Br. J. exp. Path. (1978) 59, 577
THE DETECTION OF RUBELLA-SPECIFIC IgM ANTIBODIES BY RADIOIMMUNOASSAY H. 0. KANGRO, J. R. PATTISON* AND R. B. HEATH From The Department of Virology of the Mfedical College of the London Hospital, London El 2AD, U.K. Received for publication June 12, 1978
Summary.-An indirect solid-phase radioimmunoassay (RIA) has been developed for the detection of immunoglobulin (Ig) class-specific rubella antibodies. A commercial rubella haemagglutinin is dried and fixed on to the wells of flexible microtitre plates and allowed to react with serial dilutions of whole or fractionated human sera. Class-specific rubella antibodies are then detected by determining the specific binding of 1251-labelled anti-human IgG or IgM. The RIA was first evaluated by comparison with the haemagglutination-inhibition (HI) test for the detection of rubella-specific IgM in gel-filtration fractions. RIA was found to be as specific as HI but 10-150 times more sensitive. Rubella-specific IgG antibodies did not interfere in specific IgM determinations by RIA and therefore the latter technique was applied to unfractionated sera. The results obtained indicate that RIA on unfractionated sera is a practical, sensitive and specific technique which could provide a reliable method for the diagnosis of rubella. The rubella-specific IgM titres obtained by RIA were not increased by the removal of IgG by pretreatment of sera with Staphylococcal Protein A.
THE DETECTION of rubella-specific IgM antibodies has a confirmed role in the diagnosis of both acquired and congenital rubella (Pattison and Dane, 1975; Cradock-Watson, Ridehalgh and Chantler, 1976). With acquired rubella, specific IgM determinations avoid the necessity of appropriately timed paired sera and in babies with suspected congenital rubella such determinations avoid the delay of waiting 9-12 months to see if rubella antibody persists. Techniques for the detection of rubella-specific IgM usually involve physical separation of IgG and IgM, either by sucrose-density-gradient (SDG) centrifugation or gel filtration, followed by rubella haemagglutination-inhibition (HI) tests on the fractions. Both techniques have been shown to have an appropriate sensitivity and specificity for the diagnosis of acquired rubella (Best, Banatvala and Watson, 1969; Pattison and Dane, 1975).
However, they are time-consuming procedures and are not entirely suitable for large-scale screening of sera. We have therefore investigated the possibility of using the recently developed microradioimmunoassay (RIA) technique (Rosenthal, Hayashi and Notkins, 1973) for the detection of rubella-specific IgM. MATERIALS AND METHODS Preparation of 1251-labelled anti-human IgMlf and anti-human IgG antibodies.-Anti-human IgM and anti-human IgG antibodies were purified by affinity-column chromatography. Human IgG (AMiles Laboratories Ltd, Slough) and IgM purified from a patient with Waldenstrom's macroglobulinaemia by gel filtration with BioGel A-5M (Bio-Rad Laboratories, Richmond, California) were coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) at pH 8-3 for 3 days at 4°. The immunoabsorbents were washed and columns (15 cm x 0-64 cm2) prepared and equilibrated with Tris-HCl buffer (pH 8). Two ml of rabbit anti-human IgG or
* Present address: Department of Medical Microbiology, King's College Hospital Medical School, Denmark Hill, London SE5 8R2, U.K.
45-7 8
H. 0. KANGRO, J. R. PATTISON, AND R. B. HEATH
anti-human IgM (Hoechst Pharmaceuticals) were layered on to the appropriate column and allowed to react with the top 3-4 cm of gel for 30 min. Unbound protein was eluted with TrisHCl and recycled through the column 3 times before being discarded. Anti-IgG and anti-IgM antibodies were eluted with glycine-buffered HCI (pH 2.4). The antibody-containing fraction was adjusted to neutral pH immediately following elution using powdered Tris, concentrated 7-8-fold and dialysed overnight against phosphate-buffered saline (PBS). The protein concentration was in the range 0-8-1-2 mg/ml and the preparations were stored in small aliquots at -70° until required. A modification of the chloramine T method of Greenwood, Hunter and Glover (1963) was used to prepare 125I-labelled antibodies. A sample of antibody (40-50 fig) was iodinated with 1 mCi Na 125I (The Radiochemical Centre, Amersham, England). Protein-bound radiolabel was separated from free 1251 by gel filtration with Sephadex G-50 (Pharmacia). The labelled antibody (sp. act. 15-20 ,Ci/ftg protein) was stabilized by the addition of 5% bovine serum albumin and stored at 40 with 0-1% sodium azide. Preparation of solid-phase rubella antigen.A 50,1 amount of rubella haemagglutinin (HA) (Wellcome Reagents Ltd, England) diluted in PBS containing MgC12 and CaCl2 was desiccated on to each well of flexible polyvinylchloride microtitre plates (Dynatech Laboratories Ltd, England) in a warm air flow. An antigen concentration of 8 haemagglutinating units (HAU) was used for IgG determinations and 16 HAU for IgM determinations. The adsorbed antigen was fixed with 10% formalin (pH 7 2) for 20 min at room temperature. Remaining reactive groups on the plastic surface were blocked by adding PBS containing 0.2% gelatin (PBS-gelatin) to each well and incubating the plates overnight at 4°. Excess PBS-gelatin was then shaken off and the plates dried. In control plates rubella antigen was replaced by control antigen prepared from uninfected BHK-21 monolayers by alkaline extraction (Halonen, Ryan and Stewart, 1967) followed by Tween 80/ether treatment (Norrby, 1962). The control antigen was used at dilutions giving the same protein concentration as the rubella antigen. The solid-phase antigens were stored at 40 for up to 2 weeks. RIA procedure.-Amounts of 50 pl of serum specimens or fractions serially diluted in PBS containing 5% foetal calf serum (plus 1% dextran for IgM determinations) were added to the antigen-containing wells. The plates were incubated at 370 for 1 h for IgG determinations and 3 h for IgM determinations. All dilutions were tested in duplicate. The wells were then
washed 3 times with PBS-gelatin and the plates shaken dry. A 50,1 volume of 125I-labelled indicator antibody was added to each well at a concentration of 0-05-0-06 ,ug/ml anti-human IgG or 0-02-0-03 ,ug/ml anti-human IgM, both containing 20-30,000 counts/min (cpm) per 0.05 ml. The plates were incubated for a further 1 h (IgG assay) or 2 h (IgM assay) at 370 and finally washed 15 times with tap water. Individual wells were clipped from the plates and the bound radioactivity measured in a gamma counter. The specific binding of labelled indicator antibody at each serum dilution is expressed as a binding ratio (BR). With serum fractions the BR is the mean cpm in wells exposed to test serum divided by the mean cpm in wells exposed to assay diluent in place of test serum (background binding). With unfractionated sera the BR is the mean cpm in rubellaantigen-coated wells exposed to test serum divided by the mean cpm in control-antigencoated wells. Rubella HI tests.-Rubella HI tests on unfractionated sera were carried out according to the technique of Pattison and Mace (1973) except that a single volume of 0-5% day-old chick RBC was used, and on Sephadex G-200 fractions according to the method of Pattison and Mace (1975). Gel filtration.-Sera were pretreated with MnCl2/heparin and fractionated by gel filtration with Sephadex G-200 (Pharmacia) as previously described (Pattison and Mace, 1975). Immunoglobulin concentrations of the fractions were determined by single radial immunodiffusion using LC-Partigen immunodiffusion plates (Hoechst Pharmaceuticals, Germany). Protein A treatment.-Protein A was produced on solid growth medium using the Cowan 1 strain of Staphylococcus aureus according to the method of Mallinson, Roberts and Bruce White (1976) except that dialysis membranes were not used. Sera were diluted one in 10 with PBS and treated with Protein A by the method of Ankerst et al. (1974). RESULTS
Detection of class-specific antibody in fractionated sera The ability of RIA to detect classspecific rubella antibodies was initially investigated using fractionated sera. Table I shows the results obtained on testing an early convalescent serum (taken 14 days after the onset of the rash) and a serum from a patient with no recent history of rubella. Two peaks of rubella HI activity
579
THE DETECTION OF RUBELLA-SPECIFIC IGM ANTIBODIES
TABLE I.-Rubella Antibody Titres Determined by HI and RIA in Serum Fractions after Gel Filtration with Sephadex G-200 Non-convalescent serumt
Early convalescent serum* t~
RIA titre
)
A
A
IDt
HI
RIA titre
titre , Fraction HI IgG IgM IgG IgM IgM number titre 1 140
THE DETECTION OF RUBELLA-SPECIFIC IgM ANTIBODIES BY RADIOIMMUNOASSAY H. 0. KANGRO, J. R. PATTISON* AND R. B. HEATH From The Department of Virology of the Mfedical College of the London Hospital, London El 2AD, U.K. Received for publication June 12, 1978
Summary.-An indirect solid-phase radioimmunoassay (RIA) has been developed for the detection of immunoglobulin (Ig) class-specific rubella antibodies. A commercial rubella haemagglutinin is dried and fixed on to the wells of flexible microtitre plates and allowed to react with serial dilutions of whole or fractionated human sera. Class-specific rubella antibodies are then detected by determining the specific binding of 1251-labelled anti-human IgG or IgM. The RIA was first evaluated by comparison with the haemagglutination-inhibition (HI) test for the detection of rubella-specific IgM in gel-filtration fractions. RIA was found to be as specific as HI but 10-150 times more sensitive. Rubella-specific IgG antibodies did not interfere in specific IgM determinations by RIA and therefore the latter technique was applied to unfractionated sera. The results obtained indicate that RIA on unfractionated sera is a practical, sensitive and specific technique which could provide a reliable method for the diagnosis of rubella. The rubella-specific IgM titres obtained by RIA were not increased by the removal of IgG by pretreatment of sera with Staphylococcal Protein A.
THE DETECTION of rubella-specific IgM antibodies has a confirmed role in the diagnosis of both acquired and congenital rubella (Pattison and Dane, 1975; Cradock-Watson, Ridehalgh and Chantler, 1976). With acquired rubella, specific IgM determinations avoid the necessity of appropriately timed paired sera and in babies with suspected congenital rubella such determinations avoid the delay of waiting 9-12 months to see if rubella antibody persists. Techniques for the detection of rubella-specific IgM usually involve physical separation of IgG and IgM, either by sucrose-density-gradient (SDG) centrifugation or gel filtration, followed by rubella haemagglutination-inhibition (HI) tests on the fractions. Both techniques have been shown to have an appropriate sensitivity and specificity for the diagnosis of acquired rubella (Best, Banatvala and Watson, 1969; Pattison and Dane, 1975).
However, they are time-consuming procedures and are not entirely suitable for large-scale screening of sera. We have therefore investigated the possibility of using the recently developed microradioimmunoassay (RIA) technique (Rosenthal, Hayashi and Notkins, 1973) for the detection of rubella-specific IgM. MATERIALS AND METHODS Preparation of 1251-labelled anti-human IgMlf and anti-human IgG antibodies.-Anti-human IgM and anti-human IgG antibodies were purified by affinity-column chromatography. Human IgG (AMiles Laboratories Ltd, Slough) and IgM purified from a patient with Waldenstrom's macroglobulinaemia by gel filtration with BioGel A-5M (Bio-Rad Laboratories, Richmond, California) were coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) at pH 8-3 for 3 days at 4°. The immunoabsorbents were washed and columns (15 cm x 0-64 cm2) prepared and equilibrated with Tris-HCl buffer (pH 8). Two ml of rabbit anti-human IgG or
* Present address: Department of Medical Microbiology, King's College Hospital Medical School, Denmark Hill, London SE5 8R2, U.K.
45-7 8
H. 0. KANGRO, J. R. PATTISON, AND R. B. HEATH
anti-human IgM (Hoechst Pharmaceuticals) were layered on to the appropriate column and allowed to react with the top 3-4 cm of gel for 30 min. Unbound protein was eluted with TrisHCl and recycled through the column 3 times before being discarded. Anti-IgG and anti-IgM antibodies were eluted with glycine-buffered HCI (pH 2.4). The antibody-containing fraction was adjusted to neutral pH immediately following elution using powdered Tris, concentrated 7-8-fold and dialysed overnight against phosphate-buffered saline (PBS). The protein concentration was in the range 0-8-1-2 mg/ml and the preparations were stored in small aliquots at -70° until required. A modification of the chloramine T method of Greenwood, Hunter and Glover (1963) was used to prepare 125I-labelled antibodies. A sample of antibody (40-50 fig) was iodinated with 1 mCi Na 125I (The Radiochemical Centre, Amersham, England). Protein-bound radiolabel was separated from free 1251 by gel filtration with Sephadex G-50 (Pharmacia). The labelled antibody (sp. act. 15-20 ,Ci/ftg protein) was stabilized by the addition of 5% bovine serum albumin and stored at 40 with 0-1% sodium azide. Preparation of solid-phase rubella antigen.A 50,1 amount of rubella haemagglutinin (HA) (Wellcome Reagents Ltd, England) diluted in PBS containing MgC12 and CaCl2 was desiccated on to each well of flexible polyvinylchloride microtitre plates (Dynatech Laboratories Ltd, England) in a warm air flow. An antigen concentration of 8 haemagglutinating units (HAU) was used for IgG determinations and 16 HAU for IgM determinations. The adsorbed antigen was fixed with 10% formalin (pH 7 2) for 20 min at room temperature. Remaining reactive groups on the plastic surface were blocked by adding PBS containing 0.2% gelatin (PBS-gelatin) to each well and incubating the plates overnight at 4°. Excess PBS-gelatin was then shaken off and the plates dried. In control plates rubella antigen was replaced by control antigen prepared from uninfected BHK-21 monolayers by alkaline extraction (Halonen, Ryan and Stewart, 1967) followed by Tween 80/ether treatment (Norrby, 1962). The control antigen was used at dilutions giving the same protein concentration as the rubella antigen. The solid-phase antigens were stored at 40 for up to 2 weeks. RIA procedure.-Amounts of 50 pl of serum specimens or fractions serially diluted in PBS containing 5% foetal calf serum (plus 1% dextran for IgM determinations) were added to the antigen-containing wells. The plates were incubated at 370 for 1 h for IgG determinations and 3 h for IgM determinations. All dilutions were tested in duplicate. The wells were then
washed 3 times with PBS-gelatin and the plates shaken dry. A 50,1 volume of 125I-labelled indicator antibody was added to each well at a concentration of 0-05-0-06 ,ug/ml anti-human IgG or 0-02-0-03 ,ug/ml anti-human IgM, both containing 20-30,000 counts/min (cpm) per 0.05 ml. The plates were incubated for a further 1 h (IgG assay) or 2 h (IgM assay) at 370 and finally washed 15 times with tap water. Individual wells were clipped from the plates and the bound radioactivity measured in a gamma counter. The specific binding of labelled indicator antibody at each serum dilution is expressed as a binding ratio (BR). With serum fractions the BR is the mean cpm in wells exposed to test serum divided by the mean cpm in wells exposed to assay diluent in place of test serum (background binding). With unfractionated sera the BR is the mean cpm in rubellaantigen-coated wells exposed to test serum divided by the mean cpm in control-antigencoated wells. Rubella HI tests.-Rubella HI tests on unfractionated sera were carried out according to the technique of Pattison and Mace (1973) except that a single volume of 0-5% day-old chick RBC was used, and on Sephadex G-200 fractions according to the method of Pattison and Mace (1975). Gel filtration.-Sera were pretreated with MnCl2/heparin and fractionated by gel filtration with Sephadex G-200 (Pharmacia) as previously described (Pattison and Mace, 1975). Immunoglobulin concentrations of the fractions were determined by single radial immunodiffusion using LC-Partigen immunodiffusion plates (Hoechst Pharmaceuticals, Germany). Protein A treatment.-Protein A was produced on solid growth medium using the Cowan 1 strain of Staphylococcus aureus according to the method of Mallinson, Roberts and Bruce White (1976) except that dialysis membranes were not used. Sera were diluted one in 10 with PBS and treated with Protein A by the method of Ankerst et al. (1974). RESULTS
Detection of class-specific antibody in fractionated sera The ability of RIA to detect classspecific rubella antibodies was initially investigated using fractionated sera. Table I shows the results obtained on testing an early convalescent serum (taken 14 days after the onset of the rash) and a serum from a patient with no recent history of rubella. Two peaks of rubella HI activity
579
THE DETECTION OF RUBELLA-SPECIFIC IGM ANTIBODIES
TABLE I.-Rubella Antibody Titres Determined by HI and RIA in Serum Fractions after Gel Filtration with Sephadex G-200 Non-convalescent serumt
Early convalescent serum* t~
RIA titre
)
A
A
IDt
HI
RIA titre
titre , Fraction HI IgG IgM IgG IgM IgM number titre 1 140
