The anti-human globulin test (Coombs test) has proved its importance in immuno-haematological research. Rabbits are classically used for the production of Coombs reagent. However, not all animals will produce anti-globulin antibodies in a suitable titre. Since some recent studies have demonstrated that chickens are excellent producers of precipitin [3, 4], it seemed reasonable to attempt the production of Coombs reagent by injecting chickens with human gamma globulin. Rhode Island red pullets and cockerels, six months old, were used in the experiments. The single injection method recommended by Brown and Wolfe [2] did not yield titres of sufficient strength. Several other injection schedules were tried. The best results were obtained when the chickens were injected three times (subcutaneously over the breast muscle) in four-day intervals with 200 mg. of gamma globulin per kg. On the tenth day after the last injection test bleeding were made from the wing vein in 5 ml. amounts, which were immediately centrifuged to obtain a clear, unhaemolysed serum. About 80% of the animals showed titres from 1/600 to 1/10,000, when CDe/CDe cells were sensitized against a 1 : 20 dilution of anti-D serum with a titre of 1 : 320. Ten days following the last injection, the animals were injected again subcutaneously with a similar dose and were bled eight days later. The titre showed a further increase in antibodies and in avidity, 75 % of the animals having titres of 1 : 10,000 to 1 : 20,000 or better. Normal chicken sera usually contain heterohaemagglutinins for human red cells, particularly for group 0[1]. However, the optimal dilution of chicken anti-human globulin sera obtained in these experiments varied between 1 : 100 and 1 : 350, sufficient in dilution to avoid the interference of heterophil reactions in routine work. A useful and simple screening technique is the capillary tube (1.5-2.5x100 mm.) precipitin test. A 1:40 dilution of immune chicken serum is layered over a 2 % solution of gamma globulin. This is best carried out in a buffered (0.01 M, pH 7.4) NaCl solution with eight grams per cent as the salt concentration of the reaction mixture [2]. A positive precipitin reaction after two hourse at room temperature almost always insures a titre of anti-human globulin greater than 1 : 5,000 as obtained by the Coombs method. Only one exception was observed. It has been found that chicken anti-human globulin antibodies, when used in optimal dilution, react very well with incomplete isoantibodies (anti-D, anti-C, and anti-Kell), as well as with auto-antibodies (acquired haemolytic anaemia). The simplicity of the immunization and bleeding procedures, the rapid rate of antibody production, the large percentage of positively reacting animals, and the exceptionally high titres and avidities of the antibodies thus obtained, make chickens valuable for the routine production of Coombs reagent.

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