Screening for Albuminuria: A Case for Estimation of Albumin in Urine

The usefulness of bromcresol green for estimating albumin in urine was evaluated by comparison with the Laurell "rocket" technique. In contrast to the bromcresol green method applied for urinary albumin, rather doubtful results were obtained with conventional (Microzone) electrophoresis for albumin and with precipitation techniques for total protein estimation. Albumin estimation with bromcresol green is recommended as a more reliable substitute for total-protein estimations in urine. Limitations of bromcresol green are also pointed out.

A SERUM FACTOR IN CHRONIC HYPOCOMPLEMENTEMIC NEPHRITIS DISTINCT FROM IMMUNOGLOBULINS AND ACTIVATING THE ALTERNATE PATHWAY OF COMPLEMENT

Nephritic factor (C3NeF) has been isolated from plasma of patients with hypocomplementemic chronic glomerulonephritis (HCG) by ion exchange and molecular sieve chromatography. This material was further treated with solidified anti-Ig antiserum. The purified material failed to react with antiserum to human IgG, IgG3, Fab, Fc, and kappa and lambda chains, but retained full C3NeF activity. The nonidentity of C3NeF with IgG was further demonstrated by Ouchterlony analysis using anti-IgG and anti-C3NeF. Isolated C3NeF was found to be a protein with a sedimentation coefficient of 7S and a mol wt of 150,000 daltons, which on microzone electrophoresis and gel electrophoresis at pH 8.6 behaved as a γ-globulin. C3NeF is not a C1q precipitin and does not activate the classical complement pathway. Unlike cobra venom factor, it failed to enter into a complex with C3 proactivator (C3PA) when incubated with normal human serum (NHS) and then subjected to sucrose density gradient ultracentrifugation. The action of isolated C3NeF on C3 requires C3PA, C3PA convertase (C3PAse), and properdin (P). Similarly, C3PA conversion by C3NeF requires P, C3PAse, and C3. Total hemolytic activity was lost by incubation of 64 µg of C3NeF/1 ml NHS at 37°C for 30 min. Both C3a and C5a anaphylatoxin could be generated by C3NeF in serum previously depleted of anaphylatoxin inactivator. Anti-C3NeF was found to detect an antigen in all NHS tested. Treatment of NHS with solidified anti-C3NeF caused impairment of the alternate complement pathway. It failed to sustain lysis of glutathione-treated human erythrocytes initiated by inulin. It is conceivable that the normal serum constituent which is removed by anti-C3NeF constitutes the inactive precursor of C3NeF, and a heretofore unrecognized component of the alternate pathway.

ISOLATION AND IMMUNOCHEMICAL CHARACTERIZATION OF RABBIT 7S ANTI-IGG WITH RESTRICTED HETEROGENEITY

7S anti-IgGs were isolated from four rabbit antistreptococcal antisera by the use of immunoabsorbent columns. The isolated proteins were of restricted molecular heterogeneity; they formed a monodisperse band on microzone electrophoresis and had a limited number of light chain bands when analyzed on urea polyacrylamide gel. The binding site of the 7S anti-IgGs was detected in the F(ab')2 portion of the molecule. The binding site has antibody specificity for the Fc portion of IgG. For one 7S anti-IgG the combining site on the Fc portion could further be defined. A pepsin fragment of Fc, described as Pep-III', was a potent inhibitor of the coprecipitation of 7S anti-IgG with antigen-antibody complexes. An idiotypic cross-reaction was detected between the 7S and 19S anti-IgGs isolated from the same rabbit with anti-idiotype sera prepared in guinea pigs. This idiotypic specificity was not detected in the 7S anti-IgGs of 20 other rabbits.

Changes in the Electrophoretic Patterns of Euglobulin Fractions Following Activation of the Fibrinolytic System by Exercise

A study was made of changes in blood fibrinolytic activity in a group of young male university students following varying levels of exercise. The subjects were divided into two groups. Those partaking in regular athletic programs were classified as athletes and those not as nonathletes. There was an increased fibrinolytic activity with increased severity of exercise in all subjects. Several of the nonathletic group showed a much greater maximum effect than their athletic counterparts, and all nonathletes showed the effect at much lower levels of exercise. Increase in fibrinolytic activity correlated well with increase in pulse rate. When euglobulin fractions from the plasma samples were subjected to microzone electrophoresis, there was a band change in the region of the beta globulins in many of the samples following exercise. This band change is apparent only when fibrinogen is present in the euglobulins and appears to be due to fibrinogenolysis as a result of the activation of the fibrinolytic system. These band changes were observed when the specific activity was as little as 0.058 unit of plasmin per milligram protein. Our results show that electrophoretic pattern changes of euglobulin fractions is a relatively sensitive and rapid method for detecting activation of the fibrinolytic system.

ENZYME ACTIVITIES IN CHICK EMBRYONIC CARTILAGE

Some characteristic enzymatic activities were determined in chick embryonic cartilage and compared with the analogous activities in bone and liver. Chondrocytes were isolated, broken by sonication, and subjected to subcellular fractionation to yield a nuclear pellet, the mitochondrial, lysosomal, and microsomal fractions, and the high speed supernatant solution. It was established that these fractions are characterized by enzymatic activities usually associated with similar fractions in other tissues, but with some quantitative differences. Lysozyme, a particulate-associated enzyme in other tissues, was not detected in any subcellular fraction even by the sensitive technique of microzone electrophoresis and is therefore considered to be primarily extracellular in cartilage.

RELATIONSHIPS BETWEEN RELATIVE BINDING AFFINITY AND ELECTROPHORETIC BEHAVIOR OF RABBIT ANTIBODIES TO STREPTOCOCCAL CARBOHYDRATES

After repeated intravenous injections with Group C streptococcal vaccine, most rabbit antisera were shown to contain one or more IgG antibody components, as revealed by microzone electrophoresis. A procedure for the fractionation of multiple IgG antibody components from such streptococcal antisera is described. Separation is achieved on the basis of differences in relative binding affinities of the antibody components to immunoabsorbent columns. The evidence suggests that the electrophoretic mobility, and thus the net charge of an antibody, bears a reciprocal relationship to its binding affinity for the streptococcal Group C antigens. Furthermore, the relative binding affinity affords another means to assess the functional homogeneity of streptococcal antibodies. A possible relationship between light chain variable-region subclasses and binding affinities of streptococcal antibodies is discussed.

Evaluation of the Microzone System for Simultaneous Electrophoresis of 16 Sera

We investigated the feasibility of using the "Microzone" electrophoresis system to produce 16 instead of eight electrophorograms on a single cellulose—acetate membrane by placing eight specimens at the left of center, rotating the cellulose—acetate membrane and supporting bridge 180° horizontally, and again applying eight samples to the left of center. The membrane, now with eight samples each on anodic and cathodic sides, is electrophoresed according to the usual recommended procedure. There was a statistically demonstrated association between sample application position and results (for 96 samples). This technique is nevertheless useful for determining albumin/globulin ratios and for screening large numbers of samples. Sera with borderline or abnormal results should be rerun by the usual technique.