NOTES
The Use of Frozen Cells in the Microtitre Serum Neutralization Test for Infectious Bovine Rhinotracheitis P. R. Ide*
ABSTRACT
RESUME
Some parameters of the microtitre serum neutralization test were examined when using bovine fetal kidney cells derived from stocks stored in liquid nitrogen. In replicate tests with one serum there was no significant difference (p > 0.05) between titres calculated on the basis of cytopathic effect when either two or four wells were used per serum dilution. Also, there was no significant difference between titres calculated on the basis of a cell staining method when either two or four wells were used per serum dilution. When titres obtained by the 2-well-cytopathic effect method were compared with those obtained by the 2-well-stain method differences were not significantly different and it was concluded that the latter method using frozen cells constituted a practical and reproducible test. No significant titre variation occurred in cells serially passaged after having been frozen, or in cells derived from five different fetuses. Titres of sera from 41 cattle conformed to a log normal distribution pattern.
Cette experience visait a etudier certains parametres de la micromethode de sero-neutralisation, en utilisant des cellules renales de foetus bovins pre'alablement conservees dans de l'azote liquide. La repetition d'epreuves avec un antiserum ne revela pas une difference appreciable (p > 0.05) entre les titres calcules d'apres l'effet cytopathogene, lorsqu'on utilisait deux ou quatre puits pour chacune des dilutions du serum. On ne decela pas non plus de diffe'rence appreciable entre les titres calcul6s d'apres une methode de coloration des cellules, lorsqu'on utilisait deux ou quatre puits pour chacune des dilutions du serum. Une comparaison des titres, basee sur l'effet cytopathogene obtenu en utilisant la methode de deux puits, avec ceux que donna la methode de coloration, en utilisant deux puits, ne r'vela pas de difference appreciable; on conclut donc que cette derniere methode representait, lorsqu'on utilisait des cellules congelees, une methode pratique et reproductible. Les cellules soumises a des passages en serie, apres leur congelation, ne subirent pas de variation appreciable de leur titre; on fit la meme observation avec les cellules provenant de cinq foetus. Les titres du serum de 41 bovins donne-rent une courbe logarithmique normale.
*Atlantic Area Laboratory, Animal Pathology Division, Health of Animals Branch, Agriculture Canada, P.O. Box 1410, Sackville, New Brunswick EOA 3CO.
Submitted April 10, 1974.
Vol. 39 -January, 1975
The microtitre technique has been applied to the serum neutralization (SN) test with
101
infectious bovine rhinotracheitis (IBR) virus in both primary tissue cultures and established cell lines (1, 3, 7), for which seed aliquots may be stored frozen (3). There has been little published on the reproducibility of the microtitre serum neutralization (MTSN) test in cells derived from frozen primary cultures at different passage levels or on tests conducted in frozen cells derived from different fetuses. There is also no information on the comparative reproducibility of MTSN test employing either two or four wells per serum dilution or on the comparative sensitivity of tests read either on the basis of cytopathic effect (cpe) or staining pattern. As such factors are of practical and economic importance the following work was conducted to examine such parameters. The Colorado strain (2) of IBR virus and homologous bovine antiserum (RS1) were used. Primary bovine fetal kidney (BFK) cultures were prepared as described (4) and monolayer cells were suspended in freezing medium (sodium bicarbonate and antibiotic-free Earle's salt solution' containing 10% fetal calf serum and 10% dimethyl sulfoxide2) at a concentration of approximately 3 x 106 per ml (5). Cell aliquots were stored in liquid nitrogen and for use were diluted 1:10 in medium, grown as monolayers and trypsinized (5) for use in the MTSN test which was conducted as described (1). The virus challenge was 100 TCID50 per 0.025 ml and the virus-serum mixtures were incubated for two hr at room temperature, then approximately 15,000 cells (in 0.05 ml) were added to each well and plates were sealed with an adhesive film3 and incubated at 37°C. Appropriate serum controls (as serum dilutions without virus) and a titration of the virus challenge were included in each test. Five days later plates were examined for evidence of cpe and the residual monolayer in each well was stained with 2% crystal violet in alcohol (5). Titres were calculated by the Reed-Muench method (5). In the first series of experiments replicate MTSN tests were conducted with
'Grand Island Biological Company, Grand Island, New York. 2Fisher Scientific Co., Fairlawn, New Jersey.
3Falcon Plastics, Becton Dickinson & Co. (Canada) Ltd., Mississauga, Ontario.
102
serum RS1 in two lots of cells (BFK41 and BFK45) at various passage levels (up to the seventh) after thawing, using two or four wells per serum dilution. Serum titres were calculated on the basis of both cpe and staining patterns. In the former, any cpe was interpreted as virus growth (lack of serum neutralization) and in the latter virus growth was assumed when over 75% of the cells were killed (
The Use of Frozen Cells in the Microtitre Serum Neutralization Test for Infectious Bovine Rhinotracheitis P. R. Ide*
ABSTRACT
RESUME
Some parameters of the microtitre serum neutralization test were examined when using bovine fetal kidney cells derived from stocks stored in liquid nitrogen. In replicate tests with one serum there was no significant difference (p > 0.05) between titres calculated on the basis of cytopathic effect when either two or four wells were used per serum dilution. Also, there was no significant difference between titres calculated on the basis of a cell staining method when either two or four wells were used per serum dilution. When titres obtained by the 2-well-cytopathic effect method were compared with those obtained by the 2-well-stain method differences were not significantly different and it was concluded that the latter method using frozen cells constituted a practical and reproducible test. No significant titre variation occurred in cells serially passaged after having been frozen, or in cells derived from five different fetuses. Titres of sera from 41 cattle conformed to a log normal distribution pattern.
Cette experience visait a etudier certains parametres de la micromethode de sero-neutralisation, en utilisant des cellules renales de foetus bovins pre'alablement conservees dans de l'azote liquide. La repetition d'epreuves avec un antiserum ne revela pas une difference appreciable (p > 0.05) entre les titres calcules d'apres l'effet cytopathogene, lorsqu'on utilisait deux ou quatre puits pour chacune des dilutions du serum. On ne decela pas non plus de diffe'rence appreciable entre les titres calcul6s d'apres une methode de coloration des cellules, lorsqu'on utilisait deux ou quatre puits pour chacune des dilutions du serum. Une comparaison des titres, basee sur l'effet cytopathogene obtenu en utilisant la methode de deux puits, avec ceux que donna la methode de coloration, en utilisant deux puits, ne r'vela pas de difference appreciable; on conclut donc que cette derniere methode representait, lorsqu'on utilisait des cellules congelees, une methode pratique et reproductible. Les cellules soumises a des passages en serie, apres leur congelation, ne subirent pas de variation appreciable de leur titre; on fit la meme observation avec les cellules provenant de cinq foetus. Les titres du serum de 41 bovins donne-rent une courbe logarithmique normale.
*Atlantic Area Laboratory, Animal Pathology Division, Health of Animals Branch, Agriculture Canada, P.O. Box 1410, Sackville, New Brunswick EOA 3CO.
Submitted April 10, 1974.
Vol. 39 -January, 1975
The microtitre technique has been applied to the serum neutralization (SN) test with
101
infectious bovine rhinotracheitis (IBR) virus in both primary tissue cultures and established cell lines (1, 3, 7), for which seed aliquots may be stored frozen (3). There has been little published on the reproducibility of the microtitre serum neutralization (MTSN) test in cells derived from frozen primary cultures at different passage levels or on tests conducted in frozen cells derived from different fetuses. There is also no information on the comparative reproducibility of MTSN test employing either two or four wells per serum dilution or on the comparative sensitivity of tests read either on the basis of cytopathic effect (cpe) or staining pattern. As such factors are of practical and economic importance the following work was conducted to examine such parameters. The Colorado strain (2) of IBR virus and homologous bovine antiserum (RS1) were used. Primary bovine fetal kidney (BFK) cultures were prepared as described (4) and monolayer cells were suspended in freezing medium (sodium bicarbonate and antibiotic-free Earle's salt solution' containing 10% fetal calf serum and 10% dimethyl sulfoxide2) at a concentration of approximately 3 x 106 per ml (5). Cell aliquots were stored in liquid nitrogen and for use were diluted 1:10 in medium, grown as monolayers and trypsinized (5) for use in the MTSN test which was conducted as described (1). The virus challenge was 100 TCID50 per 0.025 ml and the virus-serum mixtures were incubated for two hr at room temperature, then approximately 15,000 cells (in 0.05 ml) were added to each well and plates were sealed with an adhesive film3 and incubated at 37°C. Appropriate serum controls (as serum dilutions without virus) and a titration of the virus challenge were included in each test. Five days later plates were examined for evidence of cpe and the residual monolayer in each well was stained with 2% crystal violet in alcohol (5). Titres were calculated by the Reed-Muench method (5). In the first series of experiments replicate MTSN tests were conducted with
'Grand Island Biological Company, Grand Island, New York. 2Fisher Scientific Co., Fairlawn, New Jersey.
3Falcon Plastics, Becton Dickinson & Co. (Canada) Ltd., Mississauga, Ontario.
102
serum RS1 in two lots of cells (BFK41 and BFK45) at various passage levels (up to the seventh) after thawing, using two or four wells per serum dilution. Serum titres were calculated on the basis of both cpe and staining patterns. In the former, any cpe was interpreted as virus growth (lack of serum neutralization) and in the latter virus growth was assumed when over 75% of the cells were killed (
