Stability, antigenicity, and aggregation of Moraxella bovis cytolysin after purification and storage

Lisle W. George, John A Angelos, William W. Ruehl

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Objectives - To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas. Study Population - 8 isolates of M bovis. Procedure - Filter-sterilized broth culture supernatants of M bovis were concentrated, diafiltered, and chromatographed. The endotoxin and cytolysin activities in samples were measured. Chromatographed cytolysins of M bovis were examined by immunoblotting. Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage. Hemolysis was measured directly by use of washed bovine erythrocyte targets. Leukotoxicity was measured by use of a 51Cr release assay. Results - Cytolysin was retained by a filter with 100-kd nominal molecular weight limit. Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 × 107 d). Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxinto-protein ratio. Frozen diafiltered retentate(-80°C, 4 months) was cytolytic after thawing. Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd. Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd. Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd. Conclusions and Clinical Relevance - Diafiltered M bovis cytolysin is aggregated with endotoxin. Antigenicity and cytolytic activities in diafiltered retentate are conserved among M bovis isolates. Diafiltration could be useful for bulk semipurification of M bovis cytolysin. Cytolysin-enriched vaccines of M bovis could be contaminated by endotoxin.

Original languageEnglish (US)
Pages (from-to)977-983
Number of pages7
JournalAmerican Journal of Veterinary Research
Volume65
Issue number7
DOIs
StatePublished - Jul 2004

Fingerprint

Moraxella (Moraxella) bovis
Moraxella bovis
Perforin
endotoxins
molecular weight
Endotoxins
gels
Gels
Cytotoxins
proteins
Proteins
hemolysis
immunoblotting
thawing
Hemolysis
Immunoblotting
erythrocytes
Gel Chromatography
vaccines
Vaccines

ASJC Scopus subject areas

  • veterinary(all)

Cite this

Stability, antigenicity, and aggregation of Moraxella bovis cytolysin after purification and storage. / George, Lisle W.; Angelos, John A; Ruehl, William W.

In: American Journal of Veterinary Research, Vol. 65, No. 7, 07.2004, p. 977-983.

Research output: Contribution to journalArticle

@article{2dca987657d64ad783761d07629d8272,
title = "Stability, antigenicity, and aggregation of Moraxella bovis cytolysin after purification and storage",
abstract = "Objectives - To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas. Study Population - 8 isolates of M bovis. Procedure - Filter-sterilized broth culture supernatants of M bovis were concentrated, diafiltered, and chromatographed. The endotoxin and cytolysin activities in samples were measured. Chromatographed cytolysins of M bovis were examined by immunoblotting. Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage. Hemolysis was measured directly by use of washed bovine erythrocyte targets. Leukotoxicity was measured by use of a 51Cr release assay. Results - Cytolysin was retained by a filter with 100-kd nominal molecular weight limit. Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 × 107 d). Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxinto-protein ratio. Frozen diafiltered retentate(-80°C, 4 months) was cytolytic after thawing. Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd. Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd. Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd. Conclusions and Clinical Relevance - Diafiltered M bovis cytolysin is aggregated with endotoxin. Antigenicity and cytolytic activities in diafiltered retentate are conserved among M bovis isolates. Diafiltration could be useful for bulk semipurification of M bovis cytolysin. Cytolysin-enriched vaccines of M bovis could be contaminated by endotoxin.",
author = "George, {Lisle W.} and Angelos, {John A} and Ruehl, {William W.}",
year = "2004",
month = "7",
doi = "10.2460/ajvr.2004.65.977",
language = "English (US)",
volume = "65",
pages = "977--983",
journal = "American Journal of Veterinary Research",
issn = "0002-9645",
publisher = "American Veterinary Medical Association",
number = "7",

}

TY - JOUR

T1 - Stability, antigenicity, and aggregation of Moraxella bovis cytolysin after purification and storage

AU - George, Lisle W.

AU - Angelos, John A

AU - Ruehl, William W.

PY - 2004/7

Y1 - 2004/7

N2 - Objectives - To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas. Study Population - 8 isolates of M bovis. Procedure - Filter-sterilized broth culture supernatants of M bovis were concentrated, diafiltered, and chromatographed. The endotoxin and cytolysin activities in samples were measured. Chromatographed cytolysins of M bovis were examined by immunoblotting. Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage. Hemolysis was measured directly by use of washed bovine erythrocyte targets. Leukotoxicity was measured by use of a 51Cr release assay. Results - Cytolysin was retained by a filter with 100-kd nominal molecular weight limit. Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 × 107 d). Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxinto-protein ratio. Frozen diafiltered retentate(-80°C, 4 months) was cytolytic after thawing. Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd. Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd. Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd. Conclusions and Clinical Relevance - Diafiltered M bovis cytolysin is aggregated with endotoxin. Antigenicity and cytolytic activities in diafiltered retentate are conserved among M bovis isolates. Diafiltration could be useful for bulk semipurification of M bovis cytolysin. Cytolysin-enriched vaccines of M bovis could be contaminated by endotoxin.

AB - Objectives - To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas. Study Population - 8 isolates of M bovis. Procedure - Filter-sterilized broth culture supernatants of M bovis were concentrated, diafiltered, and chromatographed. The endotoxin and cytolysin activities in samples were measured. Chromatographed cytolysins of M bovis were examined by immunoblotting. Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage. Hemolysis was measured directly by use of washed bovine erythrocyte targets. Leukotoxicity was measured by use of a 51Cr release assay. Results - Cytolysin was retained by a filter with 100-kd nominal molecular weight limit. Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 × 107 d). Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxinto-protein ratio. Frozen diafiltered retentate(-80°C, 4 months) was cytolytic after thawing. Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd. Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd. Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd. Conclusions and Clinical Relevance - Diafiltered M bovis cytolysin is aggregated with endotoxin. Antigenicity and cytolytic activities in diafiltered retentate are conserved among M bovis isolates. Diafiltration could be useful for bulk semipurification of M bovis cytolysin. Cytolysin-enriched vaccines of M bovis could be contaminated by endotoxin.

UR - http://www.scopus.com/inward/record.url?scp=3442889703&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3442889703&partnerID=8YFLogxK

U2 - 10.2460/ajvr.2004.65.977

DO - 10.2460/ajvr.2004.65.977

M3 - Article

C2 - 15281658

AN - SCOPUS:3442889703

VL - 65

SP - 977

EP - 983

JO - American Journal of Veterinary Research

JF - American Journal of Veterinary Research

SN - 0002-9645

IS - 7

ER -