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Staphylococcal protein a fluoroimmunoassay for immune complexes.

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STAPHYLOCOCCAL PROTEIN A FLUOROIMMUNOASSAY FOR
IMMUNE COMPLEXES
ISIDORE FAIFERMAN and DAVID KOFFLER
A f l u o r o i m n o a s s a y (FIA) u t i l i z i n g f l u o r e s c e i n
l a b e l e d Staphylococcal p r o t e i n A (FSPA) was developed
f o r e v a l u a t i o n and q u a n t i t a t i o n o f c i r c u l a t i n g immune
complexes and compared w i t h a radioimmunoassay (RIA),
t h e f l u i d phase Clq b i n d i n g t e s t . The performance o f
p o l y e t h y l e n e g l y c o l (PEG) p r e c i p i t a t i o n d u r i n g t h e F I A
a l l o w a d i r e c t comparison o f t h e two t e s t s w i t h s i m i l a r
immune complex p r e c i p i t a t e s . The data derived f r a t h e
comparison o f FIA and R I A suggest t h a t these t e s t s have
comparable s e n s i t i v i t y f o r t h e d e t e c t i o n o f immune
complexes i n t h e sera o f p a t i e n t s w i t h SLE.
Sera were obtained from p a t i e n t s w i t h SLE and
rheumatoid a r t h r i t i s diagnosed on t h e b a s i s o f ARA
c r i t e r i a (1.2).
SLE sera were c l a s s i f i e d as d e r i v e d
frm p a t i e n t s w i t h a c t i v e , i n a c t i v e o r a c t i v e r e n a l
disease ( 3 ) .
The R I A was performed as p r e v i o u s l y
described ( 4 ) .
The FIA u t i l i z i n g FSPA, as m o d i f i e d
from p r e v i o u s l y described s o l i d phase 2yI staphylococcal p r o t e i n A assays. A t e s t serum was p r e c i p i t a t e d
w i t h 2.5% PEG, t h e p e l l e t was washed i n 2.5% PEG,
resuspended i n a one percent bovine serum albumin
s o l u t i o n t o which FSPA was added. F o l l o w i n g i n c u b a t i o n
a t 37°C f o r 90 minutes, a second a l i q u o t o f PEG was
added and t h e tubes reincubated a t 4°C f o r 90 minutes.
From the Department of Pathology and Laboratory Medicine, The Hahnemann Medical College and Hospital, Philadelphia. PA, and The Rockefeller University, New York. NY
Address reprint requests t o Dr. David Koffler, Department of Pathology, Hahnmann Medical College and Hospital,
230 N. Broad S t r e e t , Philadelphia, PA 19102
Supported by grant 21715 from the US Public Health
Service, by the Pennsylvania State Department o f Health, and
the Pennsylvania Lupus Foundation.
Arthritk and Rheumatism, Vol. 25, No. 7 (July 1982)
The p e l l e t s obtained by c e n t r i f u g a t i o n were washed w i t h
PEG and resuspended i n phosphate b u f f e r e d s a l i n e . The
fluorescence e m i t t e d was determined u t i l i z i n g a P e r k i n
Elmer spectrofluorometer.
Both h e a t aggregated and g l u t a r a l d e h y d e aggregated
gamma g l o b u l i n showed comparable r e a c t i v i t y i n t h e R I A
and F I A .
I n v i t r o p r e c i p i t i n curves u t i l i z i n g tetanus
t o x o i d human F a n t i t e t a n u s t o x o i d and monkey a n t i human IgG human IgG showed comparable r e a c t i v i t y f o r
R I A and FIA.
Maximal b i n d i n g was e v i d e n t f o r both
t e s t s i n t h e r e g i o n o f t h e p r e c i p i t i n curve w i t h s l i g h t
t o moderate a n t i g e n excess s o l u b l e immune complexes,
a l t h o u g h b o t h reagents showed s i g n i f i c a n t b i n d i n g t o
. s o l u b l e complexes a t 1OX a n t i g e n excess (Table 1).
A comparison o f R I A and F I A f o r immune complex
d e t e c t i o n i n SLE and rheumatoid a r t h r i t i s sera i s shown
i n Table 2.
Comparable s e n s i t i v i t y was e v i d e n t f o r
both techniques ( F i g u r e 1). S i g n i f i c a n t d i f f e r e n c e s
were found between sera obtained from i n a c t i v e SLE
p a t i e n t s and p a t i e n t s w i t h a c t i v e disease w i t h and
w i t h o u t r e n a l involvement f o r b o t h R I A and F I A . The
R I A d i d n o t d e t e c t a h i g h e r i n c i d e n c e o f immune complexes i n rheumatoid a r t h r i t i s than t h e F I A and rheumatoid a r t h r i t i s sera t h a t were p o s i t i v e by both t e s t s
exhibited a close correlation.
Evidence was obtained
u t i l i z i n g a sucrose d e n s i t y g r a d i e n t u l t r a c e n t r i f u g a t i o n t h a t s i m i l a r immune complex s i z e was detected by
t h e R I A and FIA ( F i g u r e 2 ) .
The data shown i n t h i s study a r e c o n s i s t e n t w i t h
t h e f o l l o w i n g hypotheses:
1) t h e F I A i s a f e a s i b l e
a l t e r n a t i v e t o t h e R I A f o r q u a n t i t a t i o n o f immune
complexes i n SLE sera; 2 ) t h e R I A and F I A p r i m a r i l y
d e t e c t immune complexes i n SLE sera by i n t e r a c t i o n of
Clq and Staphylococcal p r o t e i n A w t t h t h e Fc p o r t i o n o f
t h e gamma g l o b u l i n molecule; 3 ) d a t a f r a n t h i s and
previous s t u d i e s suggest t h a t t h e presence of C l q
b i n d i n g i s n o t r e l a t e d t o t h e complement f i x i n g a b i l i t y
FAIFERMAN AND KOFFLER
800
SLE INACTIVE
100
SLE ACTIVE
100
0,
QJ
C
.TI
.-C
a
.-C
0
.-C
a
dp 5c
apx
-aa
9
U
r = 0.88
p' 0.000 1
:
1c
1c
10
2
20
FIA Fluorescence units
SLE ACTIVE RENAL
100
I
10
20
FIA Fluorescence units
RHEUMATOID ARTHRITIS
lOOT
QJ
C
.TI
.-C
a
50
dp
a
a
r = 0.80
pco.00 1
10
lo
2
20
FIA Fluorescence units
Figure 1.
renal;
(D)
Correlations between R I A and F I A i n sera o f p a t i e n t s w i t h ( A )
rheumatoid a r t h r i t i s .
10
20
FIA Fluorescence units
SLE i n a c t i v e ; ( 6 ) SLE active; (C) SLE a c t i v e
STAPHYLOCOCCAL PROTEIN A ASSAY
801
Table 1. Comparison o f soluble immune caplexes
assayed by R I A and FIA
Antigen excess
Tetanus toxoid*
2x
5x
lox
RIA
% binding
Table 2. Comparison o f FIA and R I A .
No. o f
Sera
FIA
Fluorescence u n i t s
60
40
30
10
8
20
30
6
7
6
SLE i n a c t i v e
SLE a c t i v e
SLE a c t i v e renal
Rheumatoid a r t h r i t i s
Normal sera
7
25
*Human antitetanus toxoid IgG tetanus
p r e c i p i t i n curve
Monkey antihuman IgG human IgG
toxoid
of immune complexes ( 5 ) ; 4 ) based on data derived from
sucrose density gradient ul tracentrifugation studies
and precipitin curves, both R I A and F I A detect immune
complex material of comparable molecular size.
The F I A has several potential advantages over the
R I A for quantitation of immune camplexes. Staphylococcal protein A may be labeled either w i t h fluoroscein
or an enzyme and converted thereby t o an enzyme inmunoassay avoiding the utilization of a radioactive label.
The utilization of a reagent, Staphylococcal protein A,
which i s stable and commercially available i n contrast
t o C l q globulin which i s prone t o aggregation and must
be isolated from fresh human serum provides certain
advantages for laboratories performing routine assays
for i m n e complexes. Despite the need for a double
PEG precipitation with the F I A , conversion t o an enzyme
imnoassay would considerably accelerate the time
required for performance of the t e s t as well as decrease the cost and therefore make t h i s a useful clinical assay for quantitation of immune complexes.
I
36*
34
36
55
34
20
30
30
FIA
% positive
38
55
62'
58'
0
I n a c t i v e SLE sera manifested a s i g n i f i c a n t l y lower
incidence o f i m n e complexes than sera fra a c t i v e o r
i n a c t i v e renal disease (p < 0.01) by the Chi square
test.
+ 32 a c t i v e renal patients tested; 19 rheumatoid arthr i t i s patients tested.
*,The R I A d i d n o t show s i g n i f i c a n t l y higher r e a c t i v i t y
with rheumatoid a r t h r i t i s sera than the FIA ( p < O . l ) by
the Chi square test.
AHGG+
5x
lox
2 ox
RIA
% positive
.-
I S
Fraction
Figure 2. Detection o f i m n e complexes i n SLE serum 2328
by R I A and FIA. 0.5 m l serum sample was centrifuged f o r 16
h r a t 34000 rpm i n a Sw 41 Beckman rotor; 1 m l f r a c t i o n s
were collected and 100 p1 aliquots were used f o r binding
assays.
P u r i f i e d IgG and IgM were centrifuged i n p a r a l l e l
gradients as markers.
REFERENCES
1.
2.
3.
4.
5.
American Rheumatism A s s o c i a t i o n C r i t e r i a f o r diagnosis
and c l a s s i f i c a t i o n o f rheumatic disease:
Primer on
Rheumatic Diseases.
JAMA (Supplement) 224:137-138,
1973
Cohen AS, Reynolds WE, F r a n k l i n EC. Kulka PJ, Ropes MW,
Shulman LE, Wallace SL: P r e l i m i n a r y c r i t e r i a f o r t h e
c l a s s i f i c a t i o n o f systemic lupus erythematosus.
Bull
Rheum D i s 21:643-648, 1971
K o f f l e r 0. M i l l e r TE, L a h i t a R: Studies on t h e specif i c i t y and c l i n i c a l c o r r e l a t i o n o f antiribosomal a n t i bodies i n systemic lupus erythematosus sera. A r t h r i t i s
Rheum 22:463-470. 1979
Zubler RH, Lange G, Lambert PH, Miescher PA: D e t e c t i o n
o f cfrculf#ng
immune complexes i n unheated sera by a
I Clq b i n d i n g t e s t . J Immnol 116:232-235,
modified
1976
Folkerd EJ, Gardner B, Hughes-Jones NC: The r e l a t i o n s h i p between the b i n d i n g a b i l i t y and t h e r a t e o f a c t i Inmuno1 41:179v a t i o n o f t h e complement component C1.
185, 1980
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