urine. The antiserum was raised in rabbits against an immunogen of piretanide coupled to bovine serum albumin. The iodinated hydroxy derivative of piretanide was used as tracer. The separation of free and antibody-bound piretanide was performed by precipitating the antibody-tracer complex by polyethylene glycol. The limit of detection was 4 ng/ml. Studies on specificity showed less than 0.5% cross-reactivity of an identified metabolite. Interassay reproducibility showed an average coefficient of variation of 7.3%, the interassay variation was 5%. A recovery experiment yielded 100.9% recovery. There is good agreement between parallel determinations of piretanide by RIA and HPLC in both human serum and urine.

A radioimmunoassay for determination of glibenclamide and other sulfonylureas.

An antiserum was prepared for the determination of glibenclamide and for the estimation of other commercially available sulfonylureas. Rabbits were immunized with a glibenclamide-BSA conjugate. Tritiated glibenclamide was used as the tracer. The assay was performed in the presence of 8-anilinonaphthalenesulfonic acid to displace glibenclamide bound to serum protein, and free and antibody bound tracer were separated by dextran-coated charcoal. For glibenclamide determination in serum and plasma the limit of detection was 3 ng/ml. Sensitivity calculated for the whole determination range was 102 cpm for a 10 % concentration difference. Specificity studies showed a cross-reaction of less than 0.1 % for glibenclamide metabolite M1 and 9 % for metabolite M2. Other sulfonylurea drugs display cross-reactivities from 0.1% (chlorpropamide) to 190% (gliquidone). Both intra-assay and inter-assay imprecision were below 10 %. Accuracy was established by comparison of the present method with HPLC. The assay was applied to the specific determination of glibenclamide in clinical trials and for diagnosing factitious hypoglycemia caused by sulfonylureas.

[Isolation and crystallization of the Clr subunit of the first component of human complement (author's transl)]. / Isolierung und Kristallisation der Human-Komplement-Subkomponente Clr des Cl-Komplexes.

Isolation of Clr from Cohn-Fraction I of human plasma is accomplished by a series of steps which include affinity chromatography on IgG-p-azobenzyloxyethylsulfonylethoxy-Sepharose CL-4B, ion exchange chromatography and preparative zone electrophoresis. The protein is easily soluble in 0.1 M Tris/HCl buffer, pH 10.0 and is crystallized readily by means of dialysis against 0.15 M saline, pH 7.0 in the form of hexagonal dipyramides. The structural elucidation using X-ray analysis is now possible.