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Direct radioimmunoassay for the detection of IgM antibodies against mycoplasma pneumoniae
Journal o[" Immunological Methods, 32 (1980) 261--273
261
© Elsevier/North-Holland Biomedical Press
DIRECT RADIOIMMUNOASSAY FOR THE DETECTION OF IgM ANTIBODIES AGAINST M YCOPLASMA PNEUMONIAE
P.C. PRICE
Institute for Medical Microbiology, St. Gallen, Switzerland (Received 5 July 1979, accepted 2 October 1979)
A direct solid-phase radioimmunoassay is described for detecting IgM-class antibodies against Mycoplasma pneumoniae. The assay achieves a preliminary separation of IgM from other serum proteins by immunoadsorption to anti-IgM-coated wells in microtitre plates. The IgM is then tested for antigen specificity by measuring its ability to bind radiolabelled M. pneumoniae. Positive results are confirmed by retesting sera after treatment with 2-mercaptoethanol. The assay is specific for IgM-class antibodies and specific for M. pneumoniae. It is not affected by competitive inhibition from IgG and avoids the use of density gradient centrifugation or gel filtration to separate IgM from other immunoglobulins. It is sensitive, reproducible, rapid, simple and requires very little serum.
INTRODUCTION
The most c o m m o n l y used methods for detecting IgM antibodies against specified antigens are the indirect methods of immunofluorescence, radioimmunoassay (RIA) and enzyme immunoassay. These are usually two-step procedures. First, specific antibodies are allowed to bind to solid- or fluidphase antigens. The presence of IgM among the antigen-bound antibodies is then determined using anti-IgM labelled with fluorochrome, radioisotope or enzyme. A major problem associated with indirect methods is competitive inhibition of binding of IgM to antigen in the presence of antigen-specific IgG antibody. This is best overcome by separating IgM from IgG before measuring its antigen specificity. Physical methods of separation such as density gradient centrifugation or gel filtration are laborious and timeconsuming. A direct RIA for detecting IgM against specified antigens has been developed. IgM is first separated from other immunoglobulins using an immunochemical rather than physical method and is then tested directly for antigen specificity. As a model antigen, Mycoplasma pneumoniae was selected to develop the assay. It can be obtained in a clean, radioactive form (Brunner and Chanock, 1973), and serum is readily available from patients suffering from recent M. pneumoniae infections.
262 MATERIALS AND METHODS
Organisms Mycoplasma pneumoniae (Eaton agent) was obtained from Flow Laboratories, Irvine, Scotland. M. fermentans (strain PG-18) was obtained from the Research Reference Reagents Branch of the National Institute of Allergy and Infectious Diseases, Bethesda, MD, U.S.A. Growth medium and culture conditions Organisms were grown in mycoplasma broth base (BBL) containing 20% fresh horse serum, 0.5% lactalbumin hydrolysate (Difco), 1% glucose, 0.002% phenol red, 0.025% thallium acetate and 1000 U of Penicillin G/ml. This medium was adjusted to pH 8.1 and sterilized by membrane filtration. Incubation was at 37°C in rolling 1 litre transfusion bottles containing 400 ml of medium. An tigen preparation For the labelling of mycoplasma organisms, 20 pCi/ml of oleic acid-9, 10-3H (specific activity = 7.35 Ci/mmole) and 20 pCi/ml of palmitic acid-9, 10-3H (specific activity 23.5 Ci/mmole) were added to the medium on the day of inoculation. The radioactive fatty acids were obtained from New England Nuclear, Boston, MA, U.S.A. The medium was maintained at pH 8.1 -+ 0.5 by addition of 1 N NaOH. After 7--8 days, the mycoplasma were suspended in the growth medium and then centrifuged at 20,000 rev/min (Beckman rotor, 50.2 Ti) for 20 min. The pellet was washed twice in cold 0.1 M phosphate 0.15 M NaC1 pH 7.2 (PBS) using centrifugation as above. The [3H]antigens were then suspended into a volume of PBS equal to 1/50th of the original volume of medium and freeze-dried in 1 ml aliquots. Antigens were reconstituted as needed with sterile distilled water, diluted in fetal calf serum (FCS) and stored at 4 °C. Prior to use, light sonification was used to homogenize the antigen suspensions. The specific activities of the antigens were 9 X 106 cpm/mg for M. pneumoniae and 4 × 106 cpm/mg for M. fer. mentans. A lipid extract of M. pneumoniae for use as a complement-fixing (CF) antigen was prepared according to the m e t h o d of Kenny and Grayston (1965). This antigen was stabilized by the addition of bovine serum albumin (fraction V) (BSA V) (Armour, Eastbourne, U.K.) to a concentration of 2% and was freeze-dried in 1 ml amounts. A lipid extract was also obtained from [3H]M. pneumoniae using the above m e t h o d scaled down for 40 mg of [3H]organisms. N i n e t y ~ i g h t per cent of the 3H activity in the organisms was recovered in the lipid extract. Serum samples Over 35,000 serum samples submitted to the Institute for Medical Microbiology, St. Gallen, Switzerland, from patients (24,500) and healthy blood
263 donors (10,600) during the period July 1976--May 1977 were available for this study. Of these, 7447 were tested for the presence of CF antibodies against M. pneumoniae, 1141 of which were serial samples from patients. One h u n d r e d and ninety-one serum samples had CF antibodies at titres of ~>60. In this laboratory, a CF titre of ~>60 is considered to be a possible indication o f a recent infection with M. pneumoniae. Serial serum samples from 17 patients showed an increase in CF titre of 4-fold or greater, which is diagnostically indicative of a recent infection. In most cases, a description of the clinical s y m p t o m s of the patients was available. Sera were stored for long term at --20°C and for short term at 4 ° C.
Reference sera The following reference sera were used to optimize the test procedure. A negative reference serum was obtained by pooling 70 blood d o n o r sera k n o wn to contain no detectable CF antibodies against M. pneumoniae when tested at a dilution of 1 : 5. A weakly positive reference serum was obtained f r o m a p n e u m o n i a patient 11 days after onset of clinical symptoms. This serum had no detectable CF antibodies at a dilution of 1 : 10, but was consistently positive in the IgM RIA, giving a binding ratio (see RIA procedure below) of 5.5 -+ 1.1. An intermediately positive reference serum was obtained f r o m the same pa t i e nt 92 days after onset of clinical pneumonia. This serum had a CF titre of 120 and an IgM RIA binding ratio of 27.2 -+ 6.0 (binding ratios o f 40--60 were n o t u n c o m m o n for positive sera). Purification o f IgM IgM was purified using an e x t e n d e d version of the m e t h o d of Fey et al. (1976). The euglobulin fraction of a pool of 450 blood d o n o r sera was treated with ZnSO4 to precipitate IgG, subjected to sucrose density gradient centrifugation and c h r o m a t o g r a p h e d on Sephadex G-200. After concentration to 4 mg/ml, the.purified IgM was freeze-dried. 12sI radioiodination o f IgM Purified IgM was radioiodinated with [lZSI]Na (EIR, Wuerenlingen, Switzerland) using the chloramine-T m e t h o d of H unt er and G r e e n w o o d (1962). [12sI]IgM was stabilized by the addition of an equal volume of 4% BSA V in 0.05 M T r i s - ( h y d r o x y m e t h y l ) a m i n o m e t h a n e (Tris), 0.1% NAN3, pH 7.2 (TA). Removal of free 12sI- was achieved by anion-exchange chromatogr a p h y on DEAE Sephadex A-25 pre-equilibrated with 1% BSA V in TA. Some radioactive protein could be rinsed f r o m the col um n with the equilibration buffer, while [12sI]IgM was eluted using 1% BSA V in TA containing 0.25 M NaC1. A b o u t 5 atoms of 12sI were i ncorporat ed per molecule of IgM. Coating rnicro titre wells with an ti-IgM Anti-IgM serum was diluted in 0.05 M Tris buffer containing 0.15 M NaC1 and 0.1% NaN3 pH 7.2 (TSA). One hundr ed and fifty pl aliquots of this solu-
264 tion were added to r o u n d - b o t t o m e d wells in polyvinyl chloride microtitre plates (Cooke Engineering, Alexandria, VA, U.S.A.). The plates were then covered with polyester tape (Scotch tape Y 336, 3M, St. Paul, MN, U.S.A.) and stored at 4°C until used. For cont r ol purposes, some microtitre wells were coated with dilutions of normal rabbit serum.
RIA procedure After initial studies to determine optimal conditions, the following procedure was adopted. A n t i b o d y - c o a t e d wells were rinsed 5 times with TSA directly prior to use. One hundr e d pl amounts of sera diluted 1 : 10 in 10% FCS in TSA were pipetted in quadruplicate into wells which were then covered with polyester tape. After incubation for 2 h at 45°C in a water bath, the sera dilutions were removed by aspiration and the wells were rinsed 7 times with TSA. One hundr ed pl of [3H]antigen (1.5--2 × 10 s cpm) in FCS was then pipetted into each well. The wells were covered with tape and fu rth er incubated for 1 h. After aspiration of the u n b o u n d radioactive antigen and 10 rinses with TSA, the wells were dried with a hair drier, separated from each o th er by cutting with scissors and measured for ret ent i on of 3H. The negative reference serum was tested in quadruplicate or more in every assay. Results were expressed as binding ratios obtained by dividing the average cpm minus background of a sample by the average cpm minus background o f the negative reference serum.
2-Mercaptoethanol treatment Sera were added to an equal volume of 0.2 M 2-mercaptoethanol (2-ME) in PBS and incubated at 37°C for 1 h. The 2-ME-treated samples were then tested in the RIA. In some cases, sera were treated as above and in parallel with PBS containing no 2-ME. The 2-ME-treated and PBS-treated samples were then tested in parallel in the RIA.
Complement fixation test C o m p l e m e n t fixation (CF) tests were p e r f o r m e d using the microtitre p r o c e d u r e as described by Schmidt and L e n n e t t e (1970). CF titres are re po r ted as the reciprocal of the highest serum dilution giving complete fixation o f c o m p l e m e n t .
Measurement o f 3H and 12sI R e t e n t i o n o f [~H]M. pneumoniae antigens was measured by placing the dried and separated wells into 2 ml of xyl ene containing 0.36% Permablend III (Packard, IL, U.S.A.). Scintillation was measured using a Nuclear Chicago Isocap 300 (Des Plaines, IL, U.S.A.). Samples were c o u n t e d until an accuracy o f 2% was reached but n o t longer than 10 min. 12SIodine was measured using an MR 1032 aut om a t i c gamma c o u n t e r (W and W Electronics, Basle, Switzerland). Measurements were made for 5 min or until 50,000 counts had accumulated.
265 RESULTS
Selection o f anti-IgM serum for coating microtitre wells Fourteen different anti-IgM sera were used in increasing dilutions to coat wells and compared for their ability to retain [12sI]IgM. These antisera were obtained from 7 commercial suppliers and had been raised in rabbit, goat, sheep and swine. [12sI]IgM was incubated in the coated wells and, after aspiration and thorough rinsing, the wells were measured for retained [~2sI]IgM. Fig. 1 demonstrates the varying ability of 8 commercial antisera to retain [~2sI]IgM to coated wells. Antiserum H was only slightly better at IgM retention than normal rabbit serum I, while antisera A and C retained nearly 70% of the added [12S]IgM even at coating dilutions of I : 1600. For all further studies, antiserum A (rabbit anti-human-IgM, mu-chain-specific, lot number 0105, Dakopatts A/S, Denmark) was used at a dilution of 1 : 400 to coat wells. Such wells had a binding capacity of about 100 pg of IgM.
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Fig. 1. V a r y i n g ability of c o m m e r c i a l anti-IgM sera to b i n d [ 1251 ]IgM t o c o a t e d m i c r o t i t r e wells. M i c r o t i t r e wells were c o a t e d o v e r n i g h t at r o o m t e m p e r a t u r e w i t h 150 pl of increasing d i l u t i o n s (in T S A ) o f anti-IgM sera f r o m various c o m m e r c i a l suppliers ( A - - H ) a n d o f n o r m a l r a b b i t s e r u m (I). 6 0 0 , 0 0 0 c p m o f [12sI]IgM in 100 ~l T S A were t h e n i n c u b a t e d in the c o a t e d wells at r o o m t e m p e r a t u r e o v e r n i g h t . Wells were rinsed 10 t i m e s w i t h T S A a n d m e a s u r e d for b o u n d 12si.
266 Optimal incubation time and temperature for sera T h e o p t i m a l i n c u b a t i o n t i m e and t e m p e r a t u r e f o r s e r u m s a m p l e s in react i o n I ( s e p a r a t i o n o f IgM f r o m sera via i m m u n o a d s o r p t i o n t o a n t i - I g M - c o a t e d wells) (see Fig. 2) w e r e d e t e r m i n e d in t h e f o l l o w i n g m a n n e r . T h e r e f e r e n c e sera (see Materials a n d M e t h o d s ) w e r e d i l u t e d 1 : 10 in 20% FCS in T S A and i n c u b a t e d in c o a t e d wells f o r v a r y i n g p e r i o d s o f t i m e ( 1 - - 1 8 h) at various t e m p e r a t u r e s ( 4 - - 4 5 ° C ) . A f t e r rinsing, t h e wells w e r e i n c u b a t e d w i t h [3HIa n t i g e n . I t was generally f o u n d t h a t b i n d i n g ratios increased with increasing t e m p e r a t u r e a n d were less a f f e c t e d b y p r o l o n g i n g i n c u b a t i o n t i m e (Price, 1 9 7 7 ) . I n c u b a t i o n f o r 2 h at 4 5 ° C was used f o r r e a c t i o n I in all s u b s e q u e n t tests.
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267
Optimal dilution of sera T o d e t e r m i n e the best d i l u t i o n o f p a t i e n t s ' sera t o use r o u t i n e l y in the IgM RIA, 28 sera with CF titres ranging f r o m < 5 t o ~>240 were t e s t e d at serial 10-fold dilutions f r o m I : I to 1 : l 0 s (Price, 1 9 7 7 ) . Binding ratios were always highest at serum dilutions o f 1 : 10.
Optimal diluent for sera T h e r e f e r e n c e sera were t e s t e d at a dilution o f 1 : 10 in T S A which had been s u p p l e m e n t e d with 4% BSA V or with 20% calf serum, 20% h u m a n serum (M. pneumoniae sero-negative), 20% r a b b i t serum or 20% FCS. Addition o f 20% FCS to the serum d i l u e n t had a p r o n o u n c e d e f f e c t in raising binding ratios b y lowering the non-specific r e t e n t i o n o f [3H]antigen b y the negative r e f e r e n c e serum. T o d e t e r m i n e the best c o n c e n t r a t i o n o f FCS to be used in the serum diluent, the r e f e r e n c e sera were t e s t e d at a d i l u t i o n o f 1 : 10 in T S A s u p p l e m e n t e d with increasing c o n c e n t r a t i o n s o f FCS f r o m 1% to 100%. T e n per c e n t FCS in T S A was selected as the d i l u e n t for serum samples.
Optimal incubation time and temperature for [3H]antigen T h e o p t i m a l i n c u b a t i o n t i m e and t e m p e r a t u r e for the [3H]antigen in r e a c t i o n II ( d e t e r m i n a t i o n o f IgM specificity) (see Fig. 2) was d e t e r m i n e d in in the f o l l o w i n g m a n n e r . [3H]antigen was i n c u b a t e d for various periods o f t i m e (1--18 h) at various t e m p e r a t u r e s ( 4 - - 4 5 ° C ) in wells which had previo u s l y b e e n t r e a t e d for 2 h at 45°C with 1 : 10 dilutions o f the r e f e r e n c e sera in 10% FCS in TSA. Binding ratios reach their m a x i m u m values within 1 h at 45°C.
Optimal diluent for [3H]antigen In o r d e r to r e d u c e the non-specific binding o f [3H]antigen to wells in r e a c t i o n II, the e f f e c t o f adding FCS to the [3H]antigen d i l u e n t was studied. It was f o u n d essential t o suspend the [3H]antigen in 100% FCS t o o b t a i n m a x i m u m binding ratios.
Optimal specific activity of [3H]antigen T h e r e f e r e n c e sera were t e s t e d using [3H]antigen diluted in FCS to activities ranging f r o m 104 t o 106 c p m / t e s t . Binding ratios were highest w h e n the antigen was used diluted to a specific activity o f 2 × 10 s c p m / t e s t .
Comparison of 3H-organisms and 3H-lipid extract as antigens T h e r e f e r e n c e sera were t e s t e d using [3H]M. pneumoniae organisms and the lipid e x t r a c t o f [3H]M. pneumoniae as antigens. T h e use o f a suspension o f w h o l e organisms gives higher binding ratios t h a n o b t a i n e d using the lipid e x t r a c t as antigen. All r e f e r e n c e s to [3H]antigen in this s t u d y r e f e r t o a suspension o f [3H]M. pneumoniae organisms.
268
Reproducibility o f the IgM RIA T h e r e f e r e n c e negative serum and a serum which c o n s i s t e n t l y gave a high binding ratio were each tested 16 times. T h e positive serum yielded an average o f 1 1 , 0 0 0 + 563 c p m (10.4% o f the t o t a l c o u n t s in the test). T h e negative serum yielded an average o f 122 + 16 c p m (0.12% o f the total c o u n t s in the test).
Specificity o f the IgM RIA for M. pneumoniae One h u n d r e d and f i f t y - f o u r b l o o d d o n o r sera, which had no d e t e c t a b l e CF a n t i b o d i e s against M. pneumoniae w h e n tested at a dilution o f 1 : 5, were t e s t e d in the IgM RIA. T h e binding ratios o f these sera (Fig. 3A) are normally d i s t r i b u t e d a b o u t a value o f 1.1, and in all cases had values o f less t h a n 3.0. It was arbitrarily d e c i d e d t h a t a n y serum giving a binding ratio greater t h a n 3.0 w o u l d be c o n s i d e r e d positive. A binding ratio o f 3.0 lies 5 S.D.s above the average value o b t a i n e d f r o m these 154 sera. T h e binding ratios o b t a i n e d f r o m testing 99 sera with M. pneurnoniae CF titres ranging f r o m < 1 0 to ~>240 are s h o w n in Fig. 3B. T h e f r e q u e n c y o f binding ratios higher t h a n 3.0 increases with increasing CF titre. T w e n t y - o n e sera with binding ratios ranging f r o m 15 t o 39 were r e t e s t e d using [3HIM. fermentans as antigen. T h e resulting binding ratios were all l o w e r t h a n 3.0.
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Fig. 3. B i n d i n g r a t i o s o b t a i n e d for sera in t h e IgM R I A . S e r u m d i l u t i o n s o f 1 : 10 in 10% f o e t a l calf s e r u m in T S A w e r e i n c u b a t e d in q u a d r u p l i c a t e in a n t i - I g M - c o a t e d wells for 2 h at 4 5 ° C . A f t e r rinsing, 1.5 X 10 s c p m o f [3H]M. p n e u m o n i a e in 0.1 m l o f foetal calf s e r u m w e r e a d d e d t o e a c h well a n d i n c u b a t i o n was c o n t i n u e d for I h at 4 5 ° C . Wells were w a s h e d a n d m e a s u r e d f o r r e t a i n i n g 3H. A: b i n d i n g ratios o f 154 b l o o d d o n o r sera w h i c h h a d n o d e t e c t a b l e C F a n t i b o d i e s at a d i l u t i o n o f 1 : 5. B: c o r r e l a t i o n b e t w e e n b i n d i n g r a t i o s a n d C F a n t i b o d y titres o f 99 sera. C: e f f e c t o n b i n d i n g r a t i o s o f t r e a t m e n t o f sera w i t h 2-ME. T h e s e sera all h a d b i n d i n g ratios g r e a t e r t h a n 3.0 w h e n n o t t r e a t e d w i t h 2-ME (see B).
269
F o r t y - t w o sera with no detectable CF antibodies against M. pneumoniae b u t with high CF titres against antigens n o t related to M. pneumoniae were tested in the IgM RIA. None o f these sera gave binding ratios greater than 3.0.
Specificity o f the IgM RIA for IgM-class antibodies The effect o f 2-ME t r e a t m e n t on the binding ratios of sera can be seen in Fig. 3C. Here, all sera f r om Fig. 3B which yielded binding ratios greater than 3.0 were retested after t r e a t m e n t with 2-ME. IgM and IgG fractions of sera subjected to sucrose density gradient centrifugation were treated with 2-ME and with PBS and tested in parallel with the non-fractionated sera in the IgM RIA and in the CF test. The results obtained for two typical sera are shown in Table 1. Binding ratios obtained on testing the PBS-treated IgM fractions of sera are almost identical with those for the unf r a c t i ona t e d sera. Binding ratios for IgM fractions were always reduced to less than 3.0 after t r e a t m e n t with 2-ME. The IgG fractions were c o n t a m i n a t e d with IgM, which accounts for their high IgM RIA binding ratios which are reduced by 2-ME t r eat m e n t . The CF titres of IgM fractions were reduced by 2-ME t r e a t m e n t , but the CF titres of the IgG fractions were not.
Effect o f anti-M, pneumoniae IgG on the IgM RIA To determine the effect of specific IgG on the binding ratio of an IgMpositive serum, the following e x p e r i m e n t was perform ed. An anti-M.pneurnoniae IgM-positive serum (M) (CF titre ~>240, binding ratio, 42) was serially diluted 4-fold in a sero-negative serum (N) (CF titre <5, binding ratio, 1.0) and also in an IgG-positive, IgM-negative serum (G) (CF titre ~>240, binding ratio, 5; binding ratio n o t reduced by 2-ME treatment). The
TABLE 1 E F F E C T O F 2-ME T R E A T M E N T ON IgM R I A B I N D I N G R A T I O S A N D CF T I T R E S O F S U C R O S E D E N S I T Y G R A D I E N T IgG A N D IgM F R A C T I O N S S e r u m No.
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Fig. 4. E f f e c t o f anti-M, pneumoniae IgG on the IgM RIA. A n anti-M, pneurnoniae IgMpositive s e r u m (M) was serially diluted 4-fold in an IgG-positive, IgM-negative s e r u m (G) and in a sero-negative s e r u m (N). The sera and m i x t u r e s of sera were t h e n t r e a t e d with PBS and w i t h 2-ME and t e s t e d in the IgM RIA. Samples t r e a t e d w i t h PBS = closed symbols ( 0 , . , 4, , , A). S a m p l e s t r e a t e d with 2-ME = o p e n s y m b o l s (©, D, 4, ~, L~).
3 sera and t h e m i x t u r e s o f sera were t h e n treated w i t h 2-ME and w i t h PBS and t e s t e d in t h e IgM R I A . S e r u m G (Fig. 4) gave a l o w binding ratio w h i c h w a s n o t r e d u c e d by 2-ME t r e a t m e n t . S e r u m M gave a high binding ratio w h i c h w a s r e d u c e d to less than 3.0 by 2-ME. S e r u m N gave a binding ratio o f
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Days after onset of pneumonia Fig. 5. Persistence o f IgM-class a n t i b o d i e s against M. pneumoniae. Persistence o f IgM-class a n t i b o d i e s against M. pneumoniae in successive s e r u m samples f r o m 3 p n e u m o n i a p a t i e n t s as d e t e r m i n e d by the IgM RIA. P B S - t r e a t e d sera, A, 0, I : 2-ME-treated sera, A, o, D.
271 less than 3.0. The addition of serum G to serum M had practically no more effect on reducing the binding ratio than the effect of addition of serum N (Fig. 4).
Persistence o f anti-M, pneumoniae IgM The persistence of IgM antibodies against M. pneumoniae was studied by testing successive serum samples from 3 pneumonia patients. Fig. 5 shows the binding ratios obtained from testing these sera after 2-ME treatment and PBS treatment. Anti-M. pneumoniae IgM persisted for over 6 months when monitored by this method. DISCUSSION The direct solid-phase RIA has been tailored to the detection of antigenspecific IgM in serum by taking advantage of the following. IgM is catabolized faster than IgG (fractional catabolic rate in % intravascular pool/day for IgM --- 18.0, for IgG = 6.7) (Stanworth and Turner, 1973). A consequence of this is that following a recent infection the overall serum concentration of IgM temporarily increases (Hobbs, 1970), and a relatively high proportion of this IgM is specific for the agent causing illness. When patients' sera are incubated in anti-IgM-coated microtitre wells, even though only a small a m o u n t of the total serum IgM is retained (100 pg), a large proportion of it is specific for the agent of illness. When challenged with radiolabelled antigen, this IgM is capable of binding amounts of antigen which far exceed the a m o u n t of antigen which non-specifically binds to the solid phase. Hence, differentiation of positive and negative sera is achieved. The long half-life of IgG results in its accumulation at relatively high levels in serum (10--20 times the concentration of IgM in healthy adults). The specific IgG produced following a recent infection becomes diluted in preexisting IgG which has no specificity for the disease agent. Therefore, in the IgM RIA, traces of IgG which non-specifically bind to the solid phase should, for the most part, not be specific toward the agent causing illness. There should be little retention of radioactive antigen due to specific IgG which has bound non-specifically to the solid phase. In practice, however, it was found that some sera with high CF titres gave moderate IgM RIA binding ratios which could not be reduced by 2-ME treatment, and were probably the result of antigen retention by IgG. It was, therefore, f o u n d necessary to re-test after t r e a t m e n t with 2-ME all sera giving IgM RIA binding ratios higher than 3.0. A reduction in binding ratio of 3-fold or greater was considered proof that the detected antibody activity belonged to the IgM class. Competitive inhibition by specific IgG was n o t expected to be a problem in the direct IgM RIA, and it was not. The addition of an anti-M, pneumoniae IgG-positive serum to an IgM-positive serum had practically no more effect on reducing the IgM RIA binding ratio than addition of M. pneumoniae sero-negative serum. Even at a ratio of 1 2 8 0 : 1 (IgG-positive
272 serum : IgM-positive serum), no competitive inhibition of binding of IgM was observed. This, of course, was expected since the first reaction step of the direct m e t h o d removes IgG from the IgM to be tested. Absorption of IgM to a solid-phase anti-IgM, where the solid phase is the reaction container (i.e. microtitre wells) achieves a rapid and convenient separation of IgM from IgG. It proved to be extremely important to select a good source of anti-IgM for coating the solid phase. Most commercially available sera tested were demonstrably inferior to the antiserum selected for this study in their ability to bind IgM to the solid phase. The coated wells used in this study had a binding capacity of 100 pg of IgM. It was found essential to dilute the radioactive antigen in 100% FCS to obtain m a x i m u m binding ratios for positive sera. This resulted in a lowering of the a m o u n t of radioactive antigen non-specifically bound to the solid phase from the negative reference sera. [3H]M. pneumoniae organisms provided a better antigen than the lipid extract of organisms. This is probably an amplification effect, due to the fact that retention of one 3H-labelled organism results in an overall greater retention of 3H than is achieved by the specific retention of one [3H] glycerophospholipid miscile. The IgM RIA proved to be very sensitive. Sera which gave low binding ratios (e.g. 3.1, 3.3, 4.8) when tested undiluted continued to give binding ratios greater than 3.0 at dilutions of up to 1 : 10,000. It is unlikely, therefore, that the assay would give false negative results due to low sensitivity. The test is specific with regard to detection of antibodies against M. pneumoniae. No sera giving high binding ratios were found among 154 blood donors or 42 patients whose sera had high CF antibody titres against antigens from 10 viruses and rickettsiae. Furthermore, none of 21 sera, giving high binding ratios in the M. pneumoniae IgM RIA, gave binding ratios higher than 3.0 when tested against radioactive M. fermentans. Anti-M. pneumoniae IgM appears to persist for over 6 months when monitored by the IgM RIA. There does, however, appear to be a big drop in binding ratios within 2--3 months following onset of clinical symptoms. Perhaps the biggest advantage of indirect IgM methods is the need for only one labelled marker (anti-IgM) for m a n y different antigen systems. Likewise the biggest disadvantage of the direct RIA is the necessity of preparing a radioactive antigen for every illness studied. In view of the fact that at present only a few disease agents (e.g. rubella virus, toxoplasmosa) require routine diagnosis of IgM antibodies, this disadvantage is probably outweighed by the reliability and simplicity of the direct assay. Furthermore, the labelling of antigens with 3H for direct RIAs, or with enzymes for direct enzyme immunoassays, would enable the preparation and storage of large batches of antigens with very long shelf lives. ACKNOWLEDGEMENT I gratefully acknowledge the helpful support from Prof. Dr. med. Ulrich Krech.
273 REFERENCES Brunner, H. and R.M. Chanock, 1973, Proc. Soc. Exp. Biol. Med. 143, 97. Fey, H., H. Pfister, J. Messerli, N. Sturzenegger and F. Grolimund, 1976, Zbl. Vet. Med. B 23,269. Hobbs, J.R., 1970, Br. J. Hosp. Med. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194,495. Kenny, G.E. and J.T. Grayston, 1965, J. Immunol. 95, 19. Price, P.C., 1977, Doctoral thesis, University of Berne, Switzerland. Schmidt, N.J. and E.H. Lennette, 1970, J. Immunol. 105, 604. Stanworth, D.R. and M.W. Turner, 1973, in: Handbook of Experimental Immunology, ed. D.M. Weir (Blackwells, Oxford).
261
© Elsevier/North-Holland Biomedical Press
DIRECT RADIOIMMUNOASSAY FOR THE DETECTION OF IgM ANTIBODIES AGAINST M YCOPLASMA PNEUMONIAE
P.C. PRICE
Institute for Medical Microbiology, St. Gallen, Switzerland (Received 5 July 1979, accepted 2 October 1979)
A direct solid-phase radioimmunoassay is described for detecting IgM-class antibodies against Mycoplasma pneumoniae. The assay achieves a preliminary separation of IgM from other serum proteins by immunoadsorption to anti-IgM-coated wells in microtitre plates. The IgM is then tested for antigen specificity by measuring its ability to bind radiolabelled M. pneumoniae. Positive results are confirmed by retesting sera after treatment with 2-mercaptoethanol. The assay is specific for IgM-class antibodies and specific for M. pneumoniae. It is not affected by competitive inhibition from IgG and avoids the use of density gradient centrifugation or gel filtration to separate IgM from other immunoglobulins. It is sensitive, reproducible, rapid, simple and requires very little serum.
INTRODUCTION
The most c o m m o n l y used methods for detecting IgM antibodies against specified antigens are the indirect methods of immunofluorescence, radioimmunoassay (RIA) and enzyme immunoassay. These are usually two-step procedures. First, specific antibodies are allowed to bind to solid- or fluidphase antigens. The presence of IgM among the antigen-bound antibodies is then determined using anti-IgM labelled with fluorochrome, radioisotope or enzyme. A major problem associated with indirect methods is competitive inhibition of binding of IgM to antigen in the presence of antigen-specific IgG antibody. This is best overcome by separating IgM from IgG before measuring its antigen specificity. Physical methods of separation such as density gradient centrifugation or gel filtration are laborious and timeconsuming. A direct RIA for detecting IgM against specified antigens has been developed. IgM is first separated from other immunoglobulins using an immunochemical rather than physical method and is then tested directly for antigen specificity. As a model antigen, Mycoplasma pneumoniae was selected to develop the assay. It can be obtained in a clean, radioactive form (Brunner and Chanock, 1973), and serum is readily available from patients suffering from recent M. pneumoniae infections.
262 MATERIALS AND METHODS
Organisms Mycoplasma pneumoniae (Eaton agent) was obtained from Flow Laboratories, Irvine, Scotland. M. fermentans (strain PG-18) was obtained from the Research Reference Reagents Branch of the National Institute of Allergy and Infectious Diseases, Bethesda, MD, U.S.A. Growth medium and culture conditions Organisms were grown in mycoplasma broth base (BBL) containing 20% fresh horse serum, 0.5% lactalbumin hydrolysate (Difco), 1% glucose, 0.002% phenol red, 0.025% thallium acetate and 1000 U of Penicillin G/ml. This medium was adjusted to pH 8.1 and sterilized by membrane filtration. Incubation was at 37°C in rolling 1 litre transfusion bottles containing 400 ml of medium. An tigen preparation For the labelling of mycoplasma organisms, 20 pCi/ml of oleic acid-9, 10-3H (specific activity = 7.35 Ci/mmole) and 20 pCi/ml of palmitic acid-9, 10-3H (specific activity 23.5 Ci/mmole) were added to the medium on the day of inoculation. The radioactive fatty acids were obtained from New England Nuclear, Boston, MA, U.S.A. The medium was maintained at pH 8.1 -+ 0.5 by addition of 1 N NaOH. After 7--8 days, the mycoplasma were suspended in the growth medium and then centrifuged at 20,000 rev/min (Beckman rotor, 50.2 Ti) for 20 min. The pellet was washed twice in cold 0.1 M phosphate 0.15 M NaC1 pH 7.2 (PBS) using centrifugation as above. The [3H]antigens were then suspended into a volume of PBS equal to 1/50th of the original volume of medium and freeze-dried in 1 ml aliquots. Antigens were reconstituted as needed with sterile distilled water, diluted in fetal calf serum (FCS) and stored at 4 °C. Prior to use, light sonification was used to homogenize the antigen suspensions. The specific activities of the antigens were 9 X 106 cpm/mg for M. pneumoniae and 4 × 106 cpm/mg for M. fer. mentans. A lipid extract of M. pneumoniae for use as a complement-fixing (CF) antigen was prepared according to the m e t h o d of Kenny and Grayston (1965). This antigen was stabilized by the addition of bovine serum albumin (fraction V) (BSA V) (Armour, Eastbourne, U.K.) to a concentration of 2% and was freeze-dried in 1 ml amounts. A lipid extract was also obtained from [3H]M. pneumoniae using the above m e t h o d scaled down for 40 mg of [3H]organisms. N i n e t y ~ i g h t per cent of the 3H activity in the organisms was recovered in the lipid extract. Serum samples Over 35,000 serum samples submitted to the Institute for Medical Microbiology, St. Gallen, Switzerland, from patients (24,500) and healthy blood
263 donors (10,600) during the period July 1976--May 1977 were available for this study. Of these, 7447 were tested for the presence of CF antibodies against M. pneumoniae, 1141 of which were serial samples from patients. One h u n d r e d and ninety-one serum samples had CF antibodies at titres of ~>60. In this laboratory, a CF titre of ~>60 is considered to be a possible indication o f a recent infection with M. pneumoniae. Serial serum samples from 17 patients showed an increase in CF titre of 4-fold or greater, which is diagnostically indicative of a recent infection. In most cases, a description of the clinical s y m p t o m s of the patients was available. Sera were stored for long term at --20°C and for short term at 4 ° C.
Reference sera The following reference sera were used to optimize the test procedure. A negative reference serum was obtained by pooling 70 blood d o n o r sera k n o wn to contain no detectable CF antibodies against M. pneumoniae when tested at a dilution of 1 : 5. A weakly positive reference serum was obtained f r o m a p n e u m o n i a patient 11 days after onset of clinical symptoms. This serum had no detectable CF antibodies at a dilution of 1 : 10, but was consistently positive in the IgM RIA, giving a binding ratio (see RIA procedure below) of 5.5 -+ 1.1. An intermediately positive reference serum was obtained f r o m the same pa t i e nt 92 days after onset of clinical pneumonia. This serum had a CF titre of 120 and an IgM RIA binding ratio of 27.2 -+ 6.0 (binding ratios o f 40--60 were n o t u n c o m m o n for positive sera). Purification o f IgM IgM was purified using an e x t e n d e d version of the m e t h o d of Fey et al. (1976). The euglobulin fraction of a pool of 450 blood d o n o r sera was treated with ZnSO4 to precipitate IgG, subjected to sucrose density gradient centrifugation and c h r o m a t o g r a p h e d on Sephadex G-200. After concentration to 4 mg/ml, the.purified IgM was freeze-dried. 12sI radioiodination o f IgM Purified IgM was radioiodinated with [lZSI]Na (EIR, Wuerenlingen, Switzerland) using the chloramine-T m e t h o d of H unt er and G r e e n w o o d (1962). [12sI]IgM was stabilized by the addition of an equal volume of 4% BSA V in 0.05 M T r i s - ( h y d r o x y m e t h y l ) a m i n o m e t h a n e (Tris), 0.1% NAN3, pH 7.2 (TA). Removal of free 12sI- was achieved by anion-exchange chromatogr a p h y on DEAE Sephadex A-25 pre-equilibrated with 1% BSA V in TA. Some radioactive protein could be rinsed f r o m the col um n with the equilibration buffer, while [12sI]IgM was eluted using 1% BSA V in TA containing 0.25 M NaC1. A b o u t 5 atoms of 12sI were i ncorporat ed per molecule of IgM. Coating rnicro titre wells with an ti-IgM Anti-IgM serum was diluted in 0.05 M Tris buffer containing 0.15 M NaC1 and 0.1% NaN3 pH 7.2 (TSA). One hundr ed and fifty pl aliquots of this solu-
264 tion were added to r o u n d - b o t t o m e d wells in polyvinyl chloride microtitre plates (Cooke Engineering, Alexandria, VA, U.S.A.). The plates were then covered with polyester tape (Scotch tape Y 336, 3M, St. Paul, MN, U.S.A.) and stored at 4°C until used. For cont r ol purposes, some microtitre wells were coated with dilutions of normal rabbit serum.
RIA procedure After initial studies to determine optimal conditions, the following procedure was adopted. A n t i b o d y - c o a t e d wells were rinsed 5 times with TSA directly prior to use. One hundr e d pl amounts of sera diluted 1 : 10 in 10% FCS in TSA were pipetted in quadruplicate into wells which were then covered with polyester tape. After incubation for 2 h at 45°C in a water bath, the sera dilutions were removed by aspiration and the wells were rinsed 7 times with TSA. One hundr ed pl of [3H]antigen (1.5--2 × 10 s cpm) in FCS was then pipetted into each well. The wells were covered with tape and fu rth er incubated for 1 h. After aspiration of the u n b o u n d radioactive antigen and 10 rinses with TSA, the wells were dried with a hair drier, separated from each o th er by cutting with scissors and measured for ret ent i on of 3H. The negative reference serum was tested in quadruplicate or more in every assay. Results were expressed as binding ratios obtained by dividing the average cpm minus background of a sample by the average cpm minus background o f the negative reference serum.
2-Mercaptoethanol treatment Sera were added to an equal volume of 0.2 M 2-mercaptoethanol (2-ME) in PBS and incubated at 37°C for 1 h. The 2-ME-treated samples were then tested in the RIA. In some cases, sera were treated as above and in parallel with PBS containing no 2-ME. The 2-ME-treated and PBS-treated samples were then tested in parallel in the RIA.
Complement fixation test C o m p l e m e n t fixation (CF) tests were p e r f o r m e d using the microtitre p r o c e d u r e as described by Schmidt and L e n n e t t e (1970). CF titres are re po r ted as the reciprocal of the highest serum dilution giving complete fixation o f c o m p l e m e n t .
Measurement o f 3H and 12sI R e t e n t i o n o f [~H]M. pneumoniae antigens was measured by placing the dried and separated wells into 2 ml of xyl ene containing 0.36% Permablend III (Packard, IL, U.S.A.). Scintillation was measured using a Nuclear Chicago Isocap 300 (Des Plaines, IL, U.S.A.). Samples were c o u n t e d until an accuracy o f 2% was reached but n o t longer than 10 min. 12SIodine was measured using an MR 1032 aut om a t i c gamma c o u n t e r (W and W Electronics, Basle, Switzerland). Measurements were made for 5 min or until 50,000 counts had accumulated.
265 RESULTS
Selection o f anti-IgM serum for coating microtitre wells Fourteen different anti-IgM sera were used in increasing dilutions to coat wells and compared for their ability to retain [12sI]IgM. These antisera were obtained from 7 commercial suppliers and had been raised in rabbit, goat, sheep and swine. [12sI]IgM was incubated in the coated wells and, after aspiration and thorough rinsing, the wells were measured for retained [~2sI]IgM. Fig. 1 demonstrates the varying ability of 8 commercial antisera to retain [~2sI]IgM to coated wells. Antiserum H was only slightly better at IgM retention than normal rabbit serum I, while antisera A and C retained nearly 70% of the added [12S]IgM even at coating dilutions of I : 1600. For all further studies, antiserum A (rabbit anti-human-IgM, mu-chain-specific, lot number 0105, Dakopatts A/S, Denmark) was used at a dilution of 1 : 400 to coat wells. Such wells had a binding capacity of about 100 pg of IgM.
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Fig. 1. V a r y i n g ability of c o m m e r c i a l anti-IgM sera to b i n d [ 1251 ]IgM t o c o a t e d m i c r o t i t r e wells. M i c r o t i t r e wells were c o a t e d o v e r n i g h t at r o o m t e m p e r a t u r e w i t h 150 pl of increasing d i l u t i o n s (in T S A ) o f anti-IgM sera f r o m various c o m m e r c i a l suppliers ( A - - H ) a n d o f n o r m a l r a b b i t s e r u m (I). 6 0 0 , 0 0 0 c p m o f [12sI]IgM in 100 ~l T S A were t h e n i n c u b a t e d in the c o a t e d wells at r o o m t e m p e r a t u r e o v e r n i g h t . Wells were rinsed 10 t i m e s w i t h T S A a n d m e a s u r e d for b o u n d 12si.
266 Optimal incubation time and temperature for sera T h e o p t i m a l i n c u b a t i o n t i m e and t e m p e r a t u r e f o r s e r u m s a m p l e s in react i o n I ( s e p a r a t i o n o f IgM f r o m sera via i m m u n o a d s o r p t i o n t o a n t i - I g M - c o a t e d wells) (see Fig. 2) w e r e d e t e r m i n e d in t h e f o l l o w i n g m a n n e r . T h e r e f e r e n c e sera (see Materials a n d M e t h o d s ) w e r e d i l u t e d 1 : 10 in 20% FCS in T S A and i n c u b a t e d in c o a t e d wells f o r v a r y i n g p e r i o d s o f t i m e ( 1 - - 1 8 h) at various t e m p e r a t u r e s ( 4 - - 4 5 ° C ) . A f t e r rinsing, t h e wells w e r e i n c u b a t e d w i t h [3HIa n t i g e n . I t was generally f o u n d t h a t b i n d i n g ratios increased with increasing t e m p e r a t u r e a n d were less a f f e c t e d b y p r o l o n g i n g i n c u b a t i o n t i m e (Price, 1 9 7 7 ) . I n c u b a t i o n f o r 2 h at 4 5 ° C was used f o r r e a c t i o n I in all s u b s e q u e n t tests.
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267
Optimal dilution of sera T o d e t e r m i n e the best d i l u t i o n o f p a t i e n t s ' sera t o use r o u t i n e l y in the IgM RIA, 28 sera with CF titres ranging f r o m < 5 t o ~>240 were t e s t e d at serial 10-fold dilutions f r o m I : I to 1 : l 0 s (Price, 1 9 7 7 ) . Binding ratios were always highest at serum dilutions o f 1 : 10.
Optimal diluent for sera T h e r e f e r e n c e sera were t e s t e d at a dilution o f 1 : 10 in T S A which had been s u p p l e m e n t e d with 4% BSA V or with 20% calf serum, 20% h u m a n serum (M. pneumoniae sero-negative), 20% r a b b i t serum or 20% FCS. Addition o f 20% FCS to the serum d i l u e n t had a p r o n o u n c e d e f f e c t in raising binding ratios b y lowering the non-specific r e t e n t i o n o f [3H]antigen b y the negative r e f e r e n c e serum. T o d e t e r m i n e the best c o n c e n t r a t i o n o f FCS to be used in the serum diluent, the r e f e r e n c e sera were t e s t e d at a d i l u t i o n o f 1 : 10 in T S A s u p p l e m e n t e d with increasing c o n c e n t r a t i o n s o f FCS f r o m 1% to 100%. T e n per c e n t FCS in T S A was selected as the d i l u e n t for serum samples.
Optimal incubation time and temperature for [3H]antigen T h e o p t i m a l i n c u b a t i o n t i m e and t e m p e r a t u r e for the [3H]antigen in r e a c t i o n II ( d e t e r m i n a t i o n o f IgM specificity) (see Fig. 2) was d e t e r m i n e d in in the f o l l o w i n g m a n n e r . [3H]antigen was i n c u b a t e d for various periods o f t i m e (1--18 h) at various t e m p e r a t u r e s ( 4 - - 4 5 ° C ) in wells which had previo u s l y b e e n t r e a t e d for 2 h at 45°C with 1 : 10 dilutions o f the r e f e r e n c e sera in 10% FCS in TSA. Binding ratios reach their m a x i m u m values within 1 h at 45°C.
Optimal diluent for [3H]antigen In o r d e r to r e d u c e the non-specific binding o f [3H]antigen to wells in r e a c t i o n II, the e f f e c t o f adding FCS to the [3H]antigen d i l u e n t was studied. It was f o u n d essential t o suspend the [3H]antigen in 100% FCS t o o b t a i n m a x i m u m binding ratios.
Optimal specific activity of [3H]antigen T h e r e f e r e n c e sera were t e s t e d using [3H]antigen diluted in FCS to activities ranging f r o m 104 t o 106 c p m / t e s t . Binding ratios were highest w h e n the antigen was used diluted to a specific activity o f 2 × 10 s c p m / t e s t .
Comparison of 3H-organisms and 3H-lipid extract as antigens T h e r e f e r e n c e sera were t e s t e d using [3H]M. pneumoniae organisms and the lipid e x t r a c t o f [3H]M. pneumoniae as antigens. T h e use o f a suspension o f w h o l e organisms gives higher binding ratios t h a n o b t a i n e d using the lipid e x t r a c t as antigen. All r e f e r e n c e s to [3H]antigen in this s t u d y r e f e r t o a suspension o f [3H]M. pneumoniae organisms.
268
Reproducibility o f the IgM RIA T h e r e f e r e n c e negative serum and a serum which c o n s i s t e n t l y gave a high binding ratio were each tested 16 times. T h e positive serum yielded an average o f 1 1 , 0 0 0 + 563 c p m (10.4% o f the t o t a l c o u n t s in the test). T h e negative serum yielded an average o f 122 + 16 c p m (0.12% o f the total c o u n t s in the test).
Specificity o f the IgM RIA for M. pneumoniae One h u n d r e d and f i f t y - f o u r b l o o d d o n o r sera, which had no d e t e c t a b l e CF a n t i b o d i e s against M. pneumoniae w h e n tested at a dilution o f 1 : 5, were t e s t e d in the IgM RIA. T h e binding ratios o f these sera (Fig. 3A) are normally d i s t r i b u t e d a b o u t a value o f 1.1, and in all cases had values o f less t h a n 3.0. It was arbitrarily d e c i d e d t h a t a n y serum giving a binding ratio greater t h a n 3.0 w o u l d be c o n s i d e r e d positive. A binding ratio o f 3.0 lies 5 S.D.s above the average value o b t a i n e d f r o m these 154 sera. T h e binding ratios o b t a i n e d f r o m testing 99 sera with M. pneurnoniae CF titres ranging f r o m < 1 0 to ~>240 are s h o w n in Fig. 3B. T h e f r e q u e n c y o f binding ratios higher t h a n 3.0 increases with increasing CF titre. T w e n t y - o n e sera with binding ratios ranging f r o m 15 t o 39 were r e t e s t e d using [3HIM. fermentans as antigen. T h e resulting binding ratios were all l o w e r t h a n 3.0.
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Fig. 3. B i n d i n g r a t i o s o b t a i n e d for sera in t h e IgM R I A . S e r u m d i l u t i o n s o f 1 : 10 in 10% f o e t a l calf s e r u m in T S A w e r e i n c u b a t e d in q u a d r u p l i c a t e in a n t i - I g M - c o a t e d wells for 2 h at 4 5 ° C . A f t e r rinsing, 1.5 X 10 s c p m o f [3H]M. p n e u m o n i a e in 0.1 m l o f foetal calf s e r u m w e r e a d d e d t o e a c h well a n d i n c u b a t i o n was c o n t i n u e d for I h at 4 5 ° C . Wells were w a s h e d a n d m e a s u r e d f o r r e t a i n i n g 3H. A: b i n d i n g ratios o f 154 b l o o d d o n o r sera w h i c h h a d n o d e t e c t a b l e C F a n t i b o d i e s at a d i l u t i o n o f 1 : 5. B: c o r r e l a t i o n b e t w e e n b i n d i n g r a t i o s a n d C F a n t i b o d y titres o f 99 sera. C: e f f e c t o n b i n d i n g r a t i o s o f t r e a t m e n t o f sera w i t h 2-ME. T h e s e sera all h a d b i n d i n g ratios g r e a t e r t h a n 3.0 w h e n n o t t r e a t e d w i t h 2-ME (see B).
269
F o r t y - t w o sera with no detectable CF antibodies against M. pneumoniae b u t with high CF titres against antigens n o t related to M. pneumoniae were tested in the IgM RIA. None o f these sera gave binding ratios greater than 3.0.
Specificity o f the IgM RIA for IgM-class antibodies The effect o f 2-ME t r e a t m e n t on the binding ratios of sera can be seen in Fig. 3C. Here, all sera f r om Fig. 3B which yielded binding ratios greater than 3.0 were retested after t r e a t m e n t with 2-ME. IgM and IgG fractions of sera subjected to sucrose density gradient centrifugation were treated with 2-ME and with PBS and tested in parallel with the non-fractionated sera in the IgM RIA and in the CF test. The results obtained for two typical sera are shown in Table 1. Binding ratios obtained on testing the PBS-treated IgM fractions of sera are almost identical with those for the unf r a c t i ona t e d sera. Binding ratios for IgM fractions were always reduced to less than 3.0 after t r e a t m e n t with 2-ME. The IgG fractions were c o n t a m i n a t e d with IgM, which accounts for their high IgM RIA binding ratios which are reduced by 2-ME t r eat m e n t . The CF titres of IgM fractions were reduced by 2-ME t r e a t m e n t , but the CF titres of the IgG fractions were not.
Effect o f anti-M, pneumoniae IgG on the IgM RIA To determine the effect of specific IgG on the binding ratio of an IgMpositive serum, the following e x p e r i m e n t was perform ed. An anti-M.pneurnoniae IgM-positive serum (M) (CF titre ~>240, binding ratio, 42) was serially diluted 4-fold in a sero-negative serum (N) (CF titre <5, binding ratio, 1.0) and also in an IgG-positive, IgM-negative serum (G) (CF titre ~>240, binding ratio, 5; binding ratio n o t reduced by 2-ME treatment). The
TABLE 1 E F F E C T O F 2-ME T R E A T M E N T ON IgM R I A B I N D I N G R A T I O S A N D CF T I T R E S O F S U C R O S E D E N S I T Y G R A D I E N T IgG A N D IgM F R A C T I O N S S e r u m No.
Serum fraction Whole serum
IgM t r e a t e d with PBS
2-ME
IgG t r e a t e d w i t h PBS
2-ME
1
IgM R I A b i n d i n g ratio CF t i t e r
36.2 240
41.7 60
1.0 10
33.7 30
1.5 30
2
IgM R I A b i n d i n g r a t i o CF titer
62.8 240
61.5 60
1.4 10
36.1 30
1.3 30
270
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Fig. 4. E f f e c t o f anti-M, pneumoniae IgG on the IgM RIA. A n anti-M, pneurnoniae IgMpositive s e r u m (M) was serially diluted 4-fold in an IgG-positive, IgM-negative s e r u m (G) and in a sero-negative s e r u m (N). The sera and m i x t u r e s of sera were t h e n t r e a t e d with PBS and w i t h 2-ME and t e s t e d in the IgM RIA. Samples t r e a t e d w i t h PBS = closed symbols ( 0 , . , 4, , , A). S a m p l e s t r e a t e d with 2-ME = o p e n s y m b o l s (©, D, 4, ~, L~).
3 sera and t h e m i x t u r e s o f sera were t h e n treated w i t h 2-ME and w i t h PBS and t e s t e d in t h e IgM R I A . S e r u m G (Fig. 4) gave a l o w binding ratio w h i c h w a s n o t r e d u c e d by 2-ME t r e a t m e n t . S e r u m M gave a high binding ratio w h i c h w a s r e d u c e d to less than 3.0 by 2-ME. S e r u m N gave a binding ratio o f
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120
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210
240
Days after onset of pneumonia Fig. 5. Persistence o f IgM-class a n t i b o d i e s against M. pneumoniae. Persistence o f IgM-class a n t i b o d i e s against M. pneumoniae in successive s e r u m samples f r o m 3 p n e u m o n i a p a t i e n t s as d e t e r m i n e d by the IgM RIA. P B S - t r e a t e d sera, A, 0, I : 2-ME-treated sera, A, o, D.
271 less than 3.0. The addition of serum G to serum M had practically no more effect on reducing the binding ratio than the effect of addition of serum N (Fig. 4).
Persistence o f anti-M, pneumoniae IgM The persistence of IgM antibodies against M. pneumoniae was studied by testing successive serum samples from 3 pneumonia patients. Fig. 5 shows the binding ratios obtained from testing these sera after 2-ME treatment and PBS treatment. Anti-M. pneumoniae IgM persisted for over 6 months when monitored by this method. DISCUSSION The direct solid-phase RIA has been tailored to the detection of antigenspecific IgM in serum by taking advantage of the following. IgM is catabolized faster than IgG (fractional catabolic rate in % intravascular pool/day for IgM --- 18.0, for IgG = 6.7) (Stanworth and Turner, 1973). A consequence of this is that following a recent infection the overall serum concentration of IgM temporarily increases (Hobbs, 1970), and a relatively high proportion of this IgM is specific for the agent causing illness. When patients' sera are incubated in anti-IgM-coated microtitre wells, even though only a small a m o u n t of the total serum IgM is retained (100 pg), a large proportion of it is specific for the agent of illness. When challenged with radiolabelled antigen, this IgM is capable of binding amounts of antigen which far exceed the a m o u n t of antigen which non-specifically binds to the solid phase. Hence, differentiation of positive and negative sera is achieved. The long half-life of IgG results in its accumulation at relatively high levels in serum (10--20 times the concentration of IgM in healthy adults). The specific IgG produced following a recent infection becomes diluted in preexisting IgG which has no specificity for the disease agent. Therefore, in the IgM RIA, traces of IgG which non-specifically bind to the solid phase should, for the most part, not be specific toward the agent causing illness. There should be little retention of radioactive antigen due to specific IgG which has bound non-specifically to the solid phase. In practice, however, it was found that some sera with high CF titres gave moderate IgM RIA binding ratios which could not be reduced by 2-ME treatment, and were probably the result of antigen retention by IgG. It was, therefore, f o u n d necessary to re-test after t r e a t m e n t with 2-ME all sera giving IgM RIA binding ratios higher than 3.0. A reduction in binding ratio of 3-fold or greater was considered proof that the detected antibody activity belonged to the IgM class. Competitive inhibition by specific IgG was n o t expected to be a problem in the direct IgM RIA, and it was not. The addition of an anti-M, pneumoniae IgG-positive serum to an IgM-positive serum had practically no more effect on reducing the IgM RIA binding ratio than addition of M. pneumoniae sero-negative serum. Even at a ratio of 1 2 8 0 : 1 (IgG-positive
272 serum : IgM-positive serum), no competitive inhibition of binding of IgM was observed. This, of course, was expected since the first reaction step of the direct m e t h o d removes IgG from the IgM to be tested. Absorption of IgM to a solid-phase anti-IgM, where the solid phase is the reaction container (i.e. microtitre wells) achieves a rapid and convenient separation of IgM from IgG. It proved to be extremely important to select a good source of anti-IgM for coating the solid phase. Most commercially available sera tested were demonstrably inferior to the antiserum selected for this study in their ability to bind IgM to the solid phase. The coated wells used in this study had a binding capacity of 100 pg of IgM. It was found essential to dilute the radioactive antigen in 100% FCS to obtain m a x i m u m binding ratios for positive sera. This resulted in a lowering of the a m o u n t of radioactive antigen non-specifically bound to the solid phase from the negative reference sera. [3H]M. pneumoniae organisms provided a better antigen than the lipid extract of organisms. This is probably an amplification effect, due to the fact that retention of one 3H-labelled organism results in an overall greater retention of 3H than is achieved by the specific retention of one [3H] glycerophospholipid miscile. The IgM RIA proved to be very sensitive. Sera which gave low binding ratios (e.g. 3.1, 3.3, 4.8) when tested undiluted continued to give binding ratios greater than 3.0 at dilutions of up to 1 : 10,000. It is unlikely, therefore, that the assay would give false negative results due to low sensitivity. The test is specific with regard to detection of antibodies against M. pneumoniae. No sera giving high binding ratios were found among 154 blood donors or 42 patients whose sera had high CF antibody titres against antigens from 10 viruses and rickettsiae. Furthermore, none of 21 sera, giving high binding ratios in the M. pneumoniae IgM RIA, gave binding ratios higher than 3.0 when tested against radioactive M. fermentans. Anti-M. pneumoniae IgM appears to persist for over 6 months when monitored by the IgM RIA. There does, however, appear to be a big drop in binding ratios within 2--3 months following onset of clinical symptoms. Perhaps the biggest advantage of indirect IgM methods is the need for only one labelled marker (anti-IgM) for m a n y different antigen systems. Likewise the biggest disadvantage of the direct RIA is the necessity of preparing a radioactive antigen for every illness studied. In view of the fact that at present only a few disease agents (e.g. rubella virus, toxoplasmosa) require routine diagnosis of IgM antibodies, this disadvantage is probably outweighed by the reliability and simplicity of the direct assay. Furthermore, the labelling of antigens with 3H for direct RIAs, or with enzymes for direct enzyme immunoassays, would enable the preparation and storage of large batches of antigens with very long shelf lives. ACKNOWLEDGEMENT I gratefully acknowledge the helpful support from Prof. Dr. med. Ulrich Krech.
273 REFERENCES Brunner, H. and R.M. Chanock, 1973, Proc. Soc. Exp. Biol. Med. 143, 97. Fey, H., H. Pfister, J. Messerli, N. Sturzenegger and F. Grolimund, 1976, Zbl. Vet. Med. B 23,269. Hobbs, J.R., 1970, Br. J. Hosp. Med. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194,495. Kenny, G.E. and J.T. Grayston, 1965, J. Immunol. 95, 19. Price, P.C., 1977, Doctoral thesis, University of Berne, Switzerland. Schmidt, N.J. and E.H. Lennette, 1970, J. Immunol. 105, 604. Stanworth, D.R. and M.W. Turner, 1973, in: Handbook of Experimental Immunology, ed. D.M. Weir (Blackwells, Oxford).