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Passive haemagglutination test(PHA) using sheep red blood cells(SRBC) sensitized with soluble antigens by means of coupling agents, i.e., tannic acid, bis-diazotized benzidine(BDB), chromic chloride, glutaraldehyde, and so on, provide an extremely sensitive technique to detect a minute quantity of serum antibody as low as 0.01㎍. It also has advantage over agglutination and precipitation tests in that if it is transferred either to hemolysis test(PHL) or to inhibition test(PHI), soluble antigen-antibody complexes and incomplete or hapten antigen-nonprecipitating antibody complexes that are not detectable by other tests can be demonstrated. This study was undertaken to determine the optimum amount of BDB and tuberculin PPD antigen for consecutive and simultaneous coupling of SRBC in conjunction with the sensitivity of the PHA test. In addition, the sensitivities of PHA and PHL test were compared and the PHA units of antibody and diluents for tuberculin PPD antigen for PHI test were discussed. The results are summarized as follows: 1. The optimum amount of BDB for consecutive and simultaneous coupling of 0.2ml of 2.5% SRBC were 20㎍ and 13㎍, respectively, and the optimum amount of tuberculin PPD antigen were about 125㎍ and 250㎍ respectively. The optimum ratios between BDB and tuberculin PPD antigen for consecutive and simultaneous coupling were 1:6.3 and 1:19.2, respectively. 2. In the consecutive coupling, the PHA titre was linear to both BDB and tuberculin PPD concentrations, whereas in simultaneous coupling it was affected by the level of tuberculin PPD but not by that of BDB from 5㎍ to 13㎍. 3. Using SRBC sensitized consecutively with tuberculin PPD or sensitins prepared from four species of mycobacteria the homologous PHL tests were sensitive than the PHA tests by 1:2 to 1:4 dilutions, and the end-point of PHL test was more clear-cut than that of PHA test.
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