Hoyle & Fairbrother (1937) showed that tissues infected with influenza virus contained two distinct particles, the infective virus elementary body, and a smaller particle, the soluble antigen, which could be demonstrated by complement-fixation tests.

Influenza virus elementary bodies can be disintegrated by treatment with ether with the liberation of two types of smaller particle, soluble antigen and red cell agglutinin.Soluble antigen derived from the elementary body is identical in serological and chemical behaviour with soluble antigen recovered from infected tissues. Its chemical properties are those of a ribonucleoprotein.The agglutinin is an enzyme, with a protein part carrying the enzymic activity and a combining group with affinity for red cells. The agglutinin does not react in complement-fixation tests by the short fixation technique, but by the use of prolonged fixation or by indirect complement fixation it can be shown to contain a strain specific antigen and also a non-specific antigen. The specific antigen is identical with the specific complement-fixing antigen demonstrable in the intact elementary body, and has combining affinity for red blood cells. The non-specific antigen is probably similar to the protein part of the soluble antigen. No evidence has been found that the agglutinin contains either carbohydrate or nucleic acid.From the ether used to disintegrate the elementary body a serologically active lipid can be recovered which has properties suggesting that it is derived from the host cell.The author is greatly indebted to Dr L. Dmochowski for assistance in experiments involving high-speed centrifugation, to Sir Macfarlane Burnet and Dr G. L. Ada for supplies of receptor-destroying enzyme, to Dr E. S. Duthie for crystalline trypsin and chymotrypsin, and to Dr R. R. Porter for a sample of crystalline ribonuclease.

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This paper describes a study of the mechanism of the complement-fixation reaction in influenza. Its purpose was to determine the relative parts played in the reaction by the virus elementary body and the soluble antigen, and to find out whether any specific differences could be demonstrated between different strains of influenza virus A. Almost all the work has been done by means of 'chess-board' experiments of the type first used by Dean & Webb (1926) in their classical work on the complementfixation reaction. These experiments, in which a series of antigen dilutions is tested against a series of serum dilutions with a constant dose of complement, convey very much more information about the nature of a complement-fixation reaction than is obtainable by a simple titration of antigen or serum.standards of 0, 25, 50, 75 and 100% haemolysis was prepared and the tubes matched against these.A constant dose of complement (21 M.H.D.) has been used in most of the experiments. The complement was the pooled serum of several guinea-pigs preserved by the boric acid-sorbite-azide method of Richardson (1941). This preserved liquid complement has been found very satisfactory as its titre remains unchanged for weeks and enables a large number of experiments to be done with the same batch of complement.The sera used in this work were mainly human convalescent sera from the epidemic of 1943-4. They were inactivated at 560C. for 30 min. and preserved by addition of 0-08 % sodium azide. Some of the antigens used in the work have also been preserved by sodium azide.

1. When the D.S.P. strain of influenza virus A is grown in eggs into which 100 μc. of radioactive inorganic phosphate has been introduced the virus incorporates32P into its structure.2. Some 20–25% of the virus32P is found in the virus phospholipid; the remainder is combined with the virus protein and is probably present in the virus nucleic acid.3. When the virus is disintegrated by ether treatment with the liberation of separate red-cell agglutinating and complement-fixing ‘soluble antigen’ particles the non-lipid32P is found to be associated with the soluble antigen fraction and not with the haemagglutinin.4. It is suggested that the complement-fixing soluble antigen is a nucleoprotein while the haemagglutinin is a phosphorus-free protein.

METHODS of estimating indole in bacterial cultures have been devised by various workers, notably by Herter and Foster and Fellers and Clough [1925].In these methods the indole is separated from the culture by steam-distillation, the distillate being treated with Ehrlich's p-dimethylaminobenzaldehyde reagent and the indole estimated colorimetrically by comparison with the colour given by standard indole solutions. It is important not to use excess of Ehrlich's reagent since this causes a change in the colour. The methods are somewhat laborious and not very accurate, as even under optimum conditions the whole of the indole present is not recoverable in the distillate, and with unknown concentrations it is often difficult to determine the correct amount of Ehrlich's reagent to use.In the course of some investigations carried out by one of us (L. H.) it was found necessary to estimate indole present in various complex bacterial cultures, notably in cultures containing bile and bile salts, which could not be distilled on account of excessive frothing, and a study was therefore made of the possibility of estimating the indole by extraction with organic solvents.The proposed method was to extract the indole from the culture with the solvent, and estimate the indole present in the extract by the Ehrlich reaction.The various indole solvents were found to fall into three groups.(1) Solvents miscible with Ehrlich's reagent: ether, chloroform, amyl alcohol, isoamyl alcohol, capryl alcohol.(2) Solvents immiscible with Ehrlich's reagent but dissolving the rosindole body: benzene (dissolves the rosindole slowly).(3) Solvents immiscible with Ehrlich's reagent and in which the rosindole body is insoluble: light petroleum, xylene.Solvents of group (1) were found unsuitable, as it was difficult to decide the amount of Ehrlich's reagent required, and excess was found to alter the colour of the rosindole body.Solvents of group (2) were imperfect because of the unequal distribution of the rosindole body between the solvent and the Ehrlich's reagent.Solvents of group (3) were found suitable, and light petroleum, which gave the best results, was easier to handle than xylene.It was found that extraction of the culture with an equal volume of light petroleum removed about 90 % of the indole present, so that two successive extractions removed practically the whole of the indole. The extraction was equally effective whether the culture were made acid or alkaline, and it was found useful to acidify the cultures, as this facilitated separation of the liquids.

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There is much evidence to indicate that viruses, in certain cases at least, have an antigenic structure of comparable complexity to that of the bacteria. Hughes (1933) found that the serum of animals immunized with the yellow-fever virus contained two independent antibodies—precipitins and protective antibodies; the precipitinogen was distinct from the virus. Craigie & Wishart (1936) in investigations of the vaccinia virus have shown that, in addition to the elementary bodies, virus suspensions contain two soluble precipitable substances, the “L” antigen which is labile at 56° C. and the “S” antigen which is stable at 95° C. These antigens were readily demonstrated by precipitation, agglutination and complement fixation. Their nature and origin have not, however, been precisely determined. Bedson (1936), working with the psittacosis virus, prepared a soluble antigen, which was independent of the elementary bodies. It was most satisfactorily demonstrated by complement fixation.