Monika Pergande

Measurement of lysozyme in human body fluids: Comparison of various enzyme immunoassay techniques and their diagnostic application

Three variants of the immunoenzymometric assay of human lysozyme with HRP-labeled antibodies were compared. The highest sensitivity (with a detection limit of 0.2 micrograms lysozyme/L) was achieved by a one-step assay lasting 2 h. Between-batch precision for the techniques was 6-11%. Lysozyme reference values were determined in serum, cerebrospinal fluid and urine. In serum they are age-dependent and in urine sex-dependent when related to creatinine excretion. Serum lysozyme is increased in only 57% of the patients with active rheumatoid arthritis and is also unreliable for indicating remission. In Crohns disease the serum lysozyme reflects activity better, but it does not exceed the diagnostic value of alpha-1-acidic glycoprotein (orosomucoid). The lysozyme quantification in cerebrospinal fluid is useful in distinguishing between viral or bacterial meningitis.

Influence of cyclosporin A on the respiration of isolated rat kidney mitochondria

Cyclosporin A toxicity Rat kidney Mitochondrion respiration

A rapid and sensitive enzyme immunoassay for Cu/Zn superoxide dismutase with polyclonal and monoclonal antibodies.

An enzyme immunoassay for the quantification of human Cu/Zn SOD in serum, urine and erythrocytes was developed applying monoclonal and polyclonal antibodies. The one-step assay is completed within 30 min and enables the detection of 0.3 microgram Cu/Zn SOD per litre. A Cu/Zn SOD concentration of 46 +/- 21.5 micrograms/l and of 1 +/- 0.6 micrograms/mmol creatinine was determined in the serum and the urine, respectively, of healthy individuals. A content of 15 +/- 1.7 ng Cu/Zn SOD was found in 10(6) erythrocytes. Patients with Downs syndrome exhibited a 3.8-fold, a 2-fold and a 1.6-fold higher concentration of Cu/Zn SOD in their serum, urine and erythrocytes.

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA)

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.2 X 10(10) l/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, and additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 micrograms SOD/l.

A microalbuminuria assay using bromphenol blue

A simple microalbuminuria assay using bromphenol blue/glycine reagent is described. Urine samples were prepared using gel filtration on Sephadex G-50 minicolumns and absorbance was measured at 610 nm 20 s after mixing 10 parts of eluate and 1 part of reagent. The detection limit of this method was 3 mg/l; within-run and between-run precision was between 0.5 and 4.1% for borderline and raised albumin concentrations. The recovery of albumin added to samples was 98.7 +/- 2.5%. Results obtained by this method correlated closely with values obtained by radial immunodiffusion (r = 0.987). The test is cheap (reagent costs about 5 cents) and suitable for the non-specialist laboratory.

Diuresis-dependent excretions of low-molecular mass proteins in urine: β2-microglobulin, lysozyme, and ribonuclease

An investigation was carried out into how the low-molecular mass proteins beta 2-microglobulin, lysozyme, and ribonuclease were excreted over 8 h after high fluid intake (22 ml/kg of body weight in 15 min). With increasing urine flow rate the amount of lysozyme excreted per hour or per millimole creatinine increased more markedly than that of beta 2-microglobulin while at the same time the excretion rate of ribonuclease decreased. The effect of urinary flow upon the excretion rates of the various low-molecular mass proteins has to be considered as a preanalytical factor when these proteins are used as indicators of tubular dysfunction.

Influence of inorganic phosphate on the activity determination of isoenzymes of alkaline phosphatase in various buffer systems

The inhibitory effect of inorganic phosphate on the activity determination of isoenzymes of alkaline phosphatase (AP) in diethanolamine (DEA), glycine and 2-amino-2-methyl-1,3-propandiol (AMPD) buffer was studied. This effect depends on the buffer used and isoenzyme investigated. Especially the placental isoenzyme is inhibited; the inhibitory effect in DEA buffer is stronger than in the other buffers used. The requirement of purity for 4-nitrophenylphosphate with respect to its content of inorganic phosphate and conclusions for using control sera enriched with AP isoenzymes are discussed.

Further evidence for tubular dysfunction in insulin dependent diabetes

There is evidence that increased excretion of urinary enzymes and low-molecular mass proteins indicate impaired tubular function. The excretion of N-acetyl-beta-D-glucosaminidase (NAG), lysozyme, and ribonuclease in Type I diabetic patients with (n = 19) and without (n = 17) persistent proteinuria (urinary protein excretion greater than 0.5 g/day) was investigated and compared with this excretion in 30 weight- and gender-matched nondiabetic subjects without renal disease. Urinary NAG excretion was significantly higher in diabetic patients with and without persistent proteinuria (1.16 +/- 0.09 and 3.19 +/- 1.2 Umol/L creatinine, respectively) compared to controls (0.37 +/- 0.03 Umol/L creatinine p less than 0.01). In addition, the urinary excretion of lysozyme and ribonuclease was significantly increased in diabetic patients. Urinary NAG was found to correlate positively with albuminuria and proteinuria (r = 0.95 and 0.93, respectively), as well as with ribonuclease and lysozyme (r = 0.93 and 0.60; p less than 0.01) in patients with persistent proteinuria. Furthermore, NAG excretion was significantly related to the duration of diabetes (r = 0.36; p less than 0.05). No relationship existed between urinary NAG and serum creatinine, beta-2-microglobulin, and degree of metabolic control (HbA7). The lysozyme excretion, but not NAG excretion, was significantly related to hypertension in patients with clinical proteinuria. In conclusion, our results suggest a relationship between the development of tubular dysfunction and the impairment of glomerular function in diabetic nephropathy. An increased excretion of NAG and low-molecular mass proteins may indicate early nephropathy

Sex- and age-dependent reference values of alpha-1-microglobulin in urine.

Klaus Jung, Department of Experimental Organ Transplantation, University Hospital Charité, Humboldt University, Landsberger Allee 49, D-1017 Berlin (FRG) Dear Sir, There are different reference ranges of αrmicroglobulin (A-l-M) in serum and urine reported in the literature [1-11]. Reasons for these divergent data are: methodical problems caused by the heterogeneity of A-l-M, the calibration material and antisera of different specificity used in the immunochemical methods (radioimmunoassay, enzyme immu-noassay, radial immunodiffusion), inappropriate selection of reference individuals and insufficient consideration of guidelines for the statistical treatment of collected reference values [12]. Recently, Itoh and Kawai [1] described sex differences of A-l-M in body fluids in this journal. However, they could not sufficiently take into account the influence of age, because they only investigated two small groups aged up to 15 years. As we were also interested in the diagnostic validity of this lowmolecular mass protein in urine, we focused our attention on this problem analyzing the A-l-M data collected in a large group of healthy adults during the last 2 years [13]. 304 healthy adults (144 women, 160 men) were studied; they were asymptomatic, had no history of renal diseases and were not receiving medications, showed negative test results with Combur9-dipsticks and serum creatinine concentrations below 106 μmol/l. A-l-M was determined in untimed 2nd-morn-ing urine samples by single radial immunodiffusion (Behringwerke AG, Marburg, FRG). Our reference cohort was subdivided into five age groups as shown in table 1. The data were expressed as A-l-M concentration and A-1-M/urine creatinine ratio and were tested on whether they depended on sex and age (Kruskal-Wallis test; Mann-Whitney U test. A-l-M concentrations were both ageand sexdependent, whereas A-l-M/creatinine ratios did not differ between women and men and could be combined for further calculations. There were no statistical differences of the A-l-M/creatinine ratios between the three age groups of 18^40 years on one hand and between the two groups above 40 years on the other hand. A clear increase in values was observed in the group of persons older than 40 years (table 1). Since the protein/creatinine ratio allows to use random urine specimens for analysis and to consider the effect on urine flow rates, this ratio has been recommended for the interpretation of results [4, 14]. Thus, considering the age effect on this parameter as described in table 1,95% reference intervals

Suitability of commercial control sera for the quality control of activity determination of alkaline phosphatase.

The suitability of thirteen commercially available control sera for measuring alkaline phosphatase (EC 3.1.3.1; orthophosphoric acid monoester phosphohydrolase, ALP) activity in human serum was tested. Apart from differences in ALP activity observed in some reconstituted commercial sera, the behaviour of control materials towards experimental variables such as the nature and concentration of the substrate, pH and type of buffer (or PO4-acceptor) together with the composition of the isoenzymes present in human serum highlights the problems and difficulties if commercial materials are to be used as control sera. The half-saturation constants in control sera were in all cases smaller than those of ALP isoenzymes from bone and liver. The shape of substrate activity curves and the pH optimum in most of control sera differed from that of human serum. The discrepant kinetic data of control materials and human serum may mask or suggest changes relevant to commercial quality control serum but not to samples of human serum.