Journal of Immunological Methods, 13 (1976) 367--380
367
© Elsevier/North-Holland Biomedical Press
A RAPID IMMUNOFLUORESCENT PROCEDURE FOR THE D E T E C T I O N O F S P E C I F I C IgG A N D IgM A N T I B O D Y IN S E R A U S I N G S T A P H Y L O C O C C U S A U R E U S A N D L A T E X - I g G AS A B S O R B E N T S
SHIREEN CHANTLER, ELIZABETH DEVRIES, P.R. ALLEN and B.A.L. HURN Wellcome Research Laboratories, Langley Court, Beckenham, Kent, U.K.
(Received 26 July 1976, accepted 18 August 1976)
Sera from adults with toxoplasmosis and individuals with anti nuclear antibodies were examined for the presence of specific IgM and IgG antibodies and for the occurrence of anti-immunoglobulin ('RF') activity both before and after absorption with freeze-dried preparations of protein A-containing Staphylococcus aureus. Such absorption removed most of the IgG present in sera without interfering with the detection of IgM antibody by immunofluorescence, but was relatively ineffective in the removal of 'RF'. 'RF' activity, demonstrable by slide agglutination, was removed by absorption with latex coated with IgG. In order to prevent the possibility of false positive IgM staining in immunofluorescence tests, it is concluded that sequential absorption with latex-IgG particles and protein A-positive staphylococci is essential. Such absorption is technically simple and can be performed on small volumes of serum.
INTRODUCTION T h e d e m o n s t r a t i o n o f specific IgM a n t i b o d y b y i m m u n o f l u o r e s c e n c e in adult and n e o n a t a l sera has b e e n generally a c c e p t e d as indicating active or r e c e n t i n f e c t i o n (Baublis and B r o w n , 1 9 6 8 ; C o h e n et al., 1 9 6 8 ; R e m i n g t o n et al. 1 9 6 8 ; S c o t t i and L o g a n , 1 9 6 8 ; C r a d o c k - W a t s o n et al., 1 9 7 2 ; Haire and H a d d e n , 1 9 7 2 ; Matossian et al., 1 9 7 2 ; R e i m e r et al., 1 9 7 5 ) . A l t h o u g h this test is simple t o p e r f o r m and m a k e s possible the rapid analysis o f large n u m bers o f sera, several limitations have b e c o m e a p p a r e n t in r e c e n t years. Firstly, IgG a n t i b o d i e s can m a s k the d e t e c t i o n o f specific IgM a n t i b o d i e s in unf r a c t i o n a t e d s e r u m samples, p r e s u m a b l y as a result o f c o m p e t i t i v e i n h i b i t i o n b e t w e e n IgG and IgM a n t i b o d i e s for t h e same antigenic sites ( C o h e n et al., 1 9 6 7 ; R e i m e r et al., 1 9 7 5 ; C r a d o c k - W a t s o n e t al., 1 9 7 6 ) ; and s e c o n d l y , false positive IgM staining can o c c u r as a result o f IgM a n t i i m m u n o g l o b u l i n ( ' R F ' ) reacting with specific IgG a n t i b o d y w h i c h has c o m b i n e d w i t h t h e antigen u n d e r test ( H o l b o r o w a n d J o h n s o n , 1 9 6 5 ; Fraser et al., 1 9 7 1 ; Shirodaria e t al., 1 9 7 3 ; R e i m e r et al., 1 9 7 5 ) . Fraser et al. ( 1 9 7 1 ) have s h o w n t h a t a b s o r p t i o n with aggregated IgG r e m o v e s a n t i - i m m u n o g l o b u l i n activity and ' s e c o n d a r y ' IgM staining, a l t h o u g h less s a t i s f a c t o r y results have b e e n r e p o r t e d b y Gispen et al. (1975). Such ab-
368 sorption will not prevent the possibility of false negative IgM staining resulting from competitive inhibition by specific IgG antibody. Fractionation of test sera by gel filtration or sucrose density gradient centrifugation reduces the probability of both competitive inhibition and positive IgM responses due to ' R F ' activity (Cradock-Watson et al., 1972; Remington and Desmonts, 1973; Reimer et al., 1975). These techniques are, however, time consuming and severely limit the number of samples that can be handled. There is, therefore, a great need for a simple rapid procedure for separating IgG and IgM antibody activity in test sera and thereby eliminating the factors which reduce the diagnostic significance of specific tests for detection of IgM. It has been known for some years that the Fc region of human IgG combines with protein A, present on the cell wall of certain strains of Staphylococcus aureus, whereas other serum proteins do not react (Forsgren and Sjbquist, 1966; Kronvall and Williams, 1969; Kronvall and Frommel, 1970). Absorption of test sera with protein A-containing staphylococci might, therefore, provide a simple fractionation procedure. Examination of unabsorbed and absorbed sera for the presence of specific IgG and IgM by immunofluorescence might then overcome the technical limitations outlined above. The objectives of this study were firstly to examine the effect of absorption with protein A positive staphylococci upon the levels of total IgG and IgM in whole sera; secondly, to determine whether appropriate absorption was effective in removing specific IgG antibody without impairing the ease of specific IgM antibody detection and thirdly, to see whether procedures which would maximally remove IgG would also effectively remove ' R F ' by secondary absorption onto the absorbed IgG. MATERIALS AND METHODS Serum samples Samples of adult human sera known to contain anti nuclear antibodies (ANA) or anti Toxoplasma antibodies were used in this study. Preparation of staphylococcal suspensions Protein A positive S. aureus Cowen I strain and protein A negative strain, Wood 46, were grown in broth culture as described by Kronvall (1973). The cultures were formalinised, washed in saline and resuspended in phosphate buffered saline to give a 10% suspension v/v. The bacterial suspension was heated to 80°C, cooled rapidly and further washed in phosphate buffered saline (PBS). The bacteria were then resuspended in PBS containing 0.1% sodium azide and 2% mannitol to give a 10% suspension v/v, and freeze dried
369 in 1 ml or 10 ml aliquots. Freeze dried preparations were stored at 4°C until used.
Absorption of test sera with staphylococcal preparations Freeze dried preparations of staphylococci were reconstituted in distilled water to give a 10% suspension. Following centrifugation on a bench centrifuge, the supernatant was discarded and the sediment was washed in phosphate buffered saline (PBS). The sediment from 1 ml of 10% suspension (100 pl packed volume of staphylococci) was mixed with an equal volume of test serum at a dilution of 1 in 4 or 1 in 5. The co n ten ts of the tube were t h o r o u g h l y mixed on a vortex shaker and then allowed to stand at r o o m t e m p e r a t u r e for a few minutes. Following centrifugation, the supernatant serum was removed for furt her examination. The total IgG and IgM levels, the specific a n t i b o d y titres and the presence o f anti-immunoglobulin ( ' R F ' ) in unabsorbed and absorbed sera were examined.
Quantitation of IgG and IgM Quantitation was p e r f o r m e d on pr ecoat ed 8 cm glass slides by single radial diffusion in agar containing either sheep anti-human IgG or sheep antih u m a n IgM sera (Wellcome Reagents Ltd.). In all experiments, standard curves were obtained by using varying c onc e nt rat i ons of purified hum an IgG (obtained f r o m a pool of normal human serum) and purified h u m a n IgM (obtained from a pool of IgM from two m y e l o m a sera and normal human serum). Duplicate tests were p e r f o r m e d on each test sample. Following incubation at r oom t em pe r at ur e for 48--72 h the plates were t h o r o u g h l y washed and allowed to dry. The dried plates were stained in 0.3% napthalene black in 2% acetic acid. Differentation was p e r f o r m e d in 2% acetic acid, followed by washes in distilled water. The diameter of precipitation rings were read from the dried, stained plates. The c o n c e n t r a t i o n of IgG or IgM per ml o f test sample was calculated from the standard curve.
Immunofluorescent tests Anti nuclear antibodies (ANA ) Anti nuclear antibodies in test sera were d e t e c t e d by the indirect immunofluorescent p r o ce dur e utilising cryostat sections of rat liver on multispot slides. Duplicate slides were first incubated with serial dilutions of test sera. Following incubation and washing, one slide was treated with the working dilution of immunospecific fluorescein labelled anti hum an IgG and the ot her with fluorescein labelled anti h u m a n IgM (Wellcome Reagents Ltd.).
370
Anti Toxoplasma antibodies Freeze dried preparations of Toxoplasma gondii antigen {Wellcome Reagents Ltd.) were reconstituted with distilled water then pipetted ont o areas on a multispot slide. Excess antigen was removed and the remaining antigen film was air dried and fixed in m e t h a n o l for 10 min. Subsequent t r e a t m e n t was analogous to that used in the ANA test with the except i on t h a t the slides were counterstained in Evans blue solution (1 in 5000) prior to the final wash. The immunospecificity of the fluorescent reagents was established by direct and indirect i m m u n o f l u o r e s c e n t tests (Chantler and Haire, 1972). A Leitz O r t h o l u x incident light microscope equipped with a HBO 200 lamp, exciter filters 2 X KP 490, barrier filter 515 and 40 X objective NA 0.65 was used t h r o u g h o u t this study.
Detection o f anti-immunoglobulin ('RF') activity Detectio n o f R F activity in test samples was p e r f o r m e d by slide agglutination using latex coated with denat ur e d IgG (Rheum a Wellcotest). Serum dilutions were made in phos pha t e buffered saline (PBS).
Absorption o f test samples with Latex-IgG Latex particles (0.6 p diameter) were coated with denatured human IgG (2.5 mg/ml latex). The particle size of latex was greater than t hat used in R h e u m a Wellcotest in order to facilitate sedimentation. One volume of 1% suspension was centrifuged and the supernatant discarded. The sediment was washed in a large volume of PBS and recentrifuged. An equal volume of 1 in 4 or 1 in 5 diluted test serum was added to the sediment, mixed and allowed to stand at r o o m t e m p e r a t u r e for 10 min. Following a further centrifugation the supernatant serum was removed and tested for anti-immunoglobulin activity by slide agglutination and for the presence of specific ant i body activity by immunofluorescence. RESULTS
Total IgG and IgM concentration in sera following absorption with protein A containing staphylococcal preparations Eighteen d if f er ent serum samples were absorbed with an equal volume of packed protein A positive staphylococci. The concentrations of IgG and IgM in unabsorbed and absorbed sera were calculated from a total of 33 tests perf o r m e d on these sera. T he results obtained with representative sera are shown in table 1. Although the calculated c o n c e n t r a t i o n of IgG and IgM in individual sera showed some variation in experiments p e r f o r m e d on different occasions, the percentage uptake of IgG remained relatively constant. An
371 TABLE 1 Total IgG and IgM levels in unabsorbed sera and sera absorbed with protein A positive Staphylococcal preparations. Serum
AL5 AL5 AUS
Absorption
-A +re -A +ve - -
A +ve AUS SW 259 SW 710 SW 121 AL 3 H
L
*
118
- -
A -A -A -A -A
+ve +ve +ve +re +ve
- -
A +ve -A + ve
IgG
IgM
mg/ml
% uptake
mg/ml
% uptake
18.0 0.54 14.3 0.48 11.2 0.39 10.8 0.48 13.7 0.63 14.3 0.39 13.5 0.54 20.0 0.39 13.6 0.62 15.4 0.48
97.0
1.5 0.59 0.91 0.43 2.01 0.95 1.55 0.67 1.63 0.92 1.65 1.08 1.03 0.52 0.53 0.29 19.6 6.6 2.62 1.06
60.6
96.5 96.5 95.6 95.4 97.3 96.0 98.05 95.44 96.88
51.6 52.3 56.4 43.45 34.54 49.5 41.57 66.3 59.54
* Sample with high IgM level. u n e x p e c t e d f i n d i n g was t h e a p p a r e n t l y high p r o p o r t i o n o f IgM r e m o v e d b y a b s o r p t i o n . If, h o w e v e r , t h e a b s o l u t e a m o u n t r e m o v e d is c o n s i d e r e d , it is c l e a r t h a t t h e r e m o v a l o f IgG b y p r o t e i n A p o s i t i v e s t a p h y l o c o c c i is c o n s i d e r a b l y m o r e e f f e c t i v e t h a n t h a t o f IgM ( t a b l e s 1 a n d 2). In o r d e r t o see w h e t h e r IgM r e m o v a l was n o n - s p e c i f i c , f o u r sera w e r e absorbed with protein A positive and protein A negative staphylococcal p r e p a r a t i o n s . T h e IgG a n d IgM c o n t e n t o f t h e s e sera are s h o w n in fig. 1. B o t h I g G a n d IgM levels w e r e a f f e c t e d b y a b s o r p t i o n w i t h p r o t e i n A n e g a t i v e s t a p h y l o c o c c i b u t t h e a m o u n t r e m o v e d v a r i e d g r e a t l y w i t h i n d i v i d u a l sera, u n l i k e t h e s t r i k i n g a n d c o n s i s t e n t r e d u c t i o n in I g G levels t h a t r e s u l t e d f r o m a b s o r p t i o n w i t h p r o t e i n A +ve s t a p h y l o c o c c i . T h e d a t a o b t a i n e d f r o m M a n c i n i t e s t s p e r f o r m e d o n all sera are s u m m a rised in t a b l e 2.
Effect of staphylococcal absorption upon specific IgG and IgM antibody reac rio ns Since p r o t e i n A positive s t a p h y l o c o c c i had been shown to absorb approxim a t e l y 40% o f t o t a l IgM f r o m s e r u m s a m p l e s a n d p r o t e i n A n e g a t i v e p r e p a r a -
372 TABLE 2 E f f e c t o f a b s o r p t i o n w i t h p r o t e i n A positive a n d negative S t a p h y l o c o c c a l p r e p a r a t i o n s u p o n t h e u p t a k e o f IgG a n d IgM in w h o l e sera. Absorption with:-
No. o f s a m p l e s % u p t a k e o f IgG A m o u n t (rag) o f IgG r e m o v e d b y 100 ul packed staphylococci No. o f samples %uptakeofIgM A m o u n t (mg) o f IgM r e m o v e d b y 100 t~l packed staphylococci
A positive
A negative
33 94.51 1.34
4 36.8 0.45
+ 4.9 _+ 0.49
27 4 6 . 5 3 + 20.9 0 . 0 5 5 + 0.04
lgG
12
10
4 23.3 + 21.4 0.019 + 0.006
|gG
I£G
I m [
8
--
6
--
E
± 21.9 + 0.34
m \1 \1
&l \\
q 2
I~M
IgM
\\
IAM
--
SHRUM
1
SE]~UM
2
SERUM
3
SERUM
A
Fig. 1. IgG a n d IgM levels in f o u r u n a b s o r b e d sera a n d f o l l o w i n g a b s o r p t i o n w i t h p r o t e i n A positive or p r o t e i n A negative S t a p h y l o c o c c a l p r e p a r a t i o n s , cs, U n a b s o r b e d sera; [], sera a b s o r b e d w i t h p r o t e i n A - - r e S t a p h y l o c o c c i ; m, sera a b s o r b e d w i t h p r o t e i n A +re S t a p h ylococci.
373 tions had r e m o v e d variable amounts of b o t h IgM and IgG, preliminary experiments were c o n d u c t e d to see w he t he r absorption would significantly influence the titre of antibodies d e t e c t e d by i m m u n o f l o u r e s c e n t procedures. F o u r sera taken f r om adult patients with Toxoplasma infections were absorbed with either protein A positive or protein A negative staphylococcal preparations. These absorbed sera t oge t h er with unabsorbed sera were titrated for the presence o f specific IgG and IgM antibodies. The results of these tests are shown in table 3. This data shows t h a t absorption with prot ei n A positive staphylococci selectively reduces the specific IgG a n t i b o d y titre with no apparent reduction in th e IgM titre. F u r t h e r m o r e , despite the removal of variable am ount s o f IgG and IgM by protein A negative staphylococcal absorption, demonstrable by quantitative radial diffusion, there was little effect u p o n specific a n t i b o d y titres. In subsequent experiments only protein A positive staphylococci were used for absorption.
Sera from adults with anti Toxoplasma antibody T w e n t y two serum samples were absorbed with prot ei n A positive staphylococcal preparations, following which the specific a n t i b o d y titre was evaluated by i m m u n o f l u o r e s c e n c e and the presence of ' R F ' activity by slide agglutination with latex-IgG. Some of the results are tabulated in table 4. In all experiments absorption resulted in a substantial decrease in specific IgG titre w i t h o u t diminishing the IgM response. In all but two test samples the ' R F ' activity was either absent or low in the initial dilution tested. Absorp-
TABLE 3 Specific IgG and IgM Toxoplasma antibody titres in unabsorbed adult sera and in sera absorbed with eiter protein A positive or protein A negative Staphylococci. Sera
AUS KF4 AL5 AL5
Absorption
-A +ve A --ve --
A A -A A -A A
+ve --re
+re --ve +re --re
* Reciprocal of titre.
Immunofluorescence titre * IgG
IgM
512--1024
367
© Elsevier/North-Holland Biomedical Press
A RAPID IMMUNOFLUORESCENT PROCEDURE FOR THE D E T E C T I O N O F S P E C I F I C IgG A N D IgM A N T I B O D Y IN S E R A U S I N G S T A P H Y L O C O C C U S A U R E U S A N D L A T E X - I g G AS A B S O R B E N T S
SHIREEN CHANTLER, ELIZABETH DEVRIES, P.R. ALLEN and B.A.L. HURN Wellcome Research Laboratories, Langley Court, Beckenham, Kent, U.K.
(Received 26 July 1976, accepted 18 August 1976)
Sera from adults with toxoplasmosis and individuals with anti nuclear antibodies were examined for the presence of specific IgM and IgG antibodies and for the occurrence of anti-immunoglobulin ('RF') activity both before and after absorption with freeze-dried preparations of protein A-containing Staphylococcus aureus. Such absorption removed most of the IgG present in sera without interfering with the detection of IgM antibody by immunofluorescence, but was relatively ineffective in the removal of 'RF'. 'RF' activity, demonstrable by slide agglutination, was removed by absorption with latex coated with IgG. In order to prevent the possibility of false positive IgM staining in immunofluorescence tests, it is concluded that sequential absorption with latex-IgG particles and protein A-positive staphylococci is essential. Such absorption is technically simple and can be performed on small volumes of serum.
INTRODUCTION T h e d e m o n s t r a t i o n o f specific IgM a n t i b o d y b y i m m u n o f l u o r e s c e n c e in adult and n e o n a t a l sera has b e e n generally a c c e p t e d as indicating active or r e c e n t i n f e c t i o n (Baublis and B r o w n , 1 9 6 8 ; C o h e n et al., 1 9 6 8 ; R e m i n g t o n et al. 1 9 6 8 ; S c o t t i and L o g a n , 1 9 6 8 ; C r a d o c k - W a t s o n et al., 1 9 7 2 ; Haire and H a d d e n , 1 9 7 2 ; Matossian et al., 1 9 7 2 ; R e i m e r et al., 1 9 7 5 ) . A l t h o u g h this test is simple t o p e r f o r m and m a k e s possible the rapid analysis o f large n u m bers o f sera, several limitations have b e c o m e a p p a r e n t in r e c e n t years. Firstly, IgG a n t i b o d i e s can m a s k the d e t e c t i o n o f specific IgM a n t i b o d i e s in unf r a c t i o n a t e d s e r u m samples, p r e s u m a b l y as a result o f c o m p e t i t i v e i n h i b i t i o n b e t w e e n IgG and IgM a n t i b o d i e s for t h e same antigenic sites ( C o h e n et al., 1 9 6 7 ; R e i m e r et al., 1 9 7 5 ; C r a d o c k - W a t s o n e t al., 1 9 7 6 ) ; and s e c o n d l y , false positive IgM staining can o c c u r as a result o f IgM a n t i i m m u n o g l o b u l i n ( ' R F ' ) reacting with specific IgG a n t i b o d y w h i c h has c o m b i n e d w i t h t h e antigen u n d e r test ( H o l b o r o w a n d J o h n s o n , 1 9 6 5 ; Fraser et al., 1 9 7 1 ; Shirodaria e t al., 1 9 7 3 ; R e i m e r et al., 1 9 7 5 ) . Fraser et al. ( 1 9 7 1 ) have s h o w n t h a t a b s o r p t i o n with aggregated IgG r e m o v e s a n t i - i m m u n o g l o b u l i n activity and ' s e c o n d a r y ' IgM staining, a l t h o u g h less s a t i s f a c t o r y results have b e e n r e p o r t e d b y Gispen et al. (1975). Such ab-
368 sorption will not prevent the possibility of false negative IgM staining resulting from competitive inhibition by specific IgG antibody. Fractionation of test sera by gel filtration or sucrose density gradient centrifugation reduces the probability of both competitive inhibition and positive IgM responses due to ' R F ' activity (Cradock-Watson et al., 1972; Remington and Desmonts, 1973; Reimer et al., 1975). These techniques are, however, time consuming and severely limit the number of samples that can be handled. There is, therefore, a great need for a simple rapid procedure for separating IgG and IgM antibody activity in test sera and thereby eliminating the factors which reduce the diagnostic significance of specific tests for detection of IgM. It has been known for some years that the Fc region of human IgG combines with protein A, present on the cell wall of certain strains of Staphylococcus aureus, whereas other serum proteins do not react (Forsgren and Sjbquist, 1966; Kronvall and Williams, 1969; Kronvall and Frommel, 1970). Absorption of test sera with protein A-containing staphylococci might, therefore, provide a simple fractionation procedure. Examination of unabsorbed and absorbed sera for the presence of specific IgG and IgM by immunofluorescence might then overcome the technical limitations outlined above. The objectives of this study were firstly to examine the effect of absorption with protein A positive staphylococci upon the levels of total IgG and IgM in whole sera; secondly, to determine whether appropriate absorption was effective in removing specific IgG antibody without impairing the ease of specific IgM antibody detection and thirdly, to see whether procedures which would maximally remove IgG would also effectively remove ' R F ' by secondary absorption onto the absorbed IgG. MATERIALS AND METHODS Serum samples Samples of adult human sera known to contain anti nuclear antibodies (ANA) or anti Toxoplasma antibodies were used in this study. Preparation of staphylococcal suspensions Protein A positive S. aureus Cowen I strain and protein A negative strain, Wood 46, were grown in broth culture as described by Kronvall (1973). The cultures were formalinised, washed in saline and resuspended in phosphate buffered saline to give a 10% suspension v/v. The bacterial suspension was heated to 80°C, cooled rapidly and further washed in phosphate buffered saline (PBS). The bacteria were then resuspended in PBS containing 0.1% sodium azide and 2% mannitol to give a 10% suspension v/v, and freeze dried
369 in 1 ml or 10 ml aliquots. Freeze dried preparations were stored at 4°C until used.
Absorption of test sera with staphylococcal preparations Freeze dried preparations of staphylococci were reconstituted in distilled water to give a 10% suspension. Following centrifugation on a bench centrifuge, the supernatant was discarded and the sediment was washed in phosphate buffered saline (PBS). The sediment from 1 ml of 10% suspension (100 pl packed volume of staphylococci) was mixed with an equal volume of test serum at a dilution of 1 in 4 or 1 in 5. The co n ten ts of the tube were t h o r o u g h l y mixed on a vortex shaker and then allowed to stand at r o o m t e m p e r a t u r e for a few minutes. Following centrifugation, the supernatant serum was removed for furt her examination. The total IgG and IgM levels, the specific a n t i b o d y titres and the presence o f anti-immunoglobulin ( ' R F ' ) in unabsorbed and absorbed sera were examined.
Quantitation of IgG and IgM Quantitation was p e r f o r m e d on pr ecoat ed 8 cm glass slides by single radial diffusion in agar containing either sheep anti-human IgG or sheep antih u m a n IgM sera (Wellcome Reagents Ltd.). In all experiments, standard curves were obtained by using varying c onc e nt rat i ons of purified hum an IgG (obtained f r o m a pool of normal human serum) and purified h u m a n IgM (obtained from a pool of IgM from two m y e l o m a sera and normal human serum). Duplicate tests were p e r f o r m e d on each test sample. Following incubation at r oom t em pe r at ur e for 48--72 h the plates were t h o r o u g h l y washed and allowed to dry. The dried plates were stained in 0.3% napthalene black in 2% acetic acid. Differentation was p e r f o r m e d in 2% acetic acid, followed by washes in distilled water. The diameter of precipitation rings were read from the dried, stained plates. The c o n c e n t r a t i o n of IgG or IgM per ml o f test sample was calculated from the standard curve.
Immunofluorescent tests Anti nuclear antibodies (ANA ) Anti nuclear antibodies in test sera were d e t e c t e d by the indirect immunofluorescent p r o ce dur e utilising cryostat sections of rat liver on multispot slides. Duplicate slides were first incubated with serial dilutions of test sera. Following incubation and washing, one slide was treated with the working dilution of immunospecific fluorescein labelled anti hum an IgG and the ot her with fluorescein labelled anti h u m a n IgM (Wellcome Reagents Ltd.).
370
Anti Toxoplasma antibodies Freeze dried preparations of Toxoplasma gondii antigen {Wellcome Reagents Ltd.) were reconstituted with distilled water then pipetted ont o areas on a multispot slide. Excess antigen was removed and the remaining antigen film was air dried and fixed in m e t h a n o l for 10 min. Subsequent t r e a t m e n t was analogous to that used in the ANA test with the except i on t h a t the slides were counterstained in Evans blue solution (1 in 5000) prior to the final wash. The immunospecificity of the fluorescent reagents was established by direct and indirect i m m u n o f l u o r e s c e n t tests (Chantler and Haire, 1972). A Leitz O r t h o l u x incident light microscope equipped with a HBO 200 lamp, exciter filters 2 X KP 490, barrier filter 515 and 40 X objective NA 0.65 was used t h r o u g h o u t this study.
Detection o f anti-immunoglobulin ('RF') activity Detectio n o f R F activity in test samples was p e r f o r m e d by slide agglutination using latex coated with denat ur e d IgG (Rheum a Wellcotest). Serum dilutions were made in phos pha t e buffered saline (PBS).
Absorption o f test samples with Latex-IgG Latex particles (0.6 p diameter) were coated with denatured human IgG (2.5 mg/ml latex). The particle size of latex was greater than t hat used in R h e u m a Wellcotest in order to facilitate sedimentation. One volume of 1% suspension was centrifuged and the supernatant discarded. The sediment was washed in a large volume of PBS and recentrifuged. An equal volume of 1 in 4 or 1 in 5 diluted test serum was added to the sediment, mixed and allowed to stand at r o o m t e m p e r a t u r e for 10 min. Following a further centrifugation the supernatant serum was removed and tested for anti-immunoglobulin activity by slide agglutination and for the presence of specific ant i body activity by immunofluorescence. RESULTS
Total IgG and IgM concentration in sera following absorption with protein A containing staphylococcal preparations Eighteen d if f er ent serum samples were absorbed with an equal volume of packed protein A positive staphylococci. The concentrations of IgG and IgM in unabsorbed and absorbed sera were calculated from a total of 33 tests perf o r m e d on these sera. T he results obtained with representative sera are shown in table 1. Although the calculated c o n c e n t r a t i o n of IgG and IgM in individual sera showed some variation in experiments p e r f o r m e d on different occasions, the percentage uptake of IgG remained relatively constant. An
371 TABLE 1 Total IgG and IgM levels in unabsorbed sera and sera absorbed with protein A positive Staphylococcal preparations. Serum
AL5 AL5 AUS
Absorption
-A +re -A +ve - -
A +ve AUS SW 259 SW 710 SW 121 AL 3 H
L
*
118
- -
A -A -A -A -A
+ve +ve +ve +re +ve
- -
A +ve -A + ve
IgG
IgM
mg/ml
% uptake
mg/ml
% uptake
18.0 0.54 14.3 0.48 11.2 0.39 10.8 0.48 13.7 0.63 14.3 0.39 13.5 0.54 20.0 0.39 13.6 0.62 15.4 0.48
97.0
1.5 0.59 0.91 0.43 2.01 0.95 1.55 0.67 1.63 0.92 1.65 1.08 1.03 0.52 0.53 0.29 19.6 6.6 2.62 1.06
60.6
96.5 96.5 95.6 95.4 97.3 96.0 98.05 95.44 96.88
51.6 52.3 56.4 43.45 34.54 49.5 41.57 66.3 59.54
* Sample with high IgM level. u n e x p e c t e d f i n d i n g was t h e a p p a r e n t l y high p r o p o r t i o n o f IgM r e m o v e d b y a b s o r p t i o n . If, h o w e v e r , t h e a b s o l u t e a m o u n t r e m o v e d is c o n s i d e r e d , it is c l e a r t h a t t h e r e m o v a l o f IgG b y p r o t e i n A p o s i t i v e s t a p h y l o c o c c i is c o n s i d e r a b l y m o r e e f f e c t i v e t h a n t h a t o f IgM ( t a b l e s 1 a n d 2). In o r d e r t o see w h e t h e r IgM r e m o v a l was n o n - s p e c i f i c , f o u r sera w e r e absorbed with protein A positive and protein A negative staphylococcal p r e p a r a t i o n s . T h e IgG a n d IgM c o n t e n t o f t h e s e sera are s h o w n in fig. 1. B o t h I g G a n d IgM levels w e r e a f f e c t e d b y a b s o r p t i o n w i t h p r o t e i n A n e g a t i v e s t a p h y l o c o c c i b u t t h e a m o u n t r e m o v e d v a r i e d g r e a t l y w i t h i n d i v i d u a l sera, u n l i k e t h e s t r i k i n g a n d c o n s i s t e n t r e d u c t i o n in I g G levels t h a t r e s u l t e d f r o m a b s o r p t i o n w i t h p r o t e i n A +ve s t a p h y l o c o c c i . T h e d a t a o b t a i n e d f r o m M a n c i n i t e s t s p e r f o r m e d o n all sera are s u m m a rised in t a b l e 2.
Effect of staphylococcal absorption upon specific IgG and IgM antibody reac rio ns Since p r o t e i n A positive s t a p h y l o c o c c i had been shown to absorb approxim a t e l y 40% o f t o t a l IgM f r o m s e r u m s a m p l e s a n d p r o t e i n A n e g a t i v e p r e p a r a -
372 TABLE 2 E f f e c t o f a b s o r p t i o n w i t h p r o t e i n A positive a n d negative S t a p h y l o c o c c a l p r e p a r a t i o n s u p o n t h e u p t a k e o f IgG a n d IgM in w h o l e sera. Absorption with:-
No. o f s a m p l e s % u p t a k e o f IgG A m o u n t (rag) o f IgG r e m o v e d b y 100 ul packed staphylococci No. o f samples %uptakeofIgM A m o u n t (mg) o f IgM r e m o v e d b y 100 t~l packed staphylococci
A positive
A negative
33 94.51 1.34
4 36.8 0.45
+ 4.9 _+ 0.49
27 4 6 . 5 3 + 20.9 0 . 0 5 5 + 0.04
lgG
12
10
4 23.3 + 21.4 0.019 + 0.006
|gG
I£G
I m [
8
--
6
--
E
± 21.9 + 0.34
m \1 \1
&l \\
q 2
I~M
IgM
\\
IAM
--
SHRUM
1
SE]~UM
2
SERUM
3
SERUM
A
Fig. 1. IgG a n d IgM levels in f o u r u n a b s o r b e d sera a n d f o l l o w i n g a b s o r p t i o n w i t h p r o t e i n A positive or p r o t e i n A negative S t a p h y l o c o c c a l p r e p a r a t i o n s , cs, U n a b s o r b e d sera; [], sera a b s o r b e d w i t h p r o t e i n A - - r e S t a p h y l o c o c c i ; m, sera a b s o r b e d w i t h p r o t e i n A +re S t a p h ylococci.
373 tions had r e m o v e d variable amounts of b o t h IgM and IgG, preliminary experiments were c o n d u c t e d to see w he t he r absorption would significantly influence the titre of antibodies d e t e c t e d by i m m u n o f l o u r e s c e n t procedures. F o u r sera taken f r om adult patients with Toxoplasma infections were absorbed with either protein A positive or protein A negative staphylococcal preparations. These absorbed sera t oge t h er with unabsorbed sera were titrated for the presence o f specific IgG and IgM antibodies. The results of these tests are shown in table 3. This data shows t h a t absorption with prot ei n A positive staphylococci selectively reduces the specific IgG a n t i b o d y titre with no apparent reduction in th e IgM titre. F u r t h e r m o r e , despite the removal of variable am ount s o f IgG and IgM by protein A negative staphylococcal absorption, demonstrable by quantitative radial diffusion, there was little effect u p o n specific a n t i b o d y titres. In subsequent experiments only protein A positive staphylococci were used for absorption.
Sera from adults with anti Toxoplasma antibody T w e n t y two serum samples were absorbed with prot ei n A positive staphylococcal preparations, following which the specific a n t i b o d y titre was evaluated by i m m u n o f l u o r e s c e n c e and the presence of ' R F ' activity by slide agglutination with latex-IgG. Some of the results are tabulated in table 4. In all experiments absorption resulted in a substantial decrease in specific IgG titre w i t h o u t diminishing the IgM response. In all but two test samples the ' R F ' activity was either absent or low in the initial dilution tested. Absorp-
TABLE 3 Specific IgG and IgM Toxoplasma antibody titres in unabsorbed adult sera and in sera absorbed with eiter protein A positive or protein A negative Staphylococci. Sera
AUS KF4 AL5 AL5
Absorption
-A +ve A --ve --
A A -A A -A A
+ve --re
+re --ve +re --re
* Reciprocal of titre.
Immunofluorescence titre * IgG
IgM
512--1024
