JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1975, p. 287-291 Copyright C) 1975 American Society for Microbiology

Vol. 2, No. 4 Printed in U.S.A.

Production of Influenza Virus Complement-Fixing Antibody in Mouse Ascitic Fluid LENDELL A. WHITE,* WILLIAM C. GAMBLE, AND W. ADRIAN CHAPPELL Bureau of Laboratories, Center for Disease Control, Atlanta, Georgia 30333

Received for publication 17 June 1975

Suspension cultures of BHK-21/13s cells infected with either A/Hong Kong/8/68 or B/Massachusetts/3/66 strains of influenza were used to prepare type-specific influenza ribonucleoprotein-immunizing antigens. These antigens were used in comparing four immunization schedules in the production of immune mouse ascitic fluids. Large volumes of ascitic fluids were obtained by these schedules. These fluids contained type-specific complement-fixing antibody and were devoid of nonspecific host tissue or heterologous cross-reactions. Guinea pig sera containing antibodies to the enza RNP antigens as a replacement for imsoluble or ribonucleoprotein (RNP) antigen of mune guinea pig serum. the influenza virus are used in identifying influMATERIALS AND METHODS enza types A and B by the complement fixation Mice. Random-bred, 8-week-old, ICR, white, fe(CF) test. These type-specific antisera, prepared following procedures reported by Chap- male mice were used to propagate sarcoma 180/TG and to produce ascitic fluid. pell et al. (5) and Lennette and Schmidt (12), cells Sarcoma Sarcoma 180/TG cells were reserve as quality controls for the CF test in the ceived from 180/TG. Alan C. Sartorelli. They were mainserodiagnosis of influenza infections. Problems tained as a 10% cell suspension in 10% glycerol in obtaining guinea pigs without paramyxovi- saline and stored at -70 C. Immunized mice were rus CF or hemagglutination-inhibition (HAI) inoculated in the peritoneal cavity with 0.2 ml of a antibodies and in keeping these animals free of 10% suspension of a recent passage of sarcoma infection with other respiratory agents stimu- 180/TG cells to stimulate ascites formation. Adjuvant. Freund complete adjuvant (FCA) conlated a search for a more suitable method for producing influenza type-specific immune re- sisting of 85% Drakeol 6-VR (Pennsylvania Refining Co., Butler, Pa.), 15% Arlacel A (Hill Top Research, agents. Inc., Miamiville, Ohio), and 50 mg of desiccated Immune mouse ascitic fluid (IAF) has been Mycobacterium (Difco Laboratories, Deshown to be comparable to immune serum in troit, Mich.) wasbutyricum autoclaved at 15 lb/in2 at 121 C for potency and specificity of reactions in CF, HAI, 45 min and stored at 4 C until used. For immunizaand neutralization tests (15). Several investiga- tion, each antigen was mixed with an equal volume tors have described methods for producing large of FCA. A stable emulsion was prepared by vigoramounts of IAF-containing antibodies in high ously shaking the mixture in an International Centiters. Herrmann and Engle (8) demonstrated trifuge (model PR-2) equipped with shaker head no. HAI antibody levels for influenza and Newcas- 6007 and operated at 1,b00 rpm for 30 min at 4 C (9). Influenza viruses. Influenza strains A/Hong tle disease virus in sarcoma 180-induced ascitic and B/Massachusetts/3/66 were Kong/8/68 fluid. Kasel et al. (11) produced orthomyxovirus used in (H3N2) viral and RNP-immunizing antipreparing and paramyxovirus HAI antibodies and gens. The allantoic cavity of 10-day-old embryocoxsackievirus-neutralizing antibodies in nated chicken eggs was inoculated with 0.2 ml of a mouse ascitic fluids by immunzing with a viral 10-3 virus dilution in phosphate-buffered saline, antigen plus a bacterial-adjuvant mixture. Ber- pH 7.2. After incubation at 33 C for 3 days, the eggs kovich (1) and Fabiyi and Wenner (7) described were chilled at 4 C, and the allantoic fluids were CF and neutralizing antibodies to picornavi- pooled and clarified by centrifugation at 500 x g for ruses in mouse ascites formed with Erlich tu- 15 min. These allantoic fluids were used in the inmor cells. In recent years, sarcoma 180/TG cells tranasal (i.n.) inoculation of mice as well as in the in combination with Freund adjuvant have inoculum for cell cultures used in preparing RNPantigens. The hemagglutination (HA) been used extensively in the preparation of ar- immunizing titers of the allantoic fluids ranged from 1:320 to bovirus IAF containing CF, HAI, and neutraliz- 1:1280. The infectivity titers of the inocula of the ing antibodies (2, 15, 16). BHK-21/13s suspension cultures were determined by We have investigated the preparation and hemadsorption in primary rhesus kidney cell culuse of IAF prepared against type-specific influ- tures (12). These titers were 1058 mean infective 287




doses per ml for A/Hong Kong/8/68 and 107-3 mean infective doses for B/Massachusetts/3/66. Cell cultures. The BHK-21/13s suspension cultures were grown in Wistar growth medium (5) in 1liter, screw-cap, Erlenmeyer flasks on a magnetic stirrer. The Wistar growth medium was Eagle minimal essential medium prepared in Earle balanced salt solutions modified to contain a double concentration of vitamins and amino acids (except 1 x glutamine), supplemented with 0.1 mg of ferric nitrate per liter, 4.5 g of glucose per liter, 10% tryptose phosphate broth (Difco Laboratories), and 10% heatinactivated fetal calf serum. Cultures were incubated at 34 C and were subdivided every 7 days by diluting a 50-ml aliquot of cell suspension to a starting cell concentration of 5 x 105 cells per ml. Every 48 h an additional 200 ml of growth medium was added until the final volume reached 800 ml. Before inoculation, spinner cultures were centrifuged at 500 x g for 15 min, and the cells were resuspended in 200 ml of maintenance medium consisting of Eagle minimal essential medium without serum. An allantoic fluid inoculum was added to the cell suspension and allowed to adsorb with continuous agitation for 1 h at 34 C. After adsorption, the volume of medium was increased to 800 ml, giving an average cell count of 1.5 x 106 cells per ml. The suspension cultures were monitored daily for HA titer and adjusted to maintain a pH of 7.2 to 7.4. Cells were counted daily, and the viability was determined by trypan blue permeability. The cultures were harvested when the suspension contained 85 to 95% nonviable cells and were stored at -70 C until processed. Preparation of RNP antigens. The cell culture material was processed according to a modification of the technique of Rott et al. (14). The culture was concentrated to one-eighth of the original volume by using dialysis membranes and crystalline Carbowax (PEG-20M) at 4 C (3). Viral hemagglutinins were removed by exhaustive adsorption with human "O" erythrocytes. The RNP antigen was precipitated by acidification with 1 N HCl to pH 4.5 at 4 C. The precipitate was packed by centrifugation at 600 x g for 30 min and resuspended in 0.01 M tris(hydroxymethyl)aminomethane buffer, pH 8.5, at a concentration 'of one-twentieth of the original volume. Immunization of mice. Mice were immunized in groups of 25 according to the immunization schedules presented in Table 1. Influenza-infected allantoic fluids were given by the i.n. route in 0.25-ml amounts as the first inoculum of schedules 1 and 4. Emulsions of equal volumes of RNP antigen and FCA were administered intraperitoneally i.p. in 0.5ml amounts in all of the schedules. Ascitic fluid and serum. Ascitic fluids were withdrawn by paracentesis with an 18-gauge needle attached to a 30-ml syringe. Mice were initially tapped 7 to 10 days after they were inoculated with sarcoma cells, and the timing of later tappings depended upon the accumulation of fluid. The fluids from each tap were pooled and allowed to stand at room temperature for approximately 1 h. The fibrinous clot was separated from the fluid by filtration through a dou-

ble layer of sterile gauze. The fluids were centrifuged at 16,000 x g for 30 min and stored at -70 C. Bacterial contamination was eliminated by filtration through a 0.22-,um membrane filter (Millipore Corp., Bedford, Mass.). All surviving mice were bled at the end of the experiment, and the sera were pooled. Serology. The microtiter Laboratory Branch Complement Fixation technique (13) was used in performing the CF test. Block titrations were used to determine the optimal IAF, sera, and antigen dilutions. The HA titers were determined by macrotechnique, and HAI tests were performed by using the microtiter procedures described by Hierholzer et al. (10). The serologic antigens were prepared according to previously described techniques (5).

RESULTS Infected BHK-21/13s suspension cultures were monitored daily for the presence of hemagglutinins and for the percentage of viable cells remaining in the culture. The results of the culture monitorings and the CF titers of the immunizing antigens are presented in Table 2. The cultures were harvested for the preparation of RNP-immunizing antigens when more than 85% of the cells were no longer viable or on day 4 after inoculation. Daily observations of TABLE 1. Immunization schedules in producing immune mouse ascitic fluid Schedule no.

Day 0 7 14 21 23 28 30 33

35 37 40 42 47

49 57 59 69


Sarcoma IP




IP IP IP Sarcomac IP IP IP






Sarcoma IP Paracentesis


IP Paracentesis IP Sarcoma IP Paracentesis

a IN, Intranasal instillation of 0.25 ml of infectious allantoic fluids. b IP, Intraperitoneal injection of 0.5 ml of an equal volume emulsion of RNP antigen and FCA. e IP injection of 0.2 ml of a 10% suspension of sarcoma 180/TG cells.

VOL. 2, 1975



TABLE 2. Results ofdaily monitoring ofBHK-21113s suspension cultures forpreparation of

RNP-immunizing antigens

Expt no. nlo.


Virus and VuID0 ID50a titer titer

AIHK/8/68 105- ID5dml



B/mass/3/66 1073 ID5dml



Uninoculated cell cultures

Vol of inoculum (ml)

Viable cell count (x 104

HA titerb


Days after inoculation






10 20 40

150 184 138

17 14 13

Production of influenza virus complement-fixing antibody in mouse ascitic fluid.

Suspension cultures of BHK-21/13s cells infected with either A/Hong Kong/8/68 or B/Massachusetts/3/66 strains of influenza were used to prepare type-s...
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