A rapid method for detection of N-acetylglucosaminidase-type chitinase activity in crossed immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels using 4-methylumbelliferyl-N-acetyl-D-glucosaminide as substrate.

A method for the detection of N-acetylglucosaminidase (GlcNAcase) activity has been developed by using 4-methyl-umbelliferyl-N-acetyl-D-glucosaminide (4-MU-GlcNAc) as substrate in crossed immunoelectrophoresis (CIE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Visualization of the reaction with a 366 nm ultra-violet light is possible in approximately 30 min. The method is fast and sensitive in comparison with previous methods. The same band as in SDS-PAGE, showing both GlcNAcase and chitinase activity, was found in the present study; we therefore conclude that this method is also useful in a GlcNAcase-type chitinase assay.

[Immunosedimentation analysis of proteins]. / Immunosedimentatsionnyi analiz belkov.

After performing experiments with protein sedimentation the ultracentrifuge test-tube was attached to the inferior opening of the channel in the agar block with a vertical gradient of saccharose concentration. The liquid with protein zones was removed from the test-tube to the channel, forcing a 60% solution of saccharose beneath the lower layers in the test-tube. Horizontal electrophoresis was performed so that the protein zones penetrated the width of the channel walls. A piece of agar gel was cut out from the walls along the whole height of the channel. That piece was placed on the slide and embedded with a mixture of agar and antiserum. Immunoelectrophoresis was made according to Lowrell. The method permits obtaining for the first time continuous sedimentograms during immunodevelopment of the protein zones derived on ultracentrifugation.

Studies of human tear proteins--1. Analysis of tears from normal subjects by crossed immunoelectrophoresis.

Tears were collected from 10 normal subjects by two methods, 1) micropipetting after stimulation with an onion slice, and 2) Schirmer 1 test using a filter paper strip. The tears in the 5 x 5 mm part of the filter paper placed within the conjunctival sac were extracted with a 0.9% NaCl solution. The tear samples so obtained were analyzed by crossed immunoelectrophoresis. Nineteen proteins were identified by the use of monospecific antisera. Ten tear-specific proteins including the specific tear prealbumin of Bonavida et al., lactoferrin and lysozyme were identified by the intermediate gel method. Some differences were found in the pattern of the tear-specific proteins between the tears collected by the two methods, but the pattern was basically similar. Some individual differences were also found. The levels of serum proteins including albumin and IgG varied considerably, but it was thought to be due to dilution by reflex lacrimation during tear sampling. On the other hand, the pattern of the tear-specific proteins was fairly constant. The filter paper method allowed tear protein analysis, even in patients with 0 mm wetting by the Schirmer 1 test. A study of the length-volume relationship of the filter paper wetting showed that the tears contained in the 5 x 5 mm portion of the filter paper were about 1.8 microliters. The filter paper method was thought to offer a reliable clinical tool for the study of the tear proteins in various pathological conditions of the lacrimal secretion.

Two 2-dimensional electrophoreses: a combination of crossed immunoelectrophoresis and dodecyl sulfate polyacrylamide gel electrophoresis for protein analysis.

An improved method of two 2-dimensional electrophoreses that combined crossed immunoelectrophoresis and 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis was developed with a horizontal gel-electrophoretic apparatus.