War/d Journal
of Microbio/ogy
& 6iotechnotogy
11, 206-206
Application of protein-A indirect ELISA (PAI-ELBA) for the detection of anti-Smith antibodies in systemic lupus erythematosus patients B.0, Siti-Rohana, I.B. Ahmad* and B,A. Nasuruddin An indirect form of protein-A ELISA (PA&ELBA) was optimized and, when used to detect anti-Smith antibodies in sera of 31 systemic lupus erythematosus (SLE) patients, gave results comparable with those using a commercial immunodiffusion kit. The number of sera found to be positive for anti-Smith antibodies by ELISA was seven, four of which were also found positive by immunodiffusion. fiy words: Anti-Smith
antibodies,
ELISA, protein A, systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is a polymorphic autoimmune disease of idiopathic origin that is sometimes difficult to diagnose. It has therefore been suggested that, for the diagnosis of SLE, at least four of the criteria listed by the American Rheumatism Association be fulfilled (Tan ef ~1. 1982). The most commonly used criteria are the presence of anti-nuclear (AN), anti-double-stranded DNA (anti-dsDNA) and anti-Smith (anti-Sm) antibodies (Sharp ef ~1. 1976; Tan ef al. 1982). The presence of AN antibodies in the patients’ sera determines the auto-immune status of the patients’ disease. AN antibodies are reported to be present in about 96% to 99% of SLE patients (Fritzler 1986) and their assay thus remains an important screening test for SLE. Anti-dsDNA antibodies were reported to be prevalent in SLE cases (Crowe & Kushner 1977; Tan 1983). However, the presence of anti-dsDNA antibodies in other auto-immune diseases (Notman ef al. 1975) has limited the use of this assay in diagnosis of SLE. In contrast, anti-Sm antibodies are highly specific markers for this disease (Notman ef al. 1975; Reichlin 1976; Winn ef a/. 1979; Tan 1982; Nakamura ef al. 1984)
B.O. Siti-Rohana is with the Universiti Kebangsaan Malaysia (UKM), Faculty of Medicine, Koala Lumpor, Malaysia. I.B. Ahmad is with the Department of Microbiology, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, Bangi, 43600 UKM, Malaysia; fax: +603 6252698. B.A. Nasaruddin is with the Institute for Medical Research, Malaysia. Towespending author. @ 1995 Rapid Communications
of Oxford Ltd
and their presence in sera is important in the prognosis of SLE (Winn ef ul. 1979; Barada ef ~1. 1981). The immunodiffusion (ID) test is currently the most common method used for the detection of anti-Sm antibodies. The inherently low sensitivity of this method (Notman ef a/. 1975) has, however, resulted in dismissal of sera with low titres of anti-Sm antibodies as SLE negatives. Therefore, there is a need for a more sensitive serological method, such as ELISA, to detect anti-Sm antibodies in SLE sera. The high degree of sensitivity possible with ELISA, coupled with other factors such as the relative ease with which they can be performed, the short time needed before results are available and their cost-effectiveness (Engvall & Pearlmann 1971; Clark & Adam 1977) have all contributed to making an ELISA the method of choice. Subsequent modifications to the original ELISA technique have resulted in increased sensitivity and specificity. For example, the use of enzymelabelled protein-A in indirect ELISA, in place of the commonly used enzyme-labelled anti-immunoglobulins, has been shown to increase the sensitivity of the assay system (Edwards & Cooper 1985; Ahmad ef al. 1989). The present study was of the application of protein-A ELISA for the detection of anti-Sm antibodies in SLE patients.
Materials SLE
sera
and Methods
(31 samples)
were obtained from the Specialist Clinic of
PAl-ELlSA the Medical Faculty, Universiti Kebangsaan Malaysia, Kuala Lumpur. Ten normal sera from blood donors, previously tested negative for AN and anti-dsDNA antibodies (Siti-Rohana et ~1. 1992), served as negative controls and three anti-Sm-positive sera, obtained from confirmed SLE patients, served as positive controls. A4icrotitre Assays Flat-bottomed, microtitre plates were pre-treated with 5.3 M NaOH in 30% (v/v) ethanol (see Banttarri & Petersen 1983; Goodwin & Banttarri 1984). The Smith antigen, 100~1 in 50 rnM NaHCOJ/NaACOs buffer (pH 9.6) was dispensed in varying concentrations to each well of the microtitre plate and held overnight at 4OC. The antigen was from Northeast Biomedical Laboratories, USA. Coated plates were washed three times with phosphate-buffered saline (PBS)/Tween (PBS, pH 7.4, plus 0.05% Tween 20). Blocking of unoccupied surfaces was achieved by treating the wells with 3% egg albumin in PBS (pH 7.4) at 45’C for 1 h. The plates were then washed five times with PBYTween (pH 7.4). Immediately after washing, the plates were oven-dried at 37’C for I h. At this stage the plates were stored desiccated at 4OC, or used immediately for assay of serum anti-Sm antibodies.
The assay was performed according to standardized, indirect enzyme immune-assay procedures but with modifications (ZainalAbidin et a/. 1992). Briefly, serum samples were each diluted to pre-determined optimum dilution in PBS (pH 7.4), dispensed at 100 @well, then incubated either overnight at 4’C or 2 h at 37OC. After incubation, the plate was washed by rinsing it five times with PBS/Tween. Alkaline-phosphatase-labelled protein A, diluted at pre-determined concentrations in PBS (pH 7.4), was added at 100 PI/well. At the end of a 30.min incubation period at room temperature, 50 ~1 of 3 M NaOH/well was added and the absorbance (A) then read with an ELISA reader.
The optimized ELISA technique was used to determine the mean A value from IO normal sera, An A value twice this mean value for negative controls was taken to indicate a positive serum (Sutula et a/. 1986). A values determined for the three Sm-oositive control sera were used to provide comparative examples of positive A values. L
The 31 SLE sera were also tested for anti-Sm antibodies using a commercial ID test kit (ENA kit; Behring, Nippon Hoechst, Japan), according to the manufacturer’s instructions. Determination of positive and negative results was based on the reaction of identity and non-identity, respectively.
Results
and Discussion
Pretreufment of Plafes Assays using plates pre-treated with 5.3 M NaOH/30% ethanol and pretreated with PBS (control) gave A values 13% to 14% and 11% higher than untreated plates, respectively. This was consistent with the findings of Banttarri & Petersen (1983) and Goodwin & Banttarri (1984). The NaOH pretreatment enhanced the surface binding property of the microtitre wells and all subsequent experiments utilized only NaOH/ethanol-treated plates. Only plates
detection of anti-Smifh
unfibodies in SLE
with high absorption capacity should be used for effective binding of the antigen. Opfimizatioa of ELISA The optimum concentration of Sm antigen to coat the wells was found to be 3 fig/ml when dispensed at 100 pl/ well. The optimum dilution of serum was 1:50, giving a mean A of 0.808 (control mean = 0.045) and the optimum dilution of alkaline-phosphatase-labelled protein A was 1: 1000 or I pg/ml, giving an A value of 0.695 (control mean = 0.051). Overnight incubation at 4’C gave slightly higher A values than incubation at 3FC for 2 h and was used throughout the study. of Serum Samples ELISA using the optimized conditions gave 1.17 and 0205 as the mean A values for the three Sm-positive controls and the 10 normal sera (negative controls), respectively. The positive cut-off point was 0.411. Based on this cut-off point, seven Sm-positive samples were detected by ELISA, compared with four by ID. The A values for the seven antiSm-positive sera ranged from 0.509 to 1.088. The three sera which were detected as positive by ELISA, but negative by ID had A values ranging from 0.509 to 1.088. While the ID method has the advantage of being able to produce specific reactions with crude antigens, specificity and sensitivity of ELISA technique depends on the use of pure reagents. In the present study pure Smith antigen was used for the PAI-ELISA test. The Smith antigen has been characterized as an acidic, TO-kDa, non-histone nuclear protein molecule (Takano et al. 198I), although its antigenicity has been ascribed to 25 kDA and 16 kDA protein subunits (Lerner & Steizt 1979). These antigenic subunit proteins were used because the antigens may increase sensitivity and specificity of ELISA for the detection of anti-Sm antibodies. The present results indicate that PAI-ELISA could be developed into a comprehensive, cost-effective test for SLE that is rapid and simple to perform. ELISA
Acknowledgement The authors thank the Universiti Kebangsaan granting part-time study leave to BOS-R.
Malaysia,
for
References Ahmad,
LB., Anuar,
N., Salam,
F., Ahmad, N. 8s Ah, A.M. 1989 assay and diagnosis of antigen and antibodies. In Currenr Research iri Lije Sciences, eds Zakri, A.H. & Latif, A. pp. 171-183, Kuala Lumpur: Faculty of Life Sciences, Universiti Kebangsaan Malaysia. (In Malay.) Banttarri, E.E. & Petersen, A.C. 1983 A cleaning technique for cuvettes used in enzyme-linked immunosorbent assay, Hunt Disease 67, 18-20,
Serology in the detection,
B.O.
Sifi-Rohana,
LB. Ahmad
and B.A.
Nasuruddin
Barada, F.A., Andrews, B.S., Davis, JS. & Taylor, R.P. 1981 Antibodies to Sm in patients with systemic lupus erythematosus. Arthritis and Rheumatism 24, 123&1244. Clark, M.F. & Adam, A.N. 1977 Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Jowna/ of Genend Virology 34, 475-483. Crowe, W. & Kushner, I. 1977 An immunofluorescent method using Crifhidiu lucihae to detect antibodies to double-stranded DNA. Arfhritis and Rheumatism 20, 811-814. Edwards, M.L. & Cooper, J.I. 1985 Plant virus detection using a new form of indirect ELISA. ]ournul of Virological Methods 11,
30+319. Engvall, E. & Pearlmann, P. 1971 assay (ELISA): quantitative assay chernisty 8, 871-874, Fritzler, M. J. 1986 Immunofluorescent In Manual of Clinical Laboratoy N.R. Friedman, H. & Fahey, J.L. American Society for Microbiology. Goodwin, P.H. & Banttari, J.E. ELISA for potato viruses S, X, ments, additives, and a modified
Enzyme-linked immunosorbent of immunoglobulin G. 1rmrrwreantinuclear antibody tests. immunology, 3rd edn, eds Rose, pp. 733-749. Washington DC: 1984 Increased sensitivity of and Y by polystyrene pretreatassay procedure. P6ant Disease
68,944-947. Lemer, M.R. & Steizt, J.A. 1979 Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. Proceedings of the Nafionul Acudemy of Sciences of fhe United States of America 76, 5495-5499, Nakamura, R.M., Peebles, CL., Molden, D.P. 81 Tan, E.M. 1984 Advances in laboratory test for autoantibodies to nuclear antigens in systemic rheumatic disease. Laborafoy Medicine 1.5, 190-198. Notman, D.D., Kurata, N. & Tan, E.M. 1975 Profiles of antinuclear antibodies in systemic rheumatic diseases. Annuls of Infer& Medicine 83, 464-469. Reichlin, M. I976 Problems in differentiating SLE and mixed connective-tissue disease. New England journal of Medicine 298, 1194-l 196. Sharp, G., Irwin, W., May, CM. Holman, H.R., McDuffie, F.C., Hess, E.V. 81 S&mid, F.R. 1976 Association of antibodies to
208
World jomal
of Mmobiology
6 Bmtechwlogy, Vol ??, 199.5
ribonucleoprotein and Sm antigens with mixed connective tissue disease, systemic lupus erythematosus and other rheumatic diseases. New England Journal of Medicine 295, 1x491154. Siti-Rohana, B.O., Ahmad, LB. & Nasurruddin, B.A. 1992 Prevalence of antinuclear antibodies in Malaysian subjects. In: Medical Research Towards fhe 2 1st Cents y, eds Alaudden, S., Moosdeen, F., Rajikin, M.H., Saim, L. & Daud, M.Z. pp. 324-3.27. Kuala Lumpur: Medical Faculty, Universiti Kebangsaan Malaysia. Sutula, C.L., Gillett, J.M., Morissey, S.M. & Ramsdell, D.C. 1986 Interpreting ELISA data and establishing the positive-negative threshold. Plant Disease 70, 72.2-727. Takano, M., Golden, S.S., Sharp, G.C. & Agris, P.F. 1981 Molecular relationship between two nuclear antigens ribonucleoproteins and Sm: purification of active antigens and their biochemica1 characterisation. Biochemisty 20, 5929-5935. Tan, E.M. 1982 Autoantibodies to nuclear antigens (ANA); their immunobiology and medicine. In: Aduances in Immunology Vol 33, eds Kunkel, H.G. & Dixon, F.J. pp. 167-240. New York: Academic Press. Tan, E.M. 1983 Antinuclear antibodies in diagnosis and management. In The Biology of Immunologic Disease, eds Dixon, F.J. & Fisher, D.W., pp. 319-324. Sunderland, MA: Sinauer Associates. Tan, E.M., Cohen, A.S., Fries, J.F., Masi, A.T., McShane, A.J., Rothfield, N.F., Schaller, J.G., Talal, N. 81 Winchester, J.G. 1982 The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthrifis and Rheumafism 25, 1271-1277. Winn, D.M., Wolfe, J.F., Linberg, D.A., Fristoe, F.H., Kingland, L. 81 Sharp, G.C. 1979 Identification of a clinical subset of systemic lupus erythematosus by antibodies to the Sm antigen. Arthritis and Rheumatism 22, 1334-1337. Zainal-Abidin, B.A.H., Idris, S.R.H., Dolah, S. & Ahmad, 1.B. 1992 PAI-ELISA technique for the detection of antibodies against rodent malaria. Sains Malaysiana 21, 133-142. (In Malay.)
(Received
1994)
in revised form
26 Ocfober 19%; accepfed I November
of Microbio/ogy
& 6iotechnotogy
11, 206-206
Application of protein-A indirect ELISA (PAI-ELBA) for the detection of anti-Smith antibodies in systemic lupus erythematosus patients B.0, Siti-Rohana, I.B. Ahmad* and B,A. Nasuruddin An indirect form of protein-A ELISA (PA&ELBA) was optimized and, when used to detect anti-Smith antibodies in sera of 31 systemic lupus erythematosus (SLE) patients, gave results comparable with those using a commercial immunodiffusion kit. The number of sera found to be positive for anti-Smith antibodies by ELISA was seven, four of which were also found positive by immunodiffusion. fiy words: Anti-Smith
antibodies,
ELISA, protein A, systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is a polymorphic autoimmune disease of idiopathic origin that is sometimes difficult to diagnose. It has therefore been suggested that, for the diagnosis of SLE, at least four of the criteria listed by the American Rheumatism Association be fulfilled (Tan ef ~1. 1982). The most commonly used criteria are the presence of anti-nuclear (AN), anti-double-stranded DNA (anti-dsDNA) and anti-Smith (anti-Sm) antibodies (Sharp ef ~1. 1976; Tan ef al. 1982). The presence of AN antibodies in the patients’ sera determines the auto-immune status of the patients’ disease. AN antibodies are reported to be present in about 96% to 99% of SLE patients (Fritzler 1986) and their assay thus remains an important screening test for SLE. Anti-dsDNA antibodies were reported to be prevalent in SLE cases (Crowe & Kushner 1977; Tan 1983). However, the presence of anti-dsDNA antibodies in other auto-immune diseases (Notman ef al. 1975) has limited the use of this assay in diagnosis of SLE. In contrast, anti-Sm antibodies are highly specific markers for this disease (Notman ef al. 1975; Reichlin 1976; Winn ef a/. 1979; Tan 1982; Nakamura ef al. 1984)
B.O. Siti-Rohana is with the Universiti Kebangsaan Malaysia (UKM), Faculty of Medicine, Koala Lumpor, Malaysia. I.B. Ahmad is with the Department of Microbiology, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, Bangi, 43600 UKM, Malaysia; fax: +603 6252698. B.A. Nasaruddin is with the Institute for Medical Research, Malaysia. Towespending author. @ 1995 Rapid Communications
of Oxford Ltd
and their presence in sera is important in the prognosis of SLE (Winn ef ul. 1979; Barada ef ~1. 1981). The immunodiffusion (ID) test is currently the most common method used for the detection of anti-Sm antibodies. The inherently low sensitivity of this method (Notman ef a/. 1975) has, however, resulted in dismissal of sera with low titres of anti-Sm antibodies as SLE negatives. Therefore, there is a need for a more sensitive serological method, such as ELISA, to detect anti-Sm antibodies in SLE sera. The high degree of sensitivity possible with ELISA, coupled with other factors such as the relative ease with which they can be performed, the short time needed before results are available and their cost-effectiveness (Engvall & Pearlmann 1971; Clark & Adam 1977) have all contributed to making an ELISA the method of choice. Subsequent modifications to the original ELISA technique have resulted in increased sensitivity and specificity. For example, the use of enzymelabelled protein-A in indirect ELISA, in place of the commonly used enzyme-labelled anti-immunoglobulins, has been shown to increase the sensitivity of the assay system (Edwards & Cooper 1985; Ahmad ef al. 1989). The present study was of the application of protein-A ELISA for the detection of anti-Sm antibodies in SLE patients.
Materials SLE
sera
and Methods
(31 samples)
were obtained from the Specialist Clinic of
PAl-ELlSA the Medical Faculty, Universiti Kebangsaan Malaysia, Kuala Lumpur. Ten normal sera from blood donors, previously tested negative for AN and anti-dsDNA antibodies (Siti-Rohana et ~1. 1992), served as negative controls and three anti-Sm-positive sera, obtained from confirmed SLE patients, served as positive controls. A4icrotitre Assays Flat-bottomed, microtitre plates were pre-treated with 5.3 M NaOH in 30% (v/v) ethanol (see Banttarri & Petersen 1983; Goodwin & Banttarri 1984). The Smith antigen, 100~1 in 50 rnM NaHCOJ/NaACOs buffer (pH 9.6) was dispensed in varying concentrations to each well of the microtitre plate and held overnight at 4OC. The antigen was from Northeast Biomedical Laboratories, USA. Coated plates were washed three times with phosphate-buffered saline (PBS)/Tween (PBS, pH 7.4, plus 0.05% Tween 20). Blocking of unoccupied surfaces was achieved by treating the wells with 3% egg albumin in PBS (pH 7.4) at 45’C for 1 h. The plates were then washed five times with PBYTween (pH 7.4). Immediately after washing, the plates were oven-dried at 37’C for I h. At this stage the plates were stored desiccated at 4OC, or used immediately for assay of serum anti-Sm antibodies.
The assay was performed according to standardized, indirect enzyme immune-assay procedures but with modifications (ZainalAbidin et a/. 1992). Briefly, serum samples were each diluted to pre-determined optimum dilution in PBS (pH 7.4), dispensed at 100 @well, then incubated either overnight at 4’C or 2 h at 37OC. After incubation, the plate was washed by rinsing it five times with PBS/Tween. Alkaline-phosphatase-labelled protein A, diluted at pre-determined concentrations in PBS (pH 7.4), was added at 100 PI/well. At the end of a 30.min incubation period at room temperature, 50 ~1 of 3 M NaOH/well was added and the absorbance (A) then read with an ELISA reader.
The optimized ELISA technique was used to determine the mean A value from IO normal sera, An A value twice this mean value for negative controls was taken to indicate a positive serum (Sutula et a/. 1986). A values determined for the three Sm-oositive control sera were used to provide comparative examples of positive A values. L
The 31 SLE sera were also tested for anti-Sm antibodies using a commercial ID test kit (ENA kit; Behring, Nippon Hoechst, Japan), according to the manufacturer’s instructions. Determination of positive and negative results was based on the reaction of identity and non-identity, respectively.
Results
and Discussion
Pretreufment of Plafes Assays using plates pre-treated with 5.3 M NaOH/30% ethanol and pretreated with PBS (control) gave A values 13% to 14% and 11% higher than untreated plates, respectively. This was consistent with the findings of Banttarri & Petersen (1983) and Goodwin & Banttarri (1984). The NaOH pretreatment enhanced the surface binding property of the microtitre wells and all subsequent experiments utilized only NaOH/ethanol-treated plates. Only plates
detection of anti-Smifh
unfibodies in SLE
with high absorption capacity should be used for effective binding of the antigen. Opfimizatioa of ELISA The optimum concentration of Sm antigen to coat the wells was found to be 3 fig/ml when dispensed at 100 pl/ well. The optimum dilution of serum was 1:50, giving a mean A of 0.808 (control mean = 0.045) and the optimum dilution of alkaline-phosphatase-labelled protein A was 1: 1000 or I pg/ml, giving an A value of 0.695 (control mean = 0.051). Overnight incubation at 4’C gave slightly higher A values than incubation at 3FC for 2 h and was used throughout the study. of Serum Samples ELISA using the optimized conditions gave 1.17 and 0205 as the mean A values for the three Sm-positive controls and the 10 normal sera (negative controls), respectively. The positive cut-off point was 0.411. Based on this cut-off point, seven Sm-positive samples were detected by ELISA, compared with four by ID. The A values for the seven antiSm-positive sera ranged from 0.509 to 1.088. The three sera which were detected as positive by ELISA, but negative by ID had A values ranging from 0.509 to 1.088. While the ID method has the advantage of being able to produce specific reactions with crude antigens, specificity and sensitivity of ELISA technique depends on the use of pure reagents. In the present study pure Smith antigen was used for the PAI-ELISA test. The Smith antigen has been characterized as an acidic, TO-kDa, non-histone nuclear protein molecule (Takano et al. 198I), although its antigenicity has been ascribed to 25 kDA and 16 kDA protein subunits (Lerner & Steizt 1979). These antigenic subunit proteins were used because the antigens may increase sensitivity and specificity of ELISA for the detection of anti-Sm antibodies. The present results indicate that PAI-ELISA could be developed into a comprehensive, cost-effective test for SLE that is rapid and simple to perform. ELISA
Acknowledgement The authors thank the Universiti Kebangsaan granting part-time study leave to BOS-R.
Malaysia,
for
References Ahmad,
LB., Anuar,
N., Salam,
F., Ahmad, N. 8s Ah, A.M. 1989 assay and diagnosis of antigen and antibodies. In Currenr Research iri Lije Sciences, eds Zakri, A.H. & Latif, A. pp. 171-183, Kuala Lumpur: Faculty of Life Sciences, Universiti Kebangsaan Malaysia. (In Malay.) Banttarri, E.E. & Petersen, A.C. 1983 A cleaning technique for cuvettes used in enzyme-linked immunosorbent assay, Hunt Disease 67, 18-20,
Serology in the detection,
B.O.
Sifi-Rohana,
LB. Ahmad
and B.A.
Nasuruddin
Barada, F.A., Andrews, B.S., Davis, JS. & Taylor, R.P. 1981 Antibodies to Sm in patients with systemic lupus erythematosus. Arthritis and Rheumatism 24, 123&1244. Clark, M.F. & Adam, A.N. 1977 Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Jowna/ of Genend Virology 34, 475-483. Crowe, W. & Kushner, I. 1977 An immunofluorescent method using Crifhidiu lucihae to detect antibodies to double-stranded DNA. Arfhritis and Rheumatism 20, 811-814. Edwards, M.L. & Cooper, J.I. 1985 Plant virus detection using a new form of indirect ELISA. ]ournul of Virological Methods 11,
30+319. Engvall, E. & Pearlmann, P. 1971 assay (ELISA): quantitative assay chernisty 8, 871-874, Fritzler, M. J. 1986 Immunofluorescent In Manual of Clinical Laboratoy N.R. Friedman, H. & Fahey, J.L. American Society for Microbiology. Goodwin, P.H. & Banttari, J.E. ELISA for potato viruses S, X, ments, additives, and a modified
Enzyme-linked immunosorbent of immunoglobulin G. 1rmrrwreantinuclear antibody tests. immunology, 3rd edn, eds Rose, pp. 733-749. Washington DC: 1984 Increased sensitivity of and Y by polystyrene pretreatassay procedure. P6ant Disease
68,944-947. Lemer, M.R. & Steizt, J.A. 1979 Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. Proceedings of the Nafionul Acudemy of Sciences of fhe United States of America 76, 5495-5499, Nakamura, R.M., Peebles, CL., Molden, D.P. 81 Tan, E.M. 1984 Advances in laboratory test for autoantibodies to nuclear antigens in systemic rheumatic disease. Laborafoy Medicine 1.5, 190-198. Notman, D.D., Kurata, N. & Tan, E.M. 1975 Profiles of antinuclear antibodies in systemic rheumatic diseases. Annuls of Infer& Medicine 83, 464-469. Reichlin, M. I976 Problems in differentiating SLE and mixed connective-tissue disease. New England journal of Medicine 298, 1194-l 196. Sharp, G., Irwin, W., May, CM. Holman, H.R., McDuffie, F.C., Hess, E.V. 81 S&mid, F.R. 1976 Association of antibodies to
208
World jomal
of Mmobiology
6 Bmtechwlogy, Vol ??, 199.5
ribonucleoprotein and Sm antigens with mixed connective tissue disease, systemic lupus erythematosus and other rheumatic diseases. New England Journal of Medicine 295, 1x491154. Siti-Rohana, B.O., Ahmad, LB. & Nasurruddin, B.A. 1992 Prevalence of antinuclear antibodies in Malaysian subjects. In: Medical Research Towards fhe 2 1st Cents y, eds Alaudden, S., Moosdeen, F., Rajikin, M.H., Saim, L. & Daud, M.Z. pp. 324-3.27. Kuala Lumpur: Medical Faculty, Universiti Kebangsaan Malaysia. Sutula, C.L., Gillett, J.M., Morissey, S.M. & Ramsdell, D.C. 1986 Interpreting ELISA data and establishing the positive-negative threshold. Plant Disease 70, 72.2-727. Takano, M., Golden, S.S., Sharp, G.C. & Agris, P.F. 1981 Molecular relationship between two nuclear antigens ribonucleoproteins and Sm: purification of active antigens and their biochemica1 characterisation. Biochemisty 20, 5929-5935. Tan, E.M. 1982 Autoantibodies to nuclear antigens (ANA); their immunobiology and medicine. In: Aduances in Immunology Vol 33, eds Kunkel, H.G. & Dixon, F.J. pp. 167-240. New York: Academic Press. Tan, E.M. 1983 Antinuclear antibodies in diagnosis and management. In The Biology of Immunologic Disease, eds Dixon, F.J. & Fisher, D.W., pp. 319-324. Sunderland, MA: Sinauer Associates. Tan, E.M., Cohen, A.S., Fries, J.F., Masi, A.T., McShane, A.J., Rothfield, N.F., Schaller, J.G., Talal, N. 81 Winchester, J.G. 1982 The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthrifis and Rheumafism 25, 1271-1277. Winn, D.M., Wolfe, J.F., Linberg, D.A., Fristoe, F.H., Kingland, L. 81 Sharp, G.C. 1979 Identification of a clinical subset of systemic lupus erythematosus by antibodies to the Sm antigen. Arthritis and Rheumatism 22, 1334-1337. Zainal-Abidin, B.A.H., Idris, S.R.H., Dolah, S. & Ahmad, 1.B. 1992 PAI-ELISA technique for the detection of antibodies against rodent malaria. Sains Malaysiana 21, 133-142. (In Malay.)
(Received
1994)
in revised form
26 Ocfober 19%; accepfed I November
