All antisera were produced in rabbits (laboratory stock of Oryctolagus cuniculus). Three methods of injection of antigen were used in various combinations: intravenous, subcutaneous, and intraperitoneal. Injection schedules used in the production of each antiserum are listed in Table[ 1]. Both formolized and "native" antigens were used. Each rabbit received one or more series of four injections, each injection being administered on alternate days and doubling in amount: 0.5 ml., 1.0 ml., 2.0 ml., and 4.0 ml. In all but two instances more than one series of injections was necessary to produce a useful antiserum. More than two series, however, resulted in little or no improvement of the reactivity of the antiserum.
The injection-series were separated by intervals of eight days. On the eighth day after the last injection of each series, 10 ml. of blood were withdrawn from the main artery of the ear of the rabbit, and the antiserum was used in a homologous precipitin test to determine its usefulness. If the antiserum contained sufficient amounts of antibodies to conduct the projected tests, the rabbit was completely exsanguinated by cardiac puncture, by using an 18-gauge needle and a 50 ml. syringe. The whole blood was placed in clean test tubes and allowed to clot. It was allowed to stand at 2°C. for 12 to 18 hours so that most of the serum would be expressed from the clot. The serum was then decanted, centrifuged to remove all blood cells, sterilized in a Seitz filter, bottled in sterile vials, and stored at 2°C. until used.
Methods of Serological Testing
The precipitin reaction is the most successful of the serological techniques thus far devised for systematic comparisons. The reaction occurs because antigenic substances introduced into the body of an animal cause the formation of antibodies which precipitate antigens when the two are mixed. The antisera which are produced show quantitative specificities in their actions; therefore, when an antiserum containing precipitins is mixed with each of several antigens, the reaction involving the homologous antigen (that used in the production of the antiserum) is greater than those reactions involving the heterologous antigens (antigens other than those used in the production of the antiserum). Furthermore, the magnitudes of the reactions between the antiserum and the heterologous antigens vary according to the degrees of similarity of these antigens to the homologous one.
The method of precipitin testing follows that outlined by Leone (1949). The Libby (1938) Photronreflectometer was used to measure the turbidities developed by the interaction of antigen and antiserum. With this instrument parallel rays of light are passed through the turbid systems being measured. Light rays are reflected from the suspended particles to the sensitive plate of a photoelectric cell; this generates a current of electricity which causes a deflection on a galvanometer. The deflection is proportional to the amount of turbidity developed and readings may be taken directly from the scale of the instrument.
The reaction-cells of the photronreflectometer are designed to operate with a volume of 2 ml.; therefore, this volume was used in all testing. In every series of tests the amount of antiserum was held constant and the amount of antigen was varied. The volume for each antigen dilution was always 1.7 ml., and to this was added 0.3 ml. of antiserum to make up a volume of 2 ml.