1405
into account-but ciprofloxacin may relieve pressure on isolation units during institutional outbreaks especially where person-toperson spread is likely. We thank Dr S. E. J. Young, PHLS Communicable Disease Surveillance Centre, Colindale, and Dr D. Reid, Communicable Disease (Scotland) Unit, Glasgow, for outbreak data.
Regional Infectious Diseases Unit, City Hospital, Edinburgh EH10 5SB, UK, Central Microbiological Laboratories, Western General Hospital, Edinburgh, Royal Edinburgh Hospital, Department of Community Medicine, Edinburgh, and Department of Bacteriology, City Hospital, Edinburgh
LORNA J. WILLOCKS CAROLYN THOMPSON F. X. S. EMMANUEL JUDY BLIGH M. E. JONES A. C.SCOTT T. RONALD MARY HANSON P. A. MORRISON AIDA MACLEOD B. WATT AUDREY MCKENZIE J. A. GRAY
JMS. Effect of antibiotic treatment on duration of excretion of Salmonella typhimurium by children. Br Med J 1965; i: 1343-45. 2. Pichler HET, Diridl G, Stickler K, Wolf D. Clincal efficacy of ciprofloxacin compared with placebo in bacterial diarrhea. Am J Med 1987; 82 (suppl 4A): 329-32. 3. Chtistie AB. Infectious diseases: epidemiology and clinical practice, 3rd ed. Edinburgh: Churchill Livingstone, 1980: 31. 4. Pether JVS, Scott RJD. Salmonella carriers; are they dangerous? A study to identify finger contamination with salmonellae by convalescent carriers. J Infect 1982; 5:
clearly deficient. Only satisfactory methods will answer the very important questions about predictive values of ACA. was
Specialty Laboratories Inc, 2211 Michigan Avenue, Santa Monica, California 90404, USA
JAMES B. PETER
1. Loizou
S, McCrea JD, Rudge AC, Reynolds R, Boyle CC, Harris EN. Measurement of anri-cardiolipin by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results. Clin Exp Immunol 1985; 62: 738-45. 2. Harris EN, Hughes GRV. Standardising the anti-cardiolipin antibody test. Lancet 1987; i: 277. 3. Harris EN, Gharavi AE, Hughes GRV. Anticardiolipin antibody testing: the need for standardization. Arthritis Rheum 1987; 30: 835-36. 4. Harris EN, Gharavi AE, Patel SP, Hughes GRV. Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin Exp Immunol 1987; 68: 215-22. 5. Lenzi R, Rand JH, Spiera H. Anticardiolipin antibodies in pregnant patients with systemic lupus erythematosus. N Engl J Med 1986; 314: 1392-93. 6. Hughes TP, Jones P, Rozenberg MC. Hypergammaglobulinemia and the anticardiolipin antibody test. Arthritis Rheum 1989; 32: 813. 7 Fort JG, Cowchock S. Comment on the letter by Hughes et al. Arthritis Rheum 1990; 33: 607. 8. Agopian MS, Boctor FN, Peter JB. False-positive test result for IgM anticardiolipin antibody due to IgM rheumatoid factor. Arthritis Rheum 1988; 31: 1212-13.
1. Dixon
81-85.
Cardiolipin antibody assays SiR,—Dr Coulam and colleagues (April 7,
p 865) report that four laboratories gave widely divergent results for cardiolipin antibodies (ACA) in sera from 20 women with recurrent spontaneous abortions. This is hardly surprising since the laboratories were using a methodl-3of poor precision: inter-assay coefficients of variation for detection in GPL or MPL (the units used for IgG and IgM ACA, respectively) are 15-25% even in experienced hands. The sensitivity ranges are 15-100 GPL units and 6-60 MPL units, respectively, when the "standard" method is used.4 The assay, therefore, will not reliably detect antibody activity at the cut-off of 5 GPL and 3 MPL, defmed as low positive by the antiphospholipid standardisation laboratory in the department of rheumatology, University of Kentucky. The 1986 international workshop on ACA showed that even laboratories with a valid, reproducible assay could not agree on low positive samples.’ Using this "standard" method, several investigators have unwittingly ignored fundamental rules of ELISA-for example, the use of no-antigen control wells to detect misleading results due to non-specific or low affinity binding sometimes found with "sticky" sera from autoimmune and infectious diseases. They have also neglected to document linear, reproducible standard curves and to demonstrate parallelism on test samples; have used polyvalent detector antisera in a dilution at which detection of antibodies of the IgM (and IgA) isotypes is most unlikely; and have experienced false-positive results for IgM ACA when only IgG ACA and IgM rheumatoid factor are present. Other problems with the "standard" assay include widespread uncontrolled use of microtitre plates, the binding capacity of which varies from batch to batch. Some of these problems have been addressed since the introduction of the standard method. However, the standard method-even with modifications such as no-antigen controls-is still not accurate or precise enough. The answer is to redesign the format of the ELISA for ACA. The plastics used for microtitre plates have hydrophobic properties but there are alternative ways by which cardiolipin can be bound covalently, indirectly or otherwise, to the solid phase. In this way we routinely generate standard curves which are linear from 8 to 200 units (GPL and MPL), and inter-assay coefficients of variation at 8, 32, 60, and 180 GPL units are 13%, 9%, 9%, and 7%, respectively. The cause of the unacceptable laboratory-to-laboratory variation reported by Coulam and colleagues lies not with the laboratory workers but in the premature acceptance as standard of a laboratory method which
Anti-glomerular basement membrane disease after lithotripsy SiR,—Dr Guerin and colleagues (April 7, p 856) report the development of anti-glomerular basement membrane (GBM) disease after lithotripsy. They state that alterations of the structure of the GBM might favour the exposure of the globular domain NC1l of collagen, which contains the Goodpasture antigen. I report data to evaluate the physical influence on renal basement membrane structure and antigenicity on a molecular basis. In the early 1980s the GBM was defined as a structure consisting mainly of collagen type IV, laminin, and some glycosaminoglycans. This definition was based on the membrane isolated by physical means.’ Kidneys were minced and put through various sieves, and the glomeruli were isolated by use of different mesh sizes. Finally the glomeruli were sonicated by several bursts and by differential centrifugation; the membranes and Bowman’s capsules were separated. GBMs isolated by that technique showed split products of variable size and antigenicity. Collagen split products of variable molecular weights as measured by sodium dodecylsulphatepolyacrylamide gel electrophoresis were found, as well as an inconstant composition of acid glycosaminoglycans, which were destroyed by ultrasound. Another non-destructive and reproducible isolation method replaced the ultrasonic procedure, applying sodium dodecylsulphate as the disintegrating agent. Collagen was not altered by that method and the acid glycosaminoglycans proved an integral constituent of this structure .21 -his finding led to an updated definition of the GBM.3 The collagen cleaving effect is well known to those who have isolated basement membranes by ultrasound. It should, however, be added that collagen eluted from GBM does not present the Goodpasture antigen. To expose this antigen(s) proteolytic treatment is necessary. It is noteworthy that ultrasonic disruption and enzymatic treatment could expose some identical split products, one of them presenting the Goodpasture antigen. The importance of Guerin and colleagues’ observation is that a human model of anti-GBM disease was described, supplying more information on pathogenesis than did all previous reports on the development of anti-GBM-reactivity in glomerulonephritis and
poisoning. Department of Paediatrics, University of Vienna, A 1090 Vienna, Austria
G. LUBEC
NA, Meezan E, Martinez-Hernandez A, et al. Defininon of the glomerular 1981; 8: 133-34 2. Meezan E, Hjelle JT, Brendel K A simple, versatile, nondisruptive method for the 1. Kefalides
basement membrane. Connect Tiss Res
isolation of morphologically and chemically pure basement membranes from several tissues Life Sci 1975; 17: 1721-32. 3. Lubec G. Definition of the glomerular basement membrane. Renal Physiol 1984; 7: 1-2.
into account-but ciprofloxacin may relieve pressure on isolation units during institutional outbreaks especially where person-toperson spread is likely. We thank Dr S. E. J. Young, PHLS Communicable Disease Surveillance Centre, Colindale, and Dr D. Reid, Communicable Disease (Scotland) Unit, Glasgow, for outbreak data.
Regional Infectious Diseases Unit, City Hospital, Edinburgh EH10 5SB, UK, Central Microbiological Laboratories, Western General Hospital, Edinburgh, Royal Edinburgh Hospital, Department of Community Medicine, Edinburgh, and Department of Bacteriology, City Hospital, Edinburgh
LORNA J. WILLOCKS CAROLYN THOMPSON F. X. S. EMMANUEL JUDY BLIGH M. E. JONES A. C.SCOTT T. RONALD MARY HANSON P. A. MORRISON AIDA MACLEOD B. WATT AUDREY MCKENZIE J. A. GRAY
JMS. Effect of antibiotic treatment on duration of excretion of Salmonella typhimurium by children. Br Med J 1965; i: 1343-45. 2. Pichler HET, Diridl G, Stickler K, Wolf D. Clincal efficacy of ciprofloxacin compared with placebo in bacterial diarrhea. Am J Med 1987; 82 (suppl 4A): 329-32. 3. Chtistie AB. Infectious diseases: epidemiology and clinical practice, 3rd ed. Edinburgh: Churchill Livingstone, 1980: 31. 4. Pether JVS, Scott RJD. Salmonella carriers; are they dangerous? A study to identify finger contamination with salmonellae by convalescent carriers. J Infect 1982; 5:
clearly deficient. Only satisfactory methods will answer the very important questions about predictive values of ACA. was
Specialty Laboratories Inc, 2211 Michigan Avenue, Santa Monica, California 90404, USA
JAMES B. PETER
1. Loizou
S, McCrea JD, Rudge AC, Reynolds R, Boyle CC, Harris EN. Measurement of anri-cardiolipin by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results. Clin Exp Immunol 1985; 62: 738-45. 2. Harris EN, Hughes GRV. Standardising the anti-cardiolipin antibody test. Lancet 1987; i: 277. 3. Harris EN, Gharavi AE, Hughes GRV. Anticardiolipin antibody testing: the need for standardization. Arthritis Rheum 1987; 30: 835-36. 4. Harris EN, Gharavi AE, Patel SP, Hughes GRV. Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin Exp Immunol 1987; 68: 215-22. 5. Lenzi R, Rand JH, Spiera H. Anticardiolipin antibodies in pregnant patients with systemic lupus erythematosus. N Engl J Med 1986; 314: 1392-93. 6. Hughes TP, Jones P, Rozenberg MC. Hypergammaglobulinemia and the anticardiolipin antibody test. Arthritis Rheum 1989; 32: 813. 7 Fort JG, Cowchock S. Comment on the letter by Hughes et al. Arthritis Rheum 1990; 33: 607. 8. Agopian MS, Boctor FN, Peter JB. False-positive test result for IgM anticardiolipin antibody due to IgM rheumatoid factor. Arthritis Rheum 1988; 31: 1212-13.
1. Dixon
81-85.
Cardiolipin antibody assays SiR,—Dr Coulam and colleagues (April 7,
p 865) report that four laboratories gave widely divergent results for cardiolipin antibodies (ACA) in sera from 20 women with recurrent spontaneous abortions. This is hardly surprising since the laboratories were using a methodl-3of poor precision: inter-assay coefficients of variation for detection in GPL or MPL (the units used for IgG and IgM ACA, respectively) are 15-25% even in experienced hands. The sensitivity ranges are 15-100 GPL units and 6-60 MPL units, respectively, when the "standard" method is used.4 The assay, therefore, will not reliably detect antibody activity at the cut-off of 5 GPL and 3 MPL, defmed as low positive by the antiphospholipid standardisation laboratory in the department of rheumatology, University of Kentucky. The 1986 international workshop on ACA showed that even laboratories with a valid, reproducible assay could not agree on low positive samples.’ Using this "standard" method, several investigators have unwittingly ignored fundamental rules of ELISA-for example, the use of no-antigen control wells to detect misleading results due to non-specific or low affinity binding sometimes found with "sticky" sera from autoimmune and infectious diseases. They have also neglected to document linear, reproducible standard curves and to demonstrate parallelism on test samples; have used polyvalent detector antisera in a dilution at which detection of antibodies of the IgM (and IgA) isotypes is most unlikely; and have experienced false-positive results for IgM ACA when only IgG ACA and IgM rheumatoid factor are present. Other problems with the "standard" assay include widespread uncontrolled use of microtitre plates, the binding capacity of which varies from batch to batch. Some of these problems have been addressed since the introduction of the standard method. However, the standard method-even with modifications such as no-antigen controls-is still not accurate or precise enough. The answer is to redesign the format of the ELISA for ACA. The plastics used for microtitre plates have hydrophobic properties but there are alternative ways by which cardiolipin can be bound covalently, indirectly or otherwise, to the solid phase. In this way we routinely generate standard curves which are linear from 8 to 200 units (GPL and MPL), and inter-assay coefficients of variation at 8, 32, 60, and 180 GPL units are 13%, 9%, 9%, and 7%, respectively. The cause of the unacceptable laboratory-to-laboratory variation reported by Coulam and colleagues lies not with the laboratory workers but in the premature acceptance as standard of a laboratory method which
Anti-glomerular basement membrane disease after lithotripsy SiR,—Dr Guerin and colleagues (April 7, p 856) report the development of anti-glomerular basement membrane (GBM) disease after lithotripsy. They state that alterations of the structure of the GBM might favour the exposure of the globular domain NC1l of collagen, which contains the Goodpasture antigen. I report data to evaluate the physical influence on renal basement membrane structure and antigenicity on a molecular basis. In the early 1980s the GBM was defined as a structure consisting mainly of collagen type IV, laminin, and some glycosaminoglycans. This definition was based on the membrane isolated by physical means.’ Kidneys were minced and put through various sieves, and the glomeruli were isolated by use of different mesh sizes. Finally the glomeruli were sonicated by several bursts and by differential centrifugation; the membranes and Bowman’s capsules were separated. GBMs isolated by that technique showed split products of variable size and antigenicity. Collagen split products of variable molecular weights as measured by sodium dodecylsulphatepolyacrylamide gel electrophoresis were found, as well as an inconstant composition of acid glycosaminoglycans, which were destroyed by ultrasound. Another non-destructive and reproducible isolation method replaced the ultrasonic procedure, applying sodium dodecylsulphate as the disintegrating agent. Collagen was not altered by that method and the acid glycosaminoglycans proved an integral constituent of this structure .21 -his finding led to an updated definition of the GBM.3 The collagen cleaving effect is well known to those who have isolated basement membranes by ultrasound. It should, however, be added that collagen eluted from GBM does not present the Goodpasture antigen. To expose this antigen(s) proteolytic treatment is necessary. It is noteworthy that ultrasonic disruption and enzymatic treatment could expose some identical split products, one of them presenting the Goodpasture antigen. The importance of Guerin and colleagues’ observation is that a human model of anti-GBM disease was described, supplying more information on pathogenesis than did all previous reports on the development of anti-GBM-reactivity in glomerulonephritis and
poisoning. Department of Paediatrics, University of Vienna, A 1090 Vienna, Austria
G. LUBEC
NA, Meezan E, Martinez-Hernandez A, et al. Defininon of the glomerular 1981; 8: 133-34 2. Meezan E, Hjelle JT, Brendel K A simple, versatile, nondisruptive method for the 1. Kefalides
basement membrane. Connect Tiss Res
isolation of morphologically and chemically pure basement membranes from several tissues Life Sci 1975; 17: 1721-32. 3. Lubec G. Definition of the glomerular basement membrane. Renal Physiol 1984; 7: 1-2.
