1) In order to use the high-voltage paper electrophoresis of amino acids as a quantitative method, several fundamental experiments were carried out by the pure crystal of amino acid, the hydrolysate of ovalbumin and human serum.
2) Electrophoresis were performed at a potential gradient of 100 to 150v/cm and in acetic acidpyridine buffer (pH3.5).
3) Amino acid spectra which were colorized by ninhydrin reagent were altered to a series of peaks by densitometer (Filter 550mμ).
4) The relative migrativities of various amino acids, which were evaluated with respect to the position of glutamic acid, showed a good reproducibility and were constant independently of the periods of electrophoresis (12 to 60min).
5) The standard curves of 15 kinds of amino acids were obtained. As a result, in a region of 3 to 5γ of applying amount of amino acids, the straight proportionalities were found between the color yields and applying amount of various amino acids.
6) The ratio of color yields of various amino acids against that of glutamic acid were evaluated from the slopes of standard curves.
7) The quantitative determinations of amino acids were thus possible by correcting the peak areas obtained by densitometry by using these ratios of color yields of amino acids. This was verified by the electrophoretic analysis of the artificial mixture of amine acids and the hydrolysate of ovalbumin.
8) The serum (1.0ml) was deproteinized by adding methanol-acetone (3:1) mixture and supernatant was concentrated to 0.1ml in water-bath and 0.01ml of that was used for the electrophoresis.
9) In the analysis of pherograms, the ratio of each peak area for the sum of all peaks obtained by densitometry was determined. No ratios of color yields was used.
10) Although the amino acids patterns of five normal sera hardly showed any individual difference, the pathological states (carcinoma of the stomach and Banti's syndrome) gave various different patterns.
11) It was convinced that this method is very useful for the clinical application of amino acid anlyasis of human blood serum.

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1) High voltage paper electrophoresis was used for the quantitative analysis of the free amino acids of human blood serum.
2) The serum (1.0ml) was deproteinized by adding methanol-acetone (3:1) mixture and the supernatant was concentrated to 0.1ml in water bath. Electrophoresis was performed at a potential gradient of 100 volt/cm and in acetic acid-pyridine buffer (pH 3.5). The time of electrophoresis was 12 and 60 minutes.
3) The amino acid patterns of 22 normal human sera hardly showed any individual difference.
4) The amino acid patterns of gastric ulcer, cholelithiasis and tuberculosis of the lung nearly agreed with that of normal human blood serum.
5) The blood serum of patient with carcinoma gave various different amino acid patterns, but these patterns were classified by five types on carcinoma of the stomach and three types on carcinoma of the breast. It was found that these types of amino acid patterns were related to the grade of developements of carcinoma.
6) Banti's syndrome showed a particular type of amino acid pattern which was found in other diseases.

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The principle of the “crossing paper electrophoresis”: Two substances applied on two separate and inclined lines on a filter paper will cross each other with the progress of electrophoresis, provided that their mobilities are different. If the two substances react with each other to give a loosely bound addition compound, the point of crossing may show deformation. It is because the addition compound formed has a different mobility from those of its mother substances, the two reactants. Thus it will be possible to detect the formation of an addition compound by the method of “crossing paper electrophoresis”. In fact the method has been confirmed to be feasible, as shown by the application to the interaction of dyes.
The “crossing paper electrophoresis” was applied to the detection of interaction between dyes and between dye stuffs and proteins as well as amino acids:
The basic dyes, methylene blue and fuchsine were proved by the method to interact with the acidic dyes, orange GG, bromocresol green, bromophenol blue and amido black 10 B.
With proteins and dyes, the interaction was only detected between serum albumin and bromocresol green as well as bromophenol blue. The interaction between them appeared to be stoichimetric. Other dyes interacted only very weakly with proteins.
With amino acids no interaction could be detected among themselves and with dyes, egg albumin, and serum proteins. No interaction could also be detected between egg albumin and serum proteins nor between dyes.

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By the application of the crossing paper electrophoresis, antigen-antibody reactions could be detected on the filter paper. Antiserum was applied on a line on the anodic side of the filter paper and antigen was applied on a line drawn obliquely to the former on the cathodic side. By the electrophoresis, the antiserum was separated into its components and crossed over by the antigenic proteins. Antigen-antibody precipitates occurred in the zone of γ-globulin of the antiserum.
The line of precipitate formed by the reaction of egg albumin with its homologous antiserum was dissolved and moved, if excess of egg albumin had crossed with it. The antiserum obtained in the earlier stage of immunization, however, showed a line of complex, which was not dissolved by the excess of egg albumin. The complex was suspected not to be precipitate. Thus it was inferred that the anti-egg albumin antibody of the earlier stage would be “incomplete”, which would become complete precipitin afterwards.
The cross reactions of the anti-hen's egg albumin with heterogenous antigens, egg albumins of duck, goose, guinea hen, and quail, could also be deteced.
By the crossing paper electrophoresis of bovine serum and its rabbit antiserum, at least 6 lines of pricipitate were detected. Two-dimensional application of the method was also carried out: The antiserum alone was first separated on a line in the first dimension. Then the antigenic bovine serum was applied on a line vertical to the first one. And the second electrophoresis was carried out to the 2nd direction vertical to the first one. The proteins of bovine serum migrated into the zone of γ-globulin of the antiserum to form lines of precipitate. By the two-dimensional technique, however, the sensibility of the method could not be raised. A comparison with the technique of Garbar's immunoelectrophoresis was made.
The cross reactions of the anti-bovine serum rabbit antiserum with heterogenous antigens, human, swine and goat serum, were detected by the method.

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1. For the purpose of improving the ability of electrophoretic separation of the Tiselius apparatus, the author developed a method by which to form a dependable zone between solutions in cells. At the same time he obtained a particular zone electrophoretic diagram by means of an optical apparatus of his own making.
2. From the viewpoint of relative mobility, at least new 10 peaks were observed, in addition to 6 (A, α₁, α₂, β, φ and γ) which have been found.
3. Though these new peaks may be regarded as sub-fractions of the said fractions, the author now desists from forming his conclusion.

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In view of the complexity and inaccuracy of densitometery now in use, we adopted a fractional elution method as a substitute for it and made basic studies on the same with the following results:
1. Pigments are completely extracted from pieces of filter paper with 5-10cc of N/100 NaOH solution in 30 minutes at room temperature. The extinction of the extract and the amount of serum added show a linear relationship.
2. The color tone of the extract, which in prepared by removing bromphenol blue (BPB) or Amidoschwarz 10B (AS) from filter paper with N/100 NaOH solution, fades away with the passage of time; this phenomenon is especially remarkable in the extract which is prepared by removing BPB from filter paper with the said solution. Since the tendency of the color tone to fade away is true of each fraction of serum protein, no significant differences take place at least for 5 hours when the percentage of protein in serum is calculated.
3. The maximum resorption wavelengths of the extracts which are prepared by removing BPB and As from filter paper with N/100 NaOH solution are 570 and 620mμ.
4. When measurement is made by only an individual according to this method, its reproductivity is considerably high in each serum of healthy subjects and cases of nephrosis and liver cirrhosis.
5. Even when this method is performed on different individuals, it shows a reproductivity equal to that of the Tiselius method.
6. By this method we followed great changes in serum protein in cases of nephrotic syndrome that were produced during a short time by a large dosage of predonine.

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