Isolation and partial characterization of prochymosin and chymosin from cat.

Extracts of cat gastric mucosa contain a zymogen that after activation shows partial immunochemical identity with chymosin (EC 3.4.23.4) from calf. Cat prochymosin has been purified by column chromatography and gel filtration, and cat chymosin was obtained after acid activation of the zymogen. The enzyme showed the optimum of general proteolytic activity at pH 2.5. The amino acid compositions of cat prochymosin and chymosin were similar to those of the corresponding proteins from calf. The first 27 residues of both cat prochymosin and chymosin have been sequenced. Among these 54 positions only 13 differences have been observed between the proteins from cat and calf. The results support the hypothesis that the chymosins form a group of neonatal gastric proteases with high milk-clotting activity, but with such weak general proteolytic activity that postnatal uptake of IgG is not hindered.

Detection of M-components by an easy immunofixation procedure: comparison with agarose gel electrophoresis and classical immunoelectrophoresis.

Commercial anti-gamma-alpha-mu and anti-kappa-lambda (mixture of commercial anti-kappa and anti-lambda) were used for immunofixation after agarose gel electrophoresis of 100 serum samples diluted 1 : 5. This simple screening method detected M-components in 39 sera. M-components were detected in 33 sera by agarose gel electrophoresis, and in 30 sera by classical immunoelectrophoresis. More than one band was detected in 15 sera by the screening method, in 3 sera by agarose gel electrophoresis, and in 2 sera by immunoelectrophoresis. The screening method was superior to the combined use of agarose gel electrophoresis and classical immunoelectrophoresis for detection of M-components, and easier to perform and interpret.

Counter current line deflection immunoelectrophoresis. A technique for the quantification of antibodies.

Combining the electrophoretic principles of counter current immunoelectrophoresis and deflection of line precipitates in line immunoelectrophoresis provides a new technique for quantitative determination of antibodies against specific antigens (CCLD electrophoresis), even if no purified antigen or monospecific antibodies are available for construction of the detection system. We have used the method for quantitative determination of antibodies against the flagellum of Treponema phagedenis biotype Reiter and compared the diagnostic potential of this method in the diagnosis of syphilis with an ELISA method for the quantification of IgG antibodies against the flagellum. The CCLD electrophoresis could be optimized to a diagnostic performance very similar to that achieved using the ELISA method.

Computer controlled affinity chromatographical purification of soluble Plasmodium falciparum antigens from supernatants of in vitro cultures.

Affinity chromatographic procedures are difficult to scale up from the analytical to the preparative level when the ligand used for purification is a limiting factor. A versatile, computer-controlled affinity chromatographic system is described which permits automatic repetition of the purification process and sophisticated control functions based on the ultra-violet absorbance of fluid passing through the affinity column. The system has been used for automation and scaling up of the purification of Plasmodium falciparum exoantigens.

Alpha-fetoprotein-like activity in sera from patients with malignant and non-malignant disease and healthy individuals.

A new method, radio-crossed immunoelectrophoresis, demonstrates alpha-fetoprotein (AFP) in sera with a sensitivity of 1 mug/1. By this method AFP with alpha mobility was not found in sera from healthy individuals, patients with chronic active hepatitis and cirrhosis, primary biliary cirrhosis, secondary liver cancer and cystic fibrosis. In some of the sera, AFP was elevated when measured by conventional radioimmunoassay method and the sera contained an AFP-like substance with gamma mobility when analyzed by radio-crossed immunoelectrophoresis. The nature of this gamma substance is still obscure and needs further investigation.

Determination of pepsin (EC 3.4.23.1) and gastricsin (EC 3.4.23.3) in gastric juice by rocket immunoelectrophoresis.

Antisera were raised in rabbits against chromatographically purified preparations of pepsin and gastricsin. With these antisera the contents of pepsin and gastricsin in gastric juice were determined by rocket immunoelectrophoresis. The potential content of pepsin and gastricsin of a secondary standard of gastric mucosal extract was calibrated against the chromatographically purified enzymes. This secondary standard was used for routine analyses. The intra-assay and between-assay precision was 3-4% and 6-9%, respectively. Ten healthy volunteers underwent a standard pentagastrin test. The amounts of pepsin and gastricsin determined by rocket immunoelectrophoresis corresponded to the amounts observed by ion exchange chromatography of gastric juice. After stimulation with pentagastrin the secretion of both pepsin and gastricsin was increased about 10 times.

Antigen-antibody crossed electrophoresis (Laurell) applied to the study of the antigenic structure of Candida albicans.

By means of the antigen-antibody crossed electrophoresis procedure of Laurell, 68 antigens were demonstrated in Candida albicans. This is about four times the number of antigens described earlier by means of classical immunoelectrophoresis. The procedures for obtaining this result are described, including the preparation of antigen, the immunization of rabbits, and the method of N. M. G. Harboe for the production of purified and concentrated rabbit antibodies suitable for quantitative immunoelectrophoresis. The immunoplates were stained by means of the sensitive Coomassie brilliant blue R. The various quantitative immunoelectrophoretic methods offer considerable possibilities for qualitative and quantitative characterization of antigens, even in complex mixtures, and are therefore well suited for the investigation of microbial antigens.

Quantitative immunoelectrophoretic methods as tools for a polyvalent approach to standardization in the immunochemistry of Candida albicans.

Quantitative immunoelectrophoresis allows a polyvalent approach to immuno-chemical problems, i.e., identification, quantitation, and thereby standardization of antigens and antibodies in complex mixtures, for example as in crude extracts of microorganisms and their corresponding antisera. This approach is a short cut to conventional standardization of single purified substances, since the polyvalent approach gives a precise quantitative impression as described in this investigation and therefore enables the immunochemist to precisely select the right substance(s) to be purified for further standardization and characterization. To secure the precision of the results, in complex systems it is a sine qua non to select a complex antibody standard, a complex antigen standard, and to describe the complex standard precipitate pattern which again allows a quantitative study of the reproducibility of methods and procedures. The present article describes how such a standardization was approached in this laboratory within the field of Candida albicans immunochemistry. By means of crossed immunoelectrophoresis, 78 water-soluble antigens were demonstrated and enumerated in an antigen standard prepared from one strain of C. albicans A (B 311 Hasenclever). The antibody standard consisted of purified and concentrated rabbit antibodies. The migration velocity of each antigen was indicated in relation to purified human albumin. Not all precipitates could be seen in one immunoplate; therefore a standardized procedure was worked out showing reproducibly 54 precipitates. The reproducibility of quantitation by the crossed-immunoelectrophoresis procedure was determined for each of 30 antigens by repeated measurements; the relative standard deviations ranged from 2.4 to 15.4% and were below 10% for 24 antigens. A simple standardized antigen production procedure was described in great detail, and by quantitative determinations on 30 antigens the procedure was found to be satisfactorily reproducible. By means of crossed-line immunoelectrophoresis in the modification called absorption of antibodies in situ, it was found that strain B 311 contained no specific antigens in comparison to eight other strains of C. albicans. Thirty antigens were quantitated in antigen preparations made by the standardized procedure from the eight strains, and each antigen concentration was expressed as a percentage of the antigen standard. For each antigen a significant inter-strain variation was observed. Of 30 antigens, fifteen were satisfactorily stable after storage at -20 C for 1(1/2) years. The laboratory methods and procedures described in this article thus work with high precision and allow a rapid collection of quantitative data concerning many individual antigens and their corresponding antibodies without purification of antigens. A new complex antigen standard can be made with satisfactory precision from strain B 311. The production of a similar complex antibody standard is a major problem; therefore the main problem in the intra-laboratory standardization seems to be the change from one complex standard to another. In inter-laboratory standardization on the complex level, there seems to be a minimum demand that the first laboratory distributes the antibody standard and that other laboratories use the methods and procedures of the first laboratory.