488 ANTI-L.S.P. TITRES IN CHIMPANZEES IMMUNISED WITH HEPATITIS
Letters
to
B
the Editor
VACCINE*
SAFETY OF HEPATITIS-B VACCINES CONTAINING INTACT 20 nm PARTICLES
SIR,—Several groups of investigators have prepared candidate hepatitis-B vaccines by purification of 20 nm HBsAg-associated particles from the plasma of chronic carriers, followed by inactivation procedures designed to eliminate any residual infectivity. It has been suggested that host components, particularly liver-cell-membrane antigens, could theoretically be incorporated in the membrane of the HBsAg-associated particles, and that immunisation with such particles might therefore entail a risk of induction of autoimmune liver disease.1 To evaluate this possibility, we have searched for development of antibody reactive with liver-cell membrane components, as well as other autoantibodies associated with autoimmune liver disease, in two groups of chimpanzees, receiving either a candidate hepatitis B vaccine (A), or hyperimmunised by repeated injection of purified HBsAg particles suspended in Freund’s adjuvant (B). Group A comprised four chimpanzees immunised with two subcutaneous doses, 1 month apart, of an aqueous vaccine2 containing 50 µg 20 nm HBsAg particles. Sera were taken before, and 2 and 6 months after, the first immunisation. Group B included four chimpanzees, two immunised intramuscularly with 50 µg HBsAg/ay and two with HBsAg/ad, purified by two isopyknic and one rate-zonal centrifugations in cæsium-chloride gradients and suspended in Freund’s complete adjuvant. The animals received a booster injection of the same quantity of antigen in Freund’s incomplete adjuvant after one month. 6 months after the first immunisation, the HBsAg/ay immunised animals received a second booster injection containing 25 µg of HBsAg/ad and 25 µg HBsAg/ay in incomplete Freund’s adjuvant. The HBsAg/ad immunised animals received a similar booster 9 months after the first immunisation. Sera were taken for this study before immunisation, approximately 2 and 6 months after onset of immunisation, and then about 1 month after the first booster injection. At this time, these sera had anti-HBs titres by agargel diffusion ranging between 1:32 and 1:256. Serum transaminase (S.G.P.T.) remained within normal limits in weekly serum specimens taken during the year after onset of immunisation in both groups of animals. All sera were negative for antinuclear, anti-smooth-muscle, anti-mitochondrial, and anti-parietal cell autoantibodies when tested by indirect immunofluorescence.3 In addition, antibodies to liver membrane antigen (L.M.A.)4 were not detectable by indirect immunofluorescence with isolated rabbit hepatocytes. With a recently developed radioimmunoassay for detection of antibodies to the liver-specific membrane protein (L.S.P.),5,6 titres of 1:50 to 1:150 were found in tests carried out on coded samples in the preimmunisation sera of three of the chimpanzees injected with aqueous vaccine (table). These antibody levels declined in subsequent sera and probably reflect the fact that these animals had had hepatitis type A infections 5-6 months previously. Development of anti-L.S.P. was not seen in the animals hyperimmunised with HBsAg in Freund’s adjuvant. Lack of detection of liver-membrane autoantibodies in immunised animals is thus strong evidence for absence of autoimmunisation against this liver-specific membrane
antigen. 1. Zuckerman, A. J. Nature, 1975, 255, 104. 2 Purcell, R H., Gerin, J. L. Am. J med. Sci. 1975, 270, 395. 3. Walker, J. G., Doniach, D., Roitt, I. M., Sherlock, S. Lancet, 1965, i, 827. 4. Tage-Jensen, U., Arnold, W., Dietrichson, O., Hardt, F., Hopf, U., Meyer Zum Buschenfelde, K H., Nielsen,J. O Br med. J 1977, i, 206. 5 Jensen, D. M., McFarlane, I. G , Nicholson, A., Eddleston, A. L. W. F., Williams, R. J clin. Lab Immun (in the press ). 6 Jensen, D. M., McFarlane, I. G., Portmann, B S , Eddleston, A L. W. F., Williams, R. New Engl Med. 1978, 299, 1 J
*50 ag hepatitis-B Aug. 5, 1976.
vaccine
was
injected subcutaneously
on
July
8 and
The above findings do not suggest that hepatitis B vaccines containing intact 20 nm particles will give rise to autoimmune liver disease. However, surveillance to evaluate this possibility is desirable during the course of clinical trials. Faculty of Medicine, University of Lyons, Lyons, France New York Blood
Center,
New York, N.Y. 10021, U.S.A.
King’s College Hospital, and Medical School, London SE5
CHRISTIAN TREP ALFRED M. PRINCE RICARDO MELICONI
A. L. W. F. EDDLESTON ROGER WILLIAMS
Medical Department M, Rigshospitalet
Rigshospitalet, and Department ofHepatology, Hvidovre Hospital, Copenhagen, Denmarl
U. TAGE-JENSEN
JENS O. NIELSEN
FALSE-NEGATIVE TESTING FOR GALACTOSURIA
SIR,-Urine testing for sugars other than glucose, especially in the first few years of life, can be useful,’ and the presence of galactose in urine is of importance in the differential diagnosis of several clinical entities, including galactossemia and galactokinase deficiency. Galactose can be specifically identified by a galactose-oxidase method,2-4 and the assay is commercially available as a "dip-and-read test" specifically designed for testing urine (’Galactostix’, Ames).2 According to the manufacturer, the galactose oxidase dipsticks are sensitive to galactose concentrations greater than 100 mg/dl if the specific gravity of the sample is < 1-010. A recent clinical problem with this test has prompted the following report. A 3-week-old infant was admitted to hospital because of recurrent jaundice, hepatomegaly, and failure to thrive. The family and perinatal history were unremarkable. Physical examination revealed a jaundiced infant with hepatomegaly. A routine urinalysis was normal except for "2+" total reducing substances (’Clinitest’ reagent tablets, Ames). The same urine specimen was tested for glucose by glucose-oxidase method (’Labstix’, Ames) and for galactose (by galactostix) and was reported as negative for both. Two subsequent urinespecimens were tested and identical results were obtained. Thin-laver chromatography of the patient’s urine revealed significant amounts of galactose. This false-negative result with galactoseoxidase sticks led to an investigation of the sensitivity of this method. Urine was obtained from seven healthy adults and three healthy infants under sterile conditions, and all tests were done within 90 mm of collection. The urines were examined microscopically and tested biochemically for blood, protein, glucose, ketones, and nitrites and found to be normal (labstix). Every specimen was negative for reducing substances (cimitest tablets). A solution containing D(+)-galactose in distilled sterile water was prepared and allowed to stand for several hours to allow for mutarotation as suggested by the manufacturer. The standard solution was assayed by thin-layer chromatography and con1. Burton, B. K., Nadler, H. L. Pediatrics, 1978, 61, 398. 2. Roth, H., Siegel, S., Bertoli, D. Analyt. Biochem. 1965, 10, 32. 3. Tengstrom, B. Scand. J Lab. Invest 1966, 18, suppl. 92, p.104. 4. Dahlqvist, A. ibid. 1968, 22, 87.
489 RESULTS OF CLINITEST
Serial dilution of urine and galactqse were prethat the final galactose concentrations were 63, 125, 250, 300, 400, and 500 mg/dl. The specific gravity of every sample was measured by refractometry and was less than 1-010. Every sample was tested for total reducing substances (clinitst) and by galactostix. Total reducing substances are reported as negative, trace, 1+, 2+, or 3+. The presence or absence of galactose is reported as either + or -. Each sample was tested for galactose twice, using dipsticks from two different reagent bottles. Only one lot was available for testing. tamed
only galactose.
pared
so
Results are presented in the table. Galactose is accurately detected in water or in normal saline, but the dipstick method is insensitive to the same concentrations in urine. Galactose excretion is a potentially important finding which may indicate an inborn error of galactose metabolism. Our data suggest that at least one galactose-oxidase method curtest for galacrently available as a "qualitative tose in urine" usually fails to detect galactose in the range of concentrations studied. The package insert claims that concentrations of galactose greater than 100 mg/dl can be detected in urine if the specific gravity of the specimen is less than 1.010. This claim appears to be valid for galactose in water or normal saline, but invalid for urine. Preliminary data sugggst that the insensitivity of this method in urine is related at least in part to some effect of specific gravity since if galactose is inassayed in normal saline whose specific gravity creased by either adding sucrose or salt, the test sensitivity is greatly diminished. Others5 have suggested that galactoseoxidase inhibitors may be present in urine. We recommend that all babies and infants whose urine is "clinitest positive" and glucose-oxidase negative should have a urine sample analysed by other more sensitive and accurate methods, such as thin-layer or gas chromatography. If the reducing substance is galactose, galactose-1-phosphate uridyl transferase and galactokinase assays should be done. The galactose-oxidase dipstick method available is misleading and is of limited value in the routine detection of has withdrawn galactostix galactosuria. The Ames from the market.
dip-and-read
has been
currently Company
Department of Pediatrics, Northwestern University Medical School Children’s Memorial Hospital Chicago, Illinois 60614, U.S.A.
potenti
Letters
to
B
the Editor
VACCINE*
SAFETY OF HEPATITIS-B VACCINES CONTAINING INTACT 20 nm PARTICLES
SIR,—Several groups of investigators have prepared candidate hepatitis-B vaccines by purification of 20 nm HBsAg-associated particles from the plasma of chronic carriers, followed by inactivation procedures designed to eliminate any residual infectivity. It has been suggested that host components, particularly liver-cell-membrane antigens, could theoretically be incorporated in the membrane of the HBsAg-associated particles, and that immunisation with such particles might therefore entail a risk of induction of autoimmune liver disease.1 To evaluate this possibility, we have searched for development of antibody reactive with liver-cell membrane components, as well as other autoantibodies associated with autoimmune liver disease, in two groups of chimpanzees, receiving either a candidate hepatitis B vaccine (A), or hyperimmunised by repeated injection of purified HBsAg particles suspended in Freund’s adjuvant (B). Group A comprised four chimpanzees immunised with two subcutaneous doses, 1 month apart, of an aqueous vaccine2 containing 50 µg 20 nm HBsAg particles. Sera were taken before, and 2 and 6 months after, the first immunisation. Group B included four chimpanzees, two immunised intramuscularly with 50 µg HBsAg/ay and two with HBsAg/ad, purified by two isopyknic and one rate-zonal centrifugations in cæsium-chloride gradients and suspended in Freund’s complete adjuvant. The animals received a booster injection of the same quantity of antigen in Freund’s incomplete adjuvant after one month. 6 months after the first immunisation, the HBsAg/ay immunised animals received a second booster injection containing 25 µg of HBsAg/ad and 25 µg HBsAg/ay in incomplete Freund’s adjuvant. The HBsAg/ad immunised animals received a similar booster 9 months after the first immunisation. Sera were taken for this study before immunisation, approximately 2 and 6 months after onset of immunisation, and then about 1 month after the first booster injection. At this time, these sera had anti-HBs titres by agargel diffusion ranging between 1:32 and 1:256. Serum transaminase (S.G.P.T.) remained within normal limits in weekly serum specimens taken during the year after onset of immunisation in both groups of animals. All sera were negative for antinuclear, anti-smooth-muscle, anti-mitochondrial, and anti-parietal cell autoantibodies when tested by indirect immunofluorescence.3 In addition, antibodies to liver membrane antigen (L.M.A.)4 were not detectable by indirect immunofluorescence with isolated rabbit hepatocytes. With a recently developed radioimmunoassay for detection of antibodies to the liver-specific membrane protein (L.S.P.),5,6 titres of 1:50 to 1:150 were found in tests carried out on coded samples in the preimmunisation sera of three of the chimpanzees injected with aqueous vaccine (table). These antibody levels declined in subsequent sera and probably reflect the fact that these animals had had hepatitis type A infections 5-6 months previously. Development of anti-L.S.P. was not seen in the animals hyperimmunised with HBsAg in Freund’s adjuvant. Lack of detection of liver-membrane autoantibodies in immunised animals is thus strong evidence for absence of autoimmunisation against this liver-specific membrane
antigen. 1. Zuckerman, A. J. Nature, 1975, 255, 104. 2 Purcell, R H., Gerin, J. L. Am. J med. Sci. 1975, 270, 395. 3. Walker, J. G., Doniach, D., Roitt, I. M., Sherlock, S. Lancet, 1965, i, 827. 4. Tage-Jensen, U., Arnold, W., Dietrichson, O., Hardt, F., Hopf, U., Meyer Zum Buschenfelde, K H., Nielsen,J. O Br med. J 1977, i, 206. 5 Jensen, D. M., McFarlane, I. G , Nicholson, A., Eddleston, A. L. W. F., Williams, R. J clin. Lab Immun (in the press ). 6 Jensen, D. M., McFarlane, I. G., Portmann, B S , Eddleston, A L. W. F., Williams, R. New Engl Med. 1978, 299, 1 J
*50 ag hepatitis-B Aug. 5, 1976.
vaccine
was
injected subcutaneously
on
July
8 and
The above findings do not suggest that hepatitis B vaccines containing intact 20 nm particles will give rise to autoimmune liver disease. However, surveillance to evaluate this possibility is desirable during the course of clinical trials. Faculty of Medicine, University of Lyons, Lyons, France New York Blood
Center,
New York, N.Y. 10021, U.S.A.
King’s College Hospital, and Medical School, London SE5
CHRISTIAN TREP ALFRED M. PRINCE RICARDO MELICONI
A. L. W. F. EDDLESTON ROGER WILLIAMS
Medical Department M, Rigshospitalet
Rigshospitalet, and Department ofHepatology, Hvidovre Hospital, Copenhagen, Denmarl
U. TAGE-JENSEN
JENS O. NIELSEN
FALSE-NEGATIVE TESTING FOR GALACTOSURIA
SIR,-Urine testing for sugars other than glucose, especially in the first few years of life, can be useful,’ and the presence of galactose in urine is of importance in the differential diagnosis of several clinical entities, including galactossemia and galactokinase deficiency. Galactose can be specifically identified by a galactose-oxidase method,2-4 and the assay is commercially available as a "dip-and-read test" specifically designed for testing urine (’Galactostix’, Ames).2 According to the manufacturer, the galactose oxidase dipsticks are sensitive to galactose concentrations greater than 100 mg/dl if the specific gravity of the sample is < 1-010. A recent clinical problem with this test has prompted the following report. A 3-week-old infant was admitted to hospital because of recurrent jaundice, hepatomegaly, and failure to thrive. The family and perinatal history were unremarkable. Physical examination revealed a jaundiced infant with hepatomegaly. A routine urinalysis was normal except for "2+" total reducing substances (’Clinitest’ reagent tablets, Ames). The same urine specimen was tested for glucose by glucose-oxidase method (’Labstix’, Ames) and for galactose (by galactostix) and was reported as negative for both. Two subsequent urinespecimens were tested and identical results were obtained. Thin-laver chromatography of the patient’s urine revealed significant amounts of galactose. This false-negative result with galactoseoxidase sticks led to an investigation of the sensitivity of this method. Urine was obtained from seven healthy adults and three healthy infants under sterile conditions, and all tests were done within 90 mm of collection. The urines were examined microscopically and tested biochemically for blood, protein, glucose, ketones, and nitrites and found to be normal (labstix). Every specimen was negative for reducing substances (cimitest tablets). A solution containing D(+)-galactose in distilled sterile water was prepared and allowed to stand for several hours to allow for mutarotation as suggested by the manufacturer. The standard solution was assayed by thin-layer chromatography and con1. Burton, B. K., Nadler, H. L. Pediatrics, 1978, 61, 398. 2. Roth, H., Siegel, S., Bertoli, D. Analyt. Biochem. 1965, 10, 32. 3. Tengstrom, B. Scand. J Lab. Invest 1966, 18, suppl. 92, p.104. 4. Dahlqvist, A. ibid. 1968, 22, 87.
489 RESULTS OF CLINITEST
Serial dilution of urine and galactqse were prethat the final galactose concentrations were 63, 125, 250, 300, 400, and 500 mg/dl. The specific gravity of every sample was measured by refractometry and was less than 1-010. Every sample was tested for total reducing substances (clinitst) and by galactostix. Total reducing substances are reported as negative, trace, 1+, 2+, or 3+. The presence or absence of galactose is reported as either + or -. Each sample was tested for galactose twice, using dipsticks from two different reagent bottles. Only one lot was available for testing. tamed
only galactose.
pared
so
Results are presented in the table. Galactose is accurately detected in water or in normal saline, but the dipstick method is insensitive to the same concentrations in urine. Galactose excretion is a potentially important finding which may indicate an inborn error of galactose metabolism. Our data suggest that at least one galactose-oxidase method curtest for galacrently available as a "qualitative tose in urine" usually fails to detect galactose in the range of concentrations studied. The package insert claims that concentrations of galactose greater than 100 mg/dl can be detected in urine if the specific gravity of the specimen is less than 1.010. This claim appears to be valid for galactose in water or normal saline, but invalid for urine. Preliminary data sugggst that the insensitivity of this method in urine is related at least in part to some effect of specific gravity since if galactose is inassayed in normal saline whose specific gravity creased by either adding sucrose or salt, the test sensitivity is greatly diminished. Others5 have suggested that galactoseoxidase inhibitors may be present in urine. We recommend that all babies and infants whose urine is "clinitest positive" and glucose-oxidase negative should have a urine sample analysed by other more sensitive and accurate methods, such as thin-layer or gas chromatography. If the reducing substance is galactose, galactose-1-phosphate uridyl transferase and galactokinase assays should be done. The galactose-oxidase dipstick method available is misleading and is of limited value in the routine detection of has withdrawn galactostix galactosuria. The Ames from the market.
dip-and-read
has been
currently Company
Department of Pediatrics, Northwestern University Medical School Children’s Memorial Hospital Chicago, Illinois 60614, U.S.A.
potenti
