There were many reports on the neutralization test of poliomyelitis (polio) virus before 1939, as well as on the relationship between the neutralizing antibody and the illness. The animals available for the experiments of polio were only monkey before 1939, so that in any experiment animals were not used enough to estimate accurately the blood level of antibody because of their expense. However since Armstrong (1) was successful to transfer the “Lansing” strain of polio virus which had been isolated in monkey, first to the cotton rat and then to the swiss mouse, many mice were used for the neutralization test against the mouse-adapted strain, and the result would be more precise to discuss on the neutralizing antibody on the polio case.
It is well known that the higher positive rate of neutralization test of human sera against the “Lansing” strain of polio virus are found in the older age groups according to Turner (2) and Pait (3), the sera of 80% of children, less than 6 months old are capable of neutralizing the virus, while children from 6 months to 2 years of age have the positive rate in 20-30 per cent. With advances in ages an increasing percentages of individuals become positive in this test, and the sera of children between thee age of eight and twelve years have this property in 80%.
It was pointed out by Paul (4) that Japan was strongly contaminated by polio virus due to her poor sanitary condition. Well it is very important to determine how many Japanese in each age group has a neutralizing antibody against the mouse-adapted “Lansing” strain of polio virus following the subclinical infection.
The methods of neutralization test used by ntiany authors are not the same. They are summarized principally in the following :
1) by mixing the same quantities of undiluted serum and suspension of infects mouse brain and spinal cozd known to be active,
2) by mixing the same virus with sera 10, 3.2 or 2 fold diluted,
3) by mixing a series of 10 fold diluted virus with undiluted sera,
4) by mixing a series of 10 fold diluted virus with sera 10, 3.2 or 2 fold diluted.
Such mixtures are generally incubated for two hours and then injected intracerebrally into each group of mice.
Method 1) is the most simple and generally used. The point is that the virus employed in each experiment should be the same in an adequate virulence. For this purpose the virus emulsion should be kept in dry ice box in sealed ampules. If the virus is not of the same virulence it is impossible to compare the results obtained by many authors each other. For example Turner (5) used the virus suspension of 180 LD₅₀, Hammon (6) and Morgan (7) 10 LD₅₀ and Pait (3) 32 LD_50-. Method 2) is used to determine the titer of positive serum. Method 3) is for the determination of the negative serum. Method 4) isacombination of 2) and 3) . One example is shown in Table 1. Method 3) is generally used by us and sometimes combined with method 4) .

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Resistance to heat and acidity of the antibody to arsenious acid was investigated by Y. Yamasaki (2) . Serum of the guinea pigs actively sensitized by intracutaneous injections of arsenious acid was heated or acidified, and a definite amount of the treated serum was intravenously injected into normal guinea pigs, whose hypersensitivity was determined by skin reaction to arsenious acid in the manner already mentioned (1) . The serum was heated in the water-bath for 30 minutes at various degrees ; and it was acidified with weak acetic acid solution, changes of pH being measured by the colorimetric method, and neutralized with dilute sodium hydroxide solution before use. It was found that sensitizing ability of the serum was impaired very slightly at pH 5.6 and to a noticeable degree at pH 5.1, and almost completely lost at pH 4.6-4.1. As for the effect of heating it was disclosed that allergizing function was practically unaffected at 56°C, slightly impaired at 60°-65°C, markedly hindered at 70°C and almost completely lost at 75°C. Subsequently, H. Harada (3) has made like experiments in search of effects of heating and acidity upon antibodies to iodine, potassium bichromate and formaldehyde, pursuing the methods of previous workers (4, 5, 6) in sensitization and in tests of reactions. He determined hypersensitivity to iodine by the intestinal reaction and to the other substances by skin reactions, and obtained results similar to those of Y. Yamasaki.
A more extensive study was done by M. Hara (7) . Not only serum but also its globulin fraction of guinea pigs sensitized with egg albumin, picric acid or iodine (iodine-glycerine) were used in his experiments, and effects on their sensitizing abilities of heating, acidity, alkalinity, adsorption and formaldehyde were examined. Active sensitization and provocation of reactions with picric acid and iodine were accomplished by the methods used by previous authors (4, 8) . To sensitize with egg albumin, 2 doses of 0.3 cc of 5% albumin solution per 100 g of body weight were injected with the interval of one day. Globulin solution was prepared as stated by Swineford (9) . After special treatments, sera and globulin solutions were injected in the following amounts per 100 g of body weight ; 1.5 cc of anti-albumin serum, 2.0 cc of anti-picric acid serum, 2.0 cc of anti-iodine serum, 2.0 cc of anti-albumin globulin, 2.5 cc of anti-picric acid globulin, and 2.5 cc of anti-iodine globulin. And 24 hours later anaphylactic reactions were detected by the skin test for picric acid and by Dale tests for egg albumin and iodine, 1.0 cc of 0.05% solution being used in case of egg albumin. Heating and acidification were carried out as described above. For alkalification, dilute sodium hydroxide solution was added, pH was measured, and one hour later the serum or globulin solution was neutralized with acetic acid. To test the effect of formaldehyde, formalin was added to make up 0.25%, 0.5% or 1.0%, and then the serum or globulin solution was dialysed with collodion sac to remove free formaldehyde. In the treatment of adsorption, animal charcoal powder (Merck) was added to 5% or 10%, and the mixture was shaken for 20 minutes, left in the ice box for 24 hours, centrifuged, and the supernatant fluid was used.
Antibodies to three different substances did not show any difinite difference in resistance; sera and globlin solutions resisted to those treatments without noticeable difference. Experiments opt the effect of heating indicated that sensitizing function was impaired slightly by heating at 60°C for 30 minutes, moderately at 60°C for one hour, and destroyed almost completely at 60°C for 2 hours. It was affected slightly by acidity at pH 6.0, noticeably at pH 5.5-5.0 and destroyed at pH 4.0. Alkalinity impaired it slightly at pH 8.5, markedly at pH 9.0-9.5 and to a greater extent at pH 10.0.

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It has been reported (1) that papain as well as other papainase are capable of synthesizing CO-NH linkages especially in the meaning of antipodal specificity. It was desirable to see whether the ficin, a proteolytic enzyme of papain type, is capable of synthesizing peptide bonds or not.
We have now succeeded in the enzymatic synthesis of anilide or phenylhydrazide when in the presence of ficin acylated amino acid was incubated with aniline or phenylhydrazine at 37°C and pH 4.5.
The optimum pH range of the enzymatic formation of hippurylanilide has been found to be close to 4.5, at which the ficin is also able to perform hydrolysis.
It is remarkable that the antipodal specificity of the ficin as a synthesizing enzyme was revealed when acyl derivative of DL-phenylalanine was incubated with aniline and ficin. According to a very slight solubility of the anilide formed the antipodal specificity of the ficin can be utilized as a method to resolve DL-amino acids in their optical components.

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