Journal o/" lmmunolo#ical Methods, 1.1 ( 1977 ) 3 1 3 - - 3 2 3
313
© E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
RADIOIMMUNOASSAY P R O C E D U R E FOR Q U A N T I T A T I N G B A C TER I AL ANTIBODY IN HUMAN SERA
B A R B A R A A. S A N F O R D and K E N D A L L O. SMITH
Department o f Microbiology, The University o f Texas ltealth Science Cenler at San Antonio, San Antonio, Texas 78284, U.S.A. (Received 16 F e b r u a r y 1976, a c c e p t e d 15 O c t o b e r 19751
A R I A s y s t e m was d e v e l o p e d to d e t e c t a n t i b o d i e s in h u m a n sera against bacteria. S o n i c a t e s o f Escherichia coli and Fusobacterium polymovphum were used as antigens to sensitize plastic-coated beads; a n t i b o d i e s to t h e s e antigens were d e t e c t e d with 12sI-labeled a n t i h u m a n globulin. S e r u m a n t i b o d y titers against E. coli were d e t e r m i n e d by the serial dilution m e t h o d ; from the results the s t a n d a r d curve principle was applied in d e t e r m i n i n g the relative a m o u n t s o f a n t i b o d i e s against E. coli in s e r u m samples t e s t e d at a single dilution. Tbe c o e f f i c i e n t o f variation o f the RIA p r o c e d u r e was < 10%. S e r u m titers o b t a i n e d by the RIA and indirect i m m t i n o f l u o r e s e e n c e tes{ were c o m p a r e d ; R I A was m o r e sensitivo, q u a n t i t a t i v e and objective. A b s o r p t i o n studies, using E. coil and F. p o l ym o rp h u m a b s o r b e n t s , indicated the specificity o f the R I A s y s t e m in d e t e c t i n g a n t i b o d i e s against E. coli and F. polymorphum. This RIA p r o c e d u r e o f f e r s a c o m b i n a t i o n o f desirable advantages; it is sensilive, specific, objective, q u a n t i t a l i v e and easy to p e r f o r m .
INTRODUCTION Radioimmunoassay (RIA) is not a generally accepted routine procedure for detecting and quantitating immunoglobulins to bacterial antigens. However RIA has been used experimentally to measure bacterial antibodies. Hutchinson and Ziegler (1972) absorbed whole cells of Escherichia coli o n t o glass slides, reacted the bacteria with immune rabbit s e r u m and detected the primary immune reaction with ~2SI-labeled antirabbit serum. They were able to differentiate between two strains of E. coli (01 and 0119) using homologous and heterologous i m m une sera. Nassau et al. (1975) also applied an indirect RIA p r o c e dur e to the serological diagnosis of tuberculosis by using p oly s ty r en e wells sensitized with soluble culture filtrates of Mycobacterium tuberculosis H37 Rv. Human sera were reacted with the adsorbed antigen and the uptake of ant i body was det ect ed with radioiodinated antiglobulin. In their initial study the relative activity of sera correlated with the presence or absence of active tuberculosis. Recently Smith et al. (1974) developed a rapid and simple RIA system for detecting viral a nt i body in human sera which uses imitation pearls as antigen absorbents. The object of this report is to describe the successful adaptation
314 of this RIA procedure for use in quantitating specific bacterial ant i body in h u man sera. Results obtained with RIA are compared with results obtained by the more c o m m o n l y used indirect immunofluorescence test (FA). MATERIALS AND METHODS
Preparation of bacterial antigens for RIA and FA procedures Two gram-negative bacilli, Escherichia coli 075 and Fusobacterium polymorphum (American T y p e Culture Collection # 1 0 9 5 3 ) were used as antigens in this study. E. coli, a m e m b e r of the family Enterobacteriaceae, is a normal inhabitant of the gastrointestinal tract of man, while the anaerobic F. polymorphum belongs to the family Bacteroidaceae and is part of the normal flora o f the human oral cavity. Stock cultures of the two organisms were prepared by culturing the E. coli in T o d d - - H e w i t t broth at 37°C for 24 h and culturing F. polymorphum in fluid thioglycollate medium (10% fetal calf serum) in a Torbal container gassed with 10% H2 and 90% CO2 at 37°C for 48 h. Aliquots of the stock cultures were frozen at --70°C and used t h r o u g h o u t the study. Crude bacterial antigens used in the RIA procedure were prepared as follows : A fresh subculture of the stock organism was centrifuged at 2300 g for 10 min, washed 3 times with phosphate buffered saline (PBS, 0.01 M phosphate in 0.15 M saline), pH 7.2, and resuspended in 5 ml of PBS. One volume (dry weight) of powdered glass beads (25 p diameter, Heat Systems -Ultrasonics, Inc., Plainview, N.Y.) was added to 3 volumes of the bacterial suspension. The suspension was placed in an ice bath and sonicated with a microtip sonifier cell disrupter (Heat Systems -- Ultrasonics, Inc., Plainview, L.I., N.Y.) set at 7, using 10 sec bursts for a total t r e a t m e n t of 1 min. This procedure was usually repeated 5 times to obtain disruption of F. polymorphum. The suspension was centrifuged at 2600 g for 3 rain and the supernatant fluid ('crude' antigen} was removed and used immediately or stored at --70°C. Protein determinations on the 'crude' antigens were made by the L o w r y procedure (1951). Bacterial antigens for the FA procedure were prepared as follows: A fresh subculture of the stock organism was centrifuged at 2300 g for 10 min, washed 3 times with PBS, and adjusted by dilution in PBS to a reading of 50 on a Klett-Summerson photoelectric colorimeter. This dilution was experimentally established to correspond to the n u m b e r of bacteria (150 to 200 per high-power field) remaining on the slide after the washing procedures. A volume of 0.025 ml of the standardized bacterial suspension was smeared thinly on glass slides, air-dried, fixed with acetone, washed twice with PBS, and used immediately for i m m unof l uor es c e nce testing.
Source of sera Human sera were obtained from the Serology and Clinical Chemistry Laboratories, Bexar C ount y Hospital, San Antonio, Texas. In addition, several
315 patients' sera known to contain high titers of antibodies against E. eoli 075 were kindly supplied by Dr. Virginia L. Thomas in our d e p a r t m e n t . All sera were stored at --70 ° C.
RIA procedure Plastic-coated beads (6 mm diameter, Grieger's of Pasadena, California, W773-6) were used to absorb 'crude' bacterial antigens for the detection of a n t i b o d y in human sera by RIA. The n u m b e r of beads needed for each experiment were placed in a beaker and rinsed twice in distilled water. T hey were sensitized with bacterial antigen by adding a volume of soluble antigen which co mp letel y covered the beads and allowing to stand for 20 rain at 25°C. The sensitized beads were rinsed with 1% bovine serum albumin (BSA) dissolved in PBS. Duplicate beads were placed in 2 ml of test serum previously diluted in 1% BSA and incubated at 4°C for 18 h. After incubation, each sensitized bead was dipped 6 times in 1% BSA then washed in a fresh volume of 1% BSA by dipping 6 more times. Each bead was then placed in 1 ml of ~2~I-labeled polyvalent antihuman IgG + IgA + IgM globulin and incubated at 25°C for 4 h. The beads were washed as described, placed in plastic vials and measured for activity in a Beckman gamma counter. The conjugated antiserum used in the RIA procedure was prepared by fractionating the goat antihuman IgG + IgA + IgM (Hyland, Los Angeles, California) using the ammonium sulfate fractionation m e t h o d of Hebert et al. (1973). The gamma globulin fraction was labeled with J2sI by the Chloramine T Method of Daugharty et al. (1972) or t t u n t e r and G r e e n w o o d (1962) using a 1--5 mgm protein c o n c e n t r a t i o n of gamma globulin and 2 mCi Na ~2s[.
Immunofluorescence procedure Fluorescein-conjugated polyvalent antihuman immunoglobulins (goat origin) (Hyland, Los Angeles, California) were absorbed to remove any naturally-occurring antibodies against the test bacteria. The antiserum was diluted 1 : 2 in PBS and absorbed twice (25°C, 1 h) with formalin-killed subcultures o f E. coli 075 and F. polymorphum previously washed 3 times with PBS. The working dilution of the absorbed conjugate was determined by block titration using E. coli 075 antigen and two standardized sera. The conjugate had a fluorescein to protein molar ratio of 3.9, a fluorescein to protein weight ratio of 9.5 #g/rag and a protein-bound fluorescein isothiocyanate c o n cen tr atio n of 242 #g/rag. Ringed areas marked on each slide were flooded with 0.025 ml of test serum and incubated at 37°C for 30 min. The slides were washed twice in PBS and treated with the absorbed fluorescein conjugate. After incubation at 37°C for 30 min, the slides were washed twice in PBS and examined microscopically for apple-green fluorescence (AO Spencer fluorolume illuminator, model 645) with the 50× oil objective. Saline controls were included in each test.
316 RESULTS AND DISCUSSION S t a n d a r d i z a t i o n o f antigen for R I A A d u p l i c a t e b l o c k t i t r a t i o n was d o n e to d e t e r m i n e the o p t i m u m c o n c e n t r a t i o n o f bacterial antigen n e e d e d to sensitize beads. Beads were sensitized in 10-fold serial dilutions o f E. coli antigen, 1 : 101--1 : 10 ~, in PBS. The sensitized beads were reacted with 3-fold serial dilutions of an h y p e r i m m u n e serum, 1 : 10~--1 : 10 ~, in 1% BSA, and the R I A test was d o n e as described. Results were p l o t t e d as c o u n t s / m i n u t e versus the reciprocal of the antigen dilution for each d i l u t i o n o f s e r u m tested. Fig. 1 shows the curve for the 1 : 1 0 0 0 dilution o f s e r u m and d e m o n s t r a t e s a sharp decrease in c o u n t s with beads sensitized in dilutions of E. coli antigen greater than 1 : 100. At the 1 : 100 dilution the antigen c o n t a i n e d 0.16 mg p r o t e i n / m l . All s u b s e q u e n t e x p e r i m e n t s using R I A were d o n e with a c o n c e n t r a t i o n o f either 0.16 mg or 1.65 mg p r o t e i n / m l o f bacterial antigen to sensitize the beads. Use o f a standard curve Using R I A , Patterson and S m i t h ( 1 9 7 5 ) r e p o r t e d that viral a n t i b o d y titers in h u m a n sera can be d e t e r m i n e d f r o m a standard curve. Their m e t h o d elimihates the need for using the m o r e t e d i o u s and t i m e - c o n s u m i n g serial dilution m e t h o d to d e t e r m i n e serum a n t i b o d y titers. We p e r f o r m e d similar experim e n t s to see if the bacterial a n t i b o d y titers o f h u m a n sera c o u l d also be d e t e r m i n e d f r o m a s t a n d a r d curve. T e n sera were titrated by the serial dilution m e t h o d against E. coli by R I A . Results are given in table 1. Each well c o n t a i n e d an average of 2.7 × 10 ~' c o u n t s / m i n u t e / m l of 12~I-labeled a n t i s e r u m . A p p r o x i m a t e l y 6% o f the c o u n t s were a b s o r b e d in wells c o n t a i n i n g labeled a n t i s e r u m only. As can be
b6
141512
b ~o9-
7 6 5 4 3
ANTIGEN DILUTION
Fig. 1. E. coli 'crude' antigen titration against a 1 : 1,000 dilution of E. coli hyperimmune human serum by RIA. CPM, counts/min.
32,155 11,982 19,672 20,970 22,783 21,362 25,081 25,670 18,149 16,563
1:90
27,763 6,633 11,347 24,189 17,407 15,021 18,204 19,957 11,147 9,368
1:270
22,675 4,450 7,141 19,527 9,592 8,127 11,124 12,939 7,102 5,945
1:810
17,128 3,371 4,444 8,387 5,121 5,264 7,597 7,365 4,117 3,860
1:2430
9,091 2,918 3,312 3,790 3,030 3,173 5,454 3,929 3,043 2,874
1:7290
4,882 2,546 2,667 2,815 2,554 2,625 3,421 2,846 2,876 3,148
1:21,870
C o u n t s / m i n a of E. coli sensitized beads reacted with differed s e r u m d i l u t i o n s
3,279 2,608 3,133 3,696 2,604 2,087 2,550 2,480 2,915 2,991
1:65,610
2.56 2.50 2.99 2.57 2.44 2.48 2.46 2.54 2.50 2.45
3,456
Mean = 2,700
albumin c
3,580 3,727 3,877 4,009 3,100 3,604 2,752 3,067 3,037 3,816
E. coli antigen e
4,012
5,404 4,006 4,357 3,889 3,673 4,094 3,405 2,593 4,079 3,616
Non-sensitized d
Bovine s e r u m
C o u n t s / m i n a of c o n t r o l b e a d s sensitized with :
2,905 2,554 3,020 2,700 2,288 2,808 2,973 2,290 2,782 2,680
Total c o u n t s rem a i n i n g in t h e well b X 10 ¢'
a Mean c o u n t s / m i n of duplicate tests. b C o u n t s / r a i n r e m a i n i n g in the wells after r e m o v a l o f E. coli sensitized beads previously i n c u b a t e d in the 1 : 9 0 d i l u t i o n o f t h e corres p o n d i n g test serum. c I n c u b a t e d in d i l u e n t w i t h o u t s e r u m t h e n i n c u b a t e d in labeled a n t i s e r u m . d I n c u b a t e d in labeled a n t i s e r u m only.
1 2 3 4 5 6 7 8 9 10
Serum No.
Results of t i t r a t i n g the h u m a n sera for a n t i b o d i e s to E. coli 075 by RIA.
TABLE 1
318
275f 2504 225 200 "?,
t75
_0 150 125100 75 5.025 0
f
270
i
810
i
,
,
•
2430 7290 21,870 65,610 I/SERUMDILUTION Fig. '2. E. coli antibody titration of a human serum by RIA; curve was used as a standard curve in determining titers of nine other sera. 9O
seen in t a b l e 1, the c o u n t s d r o p p e d 12% a f t e r i n c u b a t i o n of sensitized b e a d s p r e v i o u s l y e x p o s e d to the 1 : 90 d i l u t i o n o f s e r u m # 5 ; this m a y p a r t l y be e x p l a i n e d b y n o n - s p e c i f i c a d s o r p t i o n o f labeled a n t i s e r u m to t h e walls o f t h e well. T h e last t h r e e c o l u m n s in t a b l e 1 indicate n o n - s p e c i f i c a d s o r p t i o n o f labeled a n t i s e r u m to (1) beads sensitized with t h e E. coli antigen and incub a t e d in d i l u e n t w i t h o u t s e r u m , (2) beads sensitized with an u n r e l a t e d antigen (BSA) and i n c u b a t e d in d i l u e n t w i t h o u t s e r u m , and (3) beads w h i c h w e r e n o t c o a t e d b u t s i m p l y placed in labeled a n t i s e r u m for t h e 4 h incubation period. Results f r o m t i t r a t i n g each o f t h e 10 sera w e r e p l o t t e d as c o u n t s / r a i n versus the r e c i p r o c a l o f t h e s e r u m dilution. Curves w i t h similar slopes w e r e o b t a i n e d ; this suggested t h a t a s t a n d a r d curve c o u l d be c o n s t r u c t e d using 1 s e r u m to which all o t h e r sera can be c o m p a r e d . This p o s s i b i l i t y was tested. T h e curve o f s e r u m # 7 (fig. 2) was t r a n s f e r r e d to tracing p a p e r and t h e n s u p e r i m p o s e d on each c o r r e s p o n d i n g p o i n t o f the curves o b t a i n e d a f t e r plotting each o f t h e r e m a i n i n g 9 sera. The titer was d e t e r m i n e d f o r each s e r u m d i l u t i o n b y e x t r a p o l a t i o n f r o m the s t a n d a r d curve; each titer was read as the r e c i p r o c a l of the d i l u t i o n giving 5 4 0 0 c o u n t s / m i n w h i c h was t w i c e the b a c k g r o u n d c o u n t s o f t h e d i l u e n t c o n t r o l (see table 1). Results are s h o w n in table 2. In using a s t a n d a r d curve to d e t e r m i n e a n t i b o d y titers it s e e m s desirable to screen each s e r u m at 2 dilutions, i.e. 1 : 1 , 0 0 0 and I : 1 0 , 0 0 0 . T h e titers o b t a i n e d using a s t a n d a r d c u r v e c o m p a r e d f a v o r a b l y with t h o s e o b t a i n e d b y the serial d i l u t i o n m e t h o d . C o m p a r i s o n o f R I A and FA titers Because i m m u n o f l u o r e s c e n c e is a w i d e l y used and sensitive m e t h o d f o r d e t e c t i n g p r i m a r y a n t i g e n - - a n t i b o d y r e a c t i o n s , we d e c i d e d to c o m p a r e this
TABLE 2
16,000 10,000
250 325 660 1,150
1,000 680 1,300 1,700 2,300 8,000 3,000 5,280
2,950 2,950 2,100 2,430
1,600 2,000 1,800 2,200 2,300
7,290 7,290 7,290 7,290 7,290 7,290 7,290
10,000 14,000 15,000 6,500 2,400
8 660 660 1,300 1,500 2,000
9
500 500 900 1,300
10
a Reciprocal of the highest dilution at which a superimposed standard curve dropped below 5,400 counts/min (twice the background counts of the diluent control).
1:90 1:270 1:810 1:2,430 1:7,290 1:21,870 1:65, 610
Extrapolation from Serum antibody titers determined by standard curve a the point at this serum dilution 1 2 3 4 5 6 7
RIA titers of human sera obtained by extrapolating from single points, using a superimposed standard curve.
¢D
50
320 TABLE 3 Comparison in sensitivities of the fluorescent antibody (FA) and the radioimmunoassay (RIA) for detecting antibody to E. coli in human sera. Serum number
Antibody titer ......................... FA a RIA b
1 2 3 4 5 6 7 8 9 10
2,048 128 64 1,024 128 128 128 64 6,1 32
18,000 520 1,700 .l,800 2,350 2,500 7,290 4,600 1,500 1,000
Fold difference 9 4 26 5 18 20 57 72 23 31 mean = 27
a Reciprocal of highest serum dilution giving positive immunofluorescence. b Determined hy plotting counts/rain versus reciprocal of serum dilution, then reading the titer at a point where the curve dropped below 5,400 counts/rain (twice the background counts of the diluent control). t e s t s y s t e m w i t h R I A in d e t e c t i n g b a c t e r i a l a n t i b o d i e s in h u m a n sera. Using t h e serial d i l u t i o n m e t h o d , a n t i b o d y t i t e r s a g a i n s t E. coli w e r e d e t e r m i n e d b y F A on t h e 10 sera p r e v i o u s l y t i t r a t e d b y R I A . T w o - f o l d serial d i l u t i o n s (1 : 4 - - 1 : 8 1 9 2 ) of t h e sera were t e s t e d . T h e r e s u l t s o f F A tests c o r r e s p o n d e d to r e s u l t s of R I A ( t a b l e 3); h o w e v e r , several i m p o r t a n t d i f f e r e n c e s b e t w e e n r e s u l t s o f t h e 2 t e s t s y s t e m s are a p p a r e n t . F i r s t , t h e t i t e r s o b t a i n e d b y R I A w e r e h i g h e r t h a n t h e F A t i t e r s b y an average d i f f e r e n c e of 2 7 - f o l d , i n d i c a t i n g t h e i n c r e a s e d s e n s i t i v i t y o f R I A . S e c o n d , t h e R I A d e t e c t e d q u a n t i t a t i v e diff e r e n c e s a m o n g t h e sera w h i c h w e r e n o t a p p a r e n t b y F A . T h e i m m u n o f l u o r e s c e n c e p r o c e d u r e is n o t r o u t i n e l y a q u a n t i t a t i v e p r o c e d u r e as is R I A . B e c a u s e i m m u n o f l u o r e s c e n c e is n o t e a s i l y q u a n t i t a t e d , t h e s t a n d a r d curve p r i n c i p l e c a n n o t be a p p l i e d , m a k i n g t h e t i t r a t i o n o f sera a n d r e a d i n g o f r e s u l t s b y F A b o t h t e d i o u s a n d t i m e - c o n s u m i n g . Also, in c o n t r a s t t o R I A , i n t e r p r e t a t i o n o f F A r e s u l t s is s u b j e c t i v e r a t h e r t h a n o b j e c t i v e , w h i c h can l e a d to i n v e s t i g a t o r bias in r e a d i n g e n d p o i n t s .
R e p r o d u c i b i l i t y o f the R I A test R e p l i c a t e R I A t e s t s w e r e p e r f o r m e d using a 1 : 1 0 0 0 d i l u t i o n o f a h u m a n s e r u m in 1% B S A a n d 1% B S A o n l y ; b o t h were r e a c t e d w i t h E. coli sensit i z e d b e a d s . R e s u l t s are given in t a b l e 4. T h e c o e f f i c i e n t of v a r i a t i o n was 5% w i t h h u m a n s e r u m a n d 6% w i t h t h e 1% B S A d i l u e n t . In p r e v i o u s e x p e r i m e n t s t h e c o e f f i c i e n t o f v a r i a t i o n has r e a c h e d 15%.
321 'FABLE 4 Results of replicate RIA tests. Test number
E. coli sensitized beads reacted with 1:1000 dilution of a human serum
1% BSA
1 2 3 4 5 6 7 8 9 10
12,671 12,555 12,833 11,803 13,411 11,626 12,798 12,991 13,520 13,9.16
1,740 1,671 1,641 1,585 1,457 1,856 1,668 1,730 1,671 1,763
mean
12,815
1,678
Standard deviation Coefficient of variation (%)
721.85 5%
107.80 6%
Proof of specificity of the RIA reaction In evaluating the usefulness o f a n y i m m u n o l o g i c a l p r o c e d u r e , especially one w h i c h shows a high degree o f sensitivity, it is necessary to d e m o n s t r a t e specificity o f the test system. We p r o p o s e d to do this by using 2 antigenically dissimilar organisms, E. eoli and F. polymorphum, as test antigens. A series o f a b s o r p t i o n e x p e r i m e n t s were d o n e using either E. coli a b s o r b e n t or F. p o l y m o r p h u m a b s o r b e n t . An h y p e r i m m u n e serum against E. coli was used as the test serum. T w o sets o f 3-fold serial dilutions o f s e r u m , 1 : 3 0 0 - 1 : 6 5 6 , 1 0 0 , were m a d e in each o f the f o l l o w i n g : (1) 1% BSA, (2) ' c r u d e ' E. coli antigen (1.6 mg p r o t e i n / m l ) in 1% BSA, and (3) ' c r u d e ' F. p o l y m o r p h u m antigen (1.5 mg p r o t e i n / m l ) in 1% BSA. A f t e r i n c u b a t i o n at 25°C for 1 h, sensitized beads were a d d e d ; I set o f serum dilutions received beads sensitized with E. coli antigen, the s e c o n d set received beads sensitized with F. polymorphurn antigen. C o n t r o l s i n c l u d e d the f o l l o w i n g : 1) 1% BSA vs. E. coli antigen. 2) 1% BSA vs. F. p o l y m o r p h u m antigen. 3) ' c r u d e ' E. coli antigen in 1% BSA vs. E. coli antigen. 4) ' c r u d e ' E. coli antigen in 1% BSA vs. F. p o l y m o r p h u m antigen. 5) ' c r u d e ' F. polymorphum antigen in 1% BSA vs. E. coli antigen. 6) ' c r u d e ' F. p o l y m o r p h u m antigen in 1% BSA vs. F. polymorphum antigen. The R I A test was carried o u t as described. Results were p l o t t e d as serum e o u n t s / m i n minus the c o r r e s p o n d i n g c o n t r o l c o u n t s / m i n versus the recipro-
322
'41
\,
I 300
900
2,700
8/00
24,300
72,900
218,700
656,100
I/SERUM DILUTION
Fig. 3. E. coli antibody titration by RIA of a human serum in 1% BSA: unabsorbed (®); absorbed with E. coli antigen ("); absorbed with F. polyrnorphum antigen (A). Control counts/rain were 1,244 (o), 1,296 (m) and 1,902 (A). cal o f the serum dilution. Titers were read at the p o i n t w h e r e the curve d r o p p e d b e l o w 1 , 0 0 0 c o u n t s / m i n . Results with E. eoli antigen-sensitized beads (fig. 3) indicate t h a t using E. eoli a b s o r b e n t r e d u c e d the serum titer 76-fold f r o m a titer o f 9 0 , 0 0 0 to a titer o f 1,190 c o m p a r e d to o n l y a 2-fold decrease in titer w h e n F. p o l y r n o r p h u r n was used as a b s o r b e n t . When F. p o l y r n o r p h u r n sensitized beads were tested, the data (fig. 4) s h o w e d t h a t the serum had a low level o f a n t i b o d y to F. p o l y r n o r p h u m which was a b s o r b e d o u t with F. p o l y m o r p h u r n a b s o r b e n t b u t was n o t a f f e c t e d by E. coli absorbent. The test serum was s h o w n to have a high titer of a n t i b o d y against E. eoli, b u t a low titer o f a n t i b o d y against a n o t h e r gram-negative bacillus, F. polyrnorphurn. In addition, a n t i b o d y titers were significantly r e d u c e d by absorbing the serum with h o m o l o g o u s b u t n o t h e t e r o l o g o u s bacterial antigens, indicating the i m m u n o l o g i c a l specificity o f the R I A .
Z
x
rrZ w 0 coo
I
~ D .,,..,.~ l
300
'~
A--
9Go
2,7~o
8,,bo
I/SERUM DILUTION
Fig. 4. F. p o l y m o r p h u m a n t i b o d y t i t r a t i o n b y R I A o f a h u m a n serum in 1% B S A : unabsorbed (o); absorbed with F. polymorphum antigen (eJ); absorbed with E. coli antigen (/,).
Control counts/rain were 2,256 (o), 2,028 (~1), and 1,453 (/~).
323
Prevalence o f a n t i b o d i e s to E. coli in h u m a n sera T h e R I A p r o c e d u r e was u s e d t o s c r e e n sera f o r t h e p r e s e n c e o f a n t i b o d i e s t o E. coll. T w e n t y - f o u r h u m a n sera w e r e t e s t e d s i m u l t a n e o u s l y a n d in d u p l i c a t e at a d i l u t i o n o f 1 : 2 0 0 f o r a n t i b o d i e s t o E. coli; c o n t r o l s i n c l u d e d b e a d s e x p o s e d t o d i l u e n t o n l y (1% B S A in PBS). T i t e r s w e r e d e t e r m i n e d on e a c h o f t h e 24 sera, using t h e s t a n d a r d curve p r i n c i p l e d e s c r i b e d . T h e serial d i l u t i o n m e t h o d was used to t i t r a t e a s e r u m f o r a n t i b o d i e s t o E. coli. R e s u l t s w e r e p l o t t e d as c o u n t s / m i n versus t h e r e c i p r o c a l o f t h e s e r u m d i l u t i o n a n d this c u r v e was u s e d as a s t a n d a r d c u r v e . T i t e r s w e r e r e a d as t h e r e c i p r o c a l o f t h e d i l u t i o n giving 6 0 1 2 c o u n t s / r a i n w h i c h was t w i c e t h e b a c k g r o u n d c o u n t s o f t h e d i l u e n t c o n t r o l . L o w t i t e r s o f a n t i b o d i e s to E. coli w e r e p r e v a l e n t in t h e s e n o r m a l sera; t h e t i t e r s r a n g e d f r o m < 9 0 t o 1400. T h e s e r e s u l t s are c o n s i s t e n t w i t h o t h e r p u b l i s h e d r e p o r t s c o n f i r m i n g t h e p r e s e n c e of l o w levels o f E. coli a n t i b o d y in n o r m a l h u m a n sera ( N e t e r et al., 1 9 5 2 ; W i n b e r g e t al., 1 9 6 3 ; T h o m a s et al., 1 9 7 4 ) . ACKNOWLEDGEMENT S u p p o r t e d in p a r t b y G r a n t R 0 1 NS 1 0 3 0 9 - 0 2 f r o m t h e N e u r o l o g i c Diseases a n d S t r o k e I n s t i t u t e , N . I . H . REFERENCES Daugharty, H., D.T. Warfield and M.L. Davis, 1972, Appl. Mierobiol. 23, 360. Hebert, G.A., P.L. Pelham and B. Pittman, 1973, Appl. Microbiol. 25,26. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194, 495. Hutchinson, H.D. and D.W. Ziegler, 1972, Appl. Microbiol. 24, 742. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Nassau, E., E.R. Parsons and G.D. Johnson, 1975, J. Immunol. Methods 6, 261. Neter, E., L. Bertram, D. Zak, M. Murdock and C. Arbesman, 1952, J. Exp. Med. 96, 1. Patterson, W.R. and K.O. Smith, 1975, J. Clin. Microbiol. 2, 130. Smith, K.O., D. Gehle and A.W. McCracken, 1974, J. Immunol. Methods 5, 337. Thomas, V., A. Shelokov and M. Forland, 1974, New England J. Med. 290, 588. Winberg, J., H.J. Andersen, L.A. Hanson and K. Lincoln, 1963, Brit. Med. J. 2, 524.
313
© E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
RADIOIMMUNOASSAY P R O C E D U R E FOR Q U A N T I T A T I N G B A C TER I AL ANTIBODY IN HUMAN SERA
B A R B A R A A. S A N F O R D and K E N D A L L O. SMITH
Department o f Microbiology, The University o f Texas ltealth Science Cenler at San Antonio, San Antonio, Texas 78284, U.S.A. (Received 16 F e b r u a r y 1976, a c c e p t e d 15 O c t o b e r 19751
A R I A s y s t e m was d e v e l o p e d to d e t e c t a n t i b o d i e s in h u m a n sera against bacteria. S o n i c a t e s o f Escherichia coli and Fusobacterium polymovphum were used as antigens to sensitize plastic-coated beads; a n t i b o d i e s to t h e s e antigens were d e t e c t e d with 12sI-labeled a n t i h u m a n globulin. S e r u m a n t i b o d y titers against E. coli were d e t e r m i n e d by the serial dilution m e t h o d ; from the results the s t a n d a r d curve principle was applied in d e t e r m i n i n g the relative a m o u n t s o f a n t i b o d i e s against E. coli in s e r u m samples t e s t e d at a single dilution. Tbe c o e f f i c i e n t o f variation o f the RIA p r o c e d u r e was < 10%. S e r u m titers o b t a i n e d by the RIA and indirect i m m t i n o f l u o r e s e e n c e tes{ were c o m p a r e d ; R I A was m o r e sensitivo, q u a n t i t a t i v e and objective. A b s o r p t i o n studies, using E. coil and F. p o l ym o rp h u m a b s o r b e n t s , indicated the specificity o f the R I A s y s t e m in d e t e c t i n g a n t i b o d i e s against E. coli and F. polymorphum. This RIA p r o c e d u r e o f f e r s a c o m b i n a t i o n o f desirable advantages; it is sensilive, specific, objective, q u a n t i t a l i v e and easy to p e r f o r m .
INTRODUCTION Radioimmunoassay (RIA) is not a generally accepted routine procedure for detecting and quantitating immunoglobulins to bacterial antigens. However RIA has been used experimentally to measure bacterial antibodies. Hutchinson and Ziegler (1972) absorbed whole cells of Escherichia coli o n t o glass slides, reacted the bacteria with immune rabbit s e r u m and detected the primary immune reaction with ~2SI-labeled antirabbit serum. They were able to differentiate between two strains of E. coli (01 and 0119) using homologous and heterologous i m m une sera. Nassau et al. (1975) also applied an indirect RIA p r o c e dur e to the serological diagnosis of tuberculosis by using p oly s ty r en e wells sensitized with soluble culture filtrates of Mycobacterium tuberculosis H37 Rv. Human sera were reacted with the adsorbed antigen and the uptake of ant i body was det ect ed with radioiodinated antiglobulin. In their initial study the relative activity of sera correlated with the presence or absence of active tuberculosis. Recently Smith et al. (1974) developed a rapid and simple RIA system for detecting viral a nt i body in human sera which uses imitation pearls as antigen absorbents. The object of this report is to describe the successful adaptation
314 of this RIA procedure for use in quantitating specific bacterial ant i body in h u man sera. Results obtained with RIA are compared with results obtained by the more c o m m o n l y used indirect immunofluorescence test (FA). MATERIALS AND METHODS
Preparation of bacterial antigens for RIA and FA procedures Two gram-negative bacilli, Escherichia coli 075 and Fusobacterium polymorphum (American T y p e Culture Collection # 1 0 9 5 3 ) were used as antigens in this study. E. coli, a m e m b e r of the family Enterobacteriaceae, is a normal inhabitant of the gastrointestinal tract of man, while the anaerobic F. polymorphum belongs to the family Bacteroidaceae and is part of the normal flora o f the human oral cavity. Stock cultures of the two organisms were prepared by culturing the E. coli in T o d d - - H e w i t t broth at 37°C for 24 h and culturing F. polymorphum in fluid thioglycollate medium (10% fetal calf serum) in a Torbal container gassed with 10% H2 and 90% CO2 at 37°C for 48 h. Aliquots of the stock cultures were frozen at --70°C and used t h r o u g h o u t the study. Crude bacterial antigens used in the RIA procedure were prepared as follows : A fresh subculture of the stock organism was centrifuged at 2300 g for 10 min, washed 3 times with phosphate buffered saline (PBS, 0.01 M phosphate in 0.15 M saline), pH 7.2, and resuspended in 5 ml of PBS. One volume (dry weight) of powdered glass beads (25 p diameter, Heat Systems -Ultrasonics, Inc., Plainview, N.Y.) was added to 3 volumes of the bacterial suspension. The suspension was placed in an ice bath and sonicated with a microtip sonifier cell disrupter (Heat Systems -- Ultrasonics, Inc., Plainview, L.I., N.Y.) set at 7, using 10 sec bursts for a total t r e a t m e n t of 1 min. This procedure was usually repeated 5 times to obtain disruption of F. polymorphum. The suspension was centrifuged at 2600 g for 3 rain and the supernatant fluid ('crude' antigen} was removed and used immediately or stored at --70°C. Protein determinations on the 'crude' antigens were made by the L o w r y procedure (1951). Bacterial antigens for the FA procedure were prepared as follows: A fresh subculture of the stock organism was centrifuged at 2300 g for 10 min, washed 3 times with PBS, and adjusted by dilution in PBS to a reading of 50 on a Klett-Summerson photoelectric colorimeter. This dilution was experimentally established to correspond to the n u m b e r of bacteria (150 to 200 per high-power field) remaining on the slide after the washing procedures. A volume of 0.025 ml of the standardized bacterial suspension was smeared thinly on glass slides, air-dried, fixed with acetone, washed twice with PBS, and used immediately for i m m unof l uor es c e nce testing.
Source of sera Human sera were obtained from the Serology and Clinical Chemistry Laboratories, Bexar C ount y Hospital, San Antonio, Texas. In addition, several
315 patients' sera known to contain high titers of antibodies against E. eoli 075 were kindly supplied by Dr. Virginia L. Thomas in our d e p a r t m e n t . All sera were stored at --70 ° C.
RIA procedure Plastic-coated beads (6 mm diameter, Grieger's of Pasadena, California, W773-6) were used to absorb 'crude' bacterial antigens for the detection of a n t i b o d y in human sera by RIA. The n u m b e r of beads needed for each experiment were placed in a beaker and rinsed twice in distilled water. T hey were sensitized with bacterial antigen by adding a volume of soluble antigen which co mp letel y covered the beads and allowing to stand for 20 rain at 25°C. The sensitized beads were rinsed with 1% bovine serum albumin (BSA) dissolved in PBS. Duplicate beads were placed in 2 ml of test serum previously diluted in 1% BSA and incubated at 4°C for 18 h. After incubation, each sensitized bead was dipped 6 times in 1% BSA then washed in a fresh volume of 1% BSA by dipping 6 more times. Each bead was then placed in 1 ml of ~2~I-labeled polyvalent antihuman IgG + IgA + IgM globulin and incubated at 25°C for 4 h. The beads were washed as described, placed in plastic vials and measured for activity in a Beckman gamma counter. The conjugated antiserum used in the RIA procedure was prepared by fractionating the goat antihuman IgG + IgA + IgM (Hyland, Los Angeles, California) using the ammonium sulfate fractionation m e t h o d of Hebert et al. (1973). The gamma globulin fraction was labeled with J2sI by the Chloramine T Method of Daugharty et al. (1972) or t t u n t e r and G r e e n w o o d (1962) using a 1--5 mgm protein c o n c e n t r a t i o n of gamma globulin and 2 mCi Na ~2s[.
Immunofluorescence procedure Fluorescein-conjugated polyvalent antihuman immunoglobulins (goat origin) (Hyland, Los Angeles, California) were absorbed to remove any naturally-occurring antibodies against the test bacteria. The antiserum was diluted 1 : 2 in PBS and absorbed twice (25°C, 1 h) with formalin-killed subcultures o f E. coli 075 and F. polymorphum previously washed 3 times with PBS. The working dilution of the absorbed conjugate was determined by block titration using E. coli 075 antigen and two standardized sera. The conjugate had a fluorescein to protein molar ratio of 3.9, a fluorescein to protein weight ratio of 9.5 #g/rag and a protein-bound fluorescein isothiocyanate c o n cen tr atio n of 242 #g/rag. Ringed areas marked on each slide were flooded with 0.025 ml of test serum and incubated at 37°C for 30 min. The slides were washed twice in PBS and treated with the absorbed fluorescein conjugate. After incubation at 37°C for 30 min, the slides were washed twice in PBS and examined microscopically for apple-green fluorescence (AO Spencer fluorolume illuminator, model 645) with the 50× oil objective. Saline controls were included in each test.
316 RESULTS AND DISCUSSION S t a n d a r d i z a t i o n o f antigen for R I A A d u p l i c a t e b l o c k t i t r a t i o n was d o n e to d e t e r m i n e the o p t i m u m c o n c e n t r a t i o n o f bacterial antigen n e e d e d to sensitize beads. Beads were sensitized in 10-fold serial dilutions o f E. coli antigen, 1 : 101--1 : 10 ~, in PBS. The sensitized beads were reacted with 3-fold serial dilutions of an h y p e r i m m u n e serum, 1 : 10~--1 : 10 ~, in 1% BSA, and the R I A test was d o n e as described. Results were p l o t t e d as c o u n t s / m i n u t e versus the reciprocal of the antigen dilution for each d i l u t i o n o f s e r u m tested. Fig. 1 shows the curve for the 1 : 1 0 0 0 dilution o f s e r u m and d e m o n s t r a t e s a sharp decrease in c o u n t s with beads sensitized in dilutions of E. coli antigen greater than 1 : 100. At the 1 : 100 dilution the antigen c o n t a i n e d 0.16 mg p r o t e i n / m l . All s u b s e q u e n t e x p e r i m e n t s using R I A were d o n e with a c o n c e n t r a t i o n o f either 0.16 mg or 1.65 mg p r o t e i n / m l o f bacterial antigen to sensitize the beads. Use o f a standard curve Using R I A , Patterson and S m i t h ( 1 9 7 5 ) r e p o r t e d that viral a n t i b o d y titers in h u m a n sera can be d e t e r m i n e d f r o m a standard curve. Their m e t h o d elimihates the need for using the m o r e t e d i o u s and t i m e - c o n s u m i n g serial dilution m e t h o d to d e t e r m i n e serum a n t i b o d y titers. We p e r f o r m e d similar experim e n t s to see if the bacterial a n t i b o d y titers o f h u m a n sera c o u l d also be d e t e r m i n e d f r o m a s t a n d a r d curve. T e n sera were titrated by the serial dilution m e t h o d against E. coli by R I A . Results are given in table 1. Each well c o n t a i n e d an average of 2.7 × 10 ~' c o u n t s / m i n u t e / m l of 12~I-labeled a n t i s e r u m . A p p r o x i m a t e l y 6% o f the c o u n t s were a b s o r b e d in wells c o n t a i n i n g labeled a n t i s e r u m only. As can be
b6
141512
b ~o9-
7 6 5 4 3
ANTIGEN DILUTION
Fig. 1. E. coli 'crude' antigen titration against a 1 : 1,000 dilution of E. coli hyperimmune human serum by RIA. CPM, counts/min.
32,155 11,982 19,672 20,970 22,783 21,362 25,081 25,670 18,149 16,563
1:90
27,763 6,633 11,347 24,189 17,407 15,021 18,204 19,957 11,147 9,368
1:270
22,675 4,450 7,141 19,527 9,592 8,127 11,124 12,939 7,102 5,945
1:810
17,128 3,371 4,444 8,387 5,121 5,264 7,597 7,365 4,117 3,860
1:2430
9,091 2,918 3,312 3,790 3,030 3,173 5,454 3,929 3,043 2,874
1:7290
4,882 2,546 2,667 2,815 2,554 2,625 3,421 2,846 2,876 3,148
1:21,870
C o u n t s / m i n a of E. coli sensitized beads reacted with differed s e r u m d i l u t i o n s
3,279 2,608 3,133 3,696 2,604 2,087 2,550 2,480 2,915 2,991
1:65,610
2.56 2.50 2.99 2.57 2.44 2.48 2.46 2.54 2.50 2.45
3,456
Mean = 2,700
albumin c
3,580 3,727 3,877 4,009 3,100 3,604 2,752 3,067 3,037 3,816
E. coli antigen e
4,012
5,404 4,006 4,357 3,889 3,673 4,094 3,405 2,593 4,079 3,616
Non-sensitized d
Bovine s e r u m
C o u n t s / m i n a of c o n t r o l b e a d s sensitized with :
2,905 2,554 3,020 2,700 2,288 2,808 2,973 2,290 2,782 2,680
Total c o u n t s rem a i n i n g in t h e well b X 10 ¢'
a Mean c o u n t s / m i n of duplicate tests. b C o u n t s / r a i n r e m a i n i n g in the wells after r e m o v a l o f E. coli sensitized beads previously i n c u b a t e d in the 1 : 9 0 d i l u t i o n o f t h e corres p o n d i n g test serum. c I n c u b a t e d in d i l u e n t w i t h o u t s e r u m t h e n i n c u b a t e d in labeled a n t i s e r u m . d I n c u b a t e d in labeled a n t i s e r u m only.
1 2 3 4 5 6 7 8 9 10
Serum No.
Results of t i t r a t i n g the h u m a n sera for a n t i b o d i e s to E. coli 075 by RIA.
TABLE 1
318
275f 2504 225 200 "?,
t75
_0 150 125100 75 5.025 0
f
270
i
810
i
,
,
•
2430 7290 21,870 65,610 I/SERUMDILUTION Fig. '2. E. coli antibody titration of a human serum by RIA; curve was used as a standard curve in determining titers of nine other sera. 9O
seen in t a b l e 1, the c o u n t s d r o p p e d 12% a f t e r i n c u b a t i o n of sensitized b e a d s p r e v i o u s l y e x p o s e d to the 1 : 90 d i l u t i o n o f s e r u m # 5 ; this m a y p a r t l y be e x p l a i n e d b y n o n - s p e c i f i c a d s o r p t i o n o f labeled a n t i s e r u m to t h e walls o f t h e well. T h e last t h r e e c o l u m n s in t a b l e 1 indicate n o n - s p e c i f i c a d s o r p t i o n o f labeled a n t i s e r u m to (1) beads sensitized with t h e E. coli antigen and incub a t e d in d i l u e n t w i t h o u t s e r u m , (2) beads sensitized with an u n r e l a t e d antigen (BSA) and i n c u b a t e d in d i l u e n t w i t h o u t s e r u m , and (3) beads w h i c h w e r e n o t c o a t e d b u t s i m p l y placed in labeled a n t i s e r u m for t h e 4 h incubation period. Results f r o m t i t r a t i n g each o f t h e 10 sera w e r e p l o t t e d as c o u n t s / r a i n versus the r e c i p r o c a l o f t h e s e r u m dilution. Curves w i t h similar slopes w e r e o b t a i n e d ; this suggested t h a t a s t a n d a r d curve c o u l d be c o n s t r u c t e d using 1 s e r u m to which all o t h e r sera can be c o m p a r e d . This p o s s i b i l i t y was tested. T h e curve o f s e r u m # 7 (fig. 2) was t r a n s f e r r e d to tracing p a p e r and t h e n s u p e r i m p o s e d on each c o r r e s p o n d i n g p o i n t o f the curves o b t a i n e d a f t e r plotting each o f t h e r e m a i n i n g 9 sera. The titer was d e t e r m i n e d f o r each s e r u m d i l u t i o n b y e x t r a p o l a t i o n f r o m the s t a n d a r d curve; each titer was read as the r e c i p r o c a l of the d i l u t i o n giving 5 4 0 0 c o u n t s / m i n w h i c h was t w i c e the b a c k g r o u n d c o u n t s o f t h e d i l u e n t c o n t r o l (see table 1). Results are s h o w n in table 2. In using a s t a n d a r d curve to d e t e r m i n e a n t i b o d y titers it s e e m s desirable to screen each s e r u m at 2 dilutions, i.e. 1 : 1 , 0 0 0 and I : 1 0 , 0 0 0 . T h e titers o b t a i n e d using a s t a n d a r d c u r v e c o m p a r e d f a v o r a b l y with t h o s e o b t a i n e d b y the serial d i l u t i o n m e t h o d . C o m p a r i s o n o f R I A and FA titers Because i m m u n o f l u o r e s c e n c e is a w i d e l y used and sensitive m e t h o d f o r d e t e c t i n g p r i m a r y a n t i g e n - - a n t i b o d y r e a c t i o n s , we d e c i d e d to c o m p a r e this
TABLE 2
16,000 10,000
250 325 660 1,150
1,000 680 1,300 1,700 2,300 8,000 3,000 5,280
2,950 2,950 2,100 2,430
1,600 2,000 1,800 2,200 2,300
7,290 7,290 7,290 7,290 7,290 7,290 7,290
10,000 14,000 15,000 6,500 2,400
8 660 660 1,300 1,500 2,000
9
500 500 900 1,300
10
a Reciprocal of the highest dilution at which a superimposed standard curve dropped below 5,400 counts/min (twice the background counts of the diluent control).
1:90 1:270 1:810 1:2,430 1:7,290 1:21,870 1:65, 610
Extrapolation from Serum antibody titers determined by standard curve a the point at this serum dilution 1 2 3 4 5 6 7
RIA titers of human sera obtained by extrapolating from single points, using a superimposed standard curve.
¢D
50
320 TABLE 3 Comparison in sensitivities of the fluorescent antibody (FA) and the radioimmunoassay (RIA) for detecting antibody to E. coli in human sera. Serum number
Antibody titer ......................... FA a RIA b
1 2 3 4 5 6 7 8 9 10
2,048 128 64 1,024 128 128 128 64 6,1 32
18,000 520 1,700 .l,800 2,350 2,500 7,290 4,600 1,500 1,000
Fold difference 9 4 26 5 18 20 57 72 23 31 mean = 27
a Reciprocal of highest serum dilution giving positive immunofluorescence. b Determined hy plotting counts/rain versus reciprocal of serum dilution, then reading the titer at a point where the curve dropped below 5,400 counts/rain (twice the background counts of the diluent control). t e s t s y s t e m w i t h R I A in d e t e c t i n g b a c t e r i a l a n t i b o d i e s in h u m a n sera. Using t h e serial d i l u t i o n m e t h o d , a n t i b o d y t i t e r s a g a i n s t E. coli w e r e d e t e r m i n e d b y F A on t h e 10 sera p r e v i o u s l y t i t r a t e d b y R I A . T w o - f o l d serial d i l u t i o n s (1 : 4 - - 1 : 8 1 9 2 ) of t h e sera were t e s t e d . T h e r e s u l t s o f F A tests c o r r e s p o n d e d to r e s u l t s of R I A ( t a b l e 3); h o w e v e r , several i m p o r t a n t d i f f e r e n c e s b e t w e e n r e s u l t s o f t h e 2 t e s t s y s t e m s are a p p a r e n t . F i r s t , t h e t i t e r s o b t a i n e d b y R I A w e r e h i g h e r t h a n t h e F A t i t e r s b y an average d i f f e r e n c e of 2 7 - f o l d , i n d i c a t i n g t h e i n c r e a s e d s e n s i t i v i t y o f R I A . S e c o n d , t h e R I A d e t e c t e d q u a n t i t a t i v e diff e r e n c e s a m o n g t h e sera w h i c h w e r e n o t a p p a r e n t b y F A . T h e i m m u n o f l u o r e s c e n c e p r o c e d u r e is n o t r o u t i n e l y a q u a n t i t a t i v e p r o c e d u r e as is R I A . B e c a u s e i m m u n o f l u o r e s c e n c e is n o t e a s i l y q u a n t i t a t e d , t h e s t a n d a r d curve p r i n c i p l e c a n n o t be a p p l i e d , m a k i n g t h e t i t r a t i o n o f sera a n d r e a d i n g o f r e s u l t s b y F A b o t h t e d i o u s a n d t i m e - c o n s u m i n g . Also, in c o n t r a s t t o R I A , i n t e r p r e t a t i o n o f F A r e s u l t s is s u b j e c t i v e r a t h e r t h a n o b j e c t i v e , w h i c h can l e a d to i n v e s t i g a t o r bias in r e a d i n g e n d p o i n t s .
R e p r o d u c i b i l i t y o f the R I A test R e p l i c a t e R I A t e s t s w e r e p e r f o r m e d using a 1 : 1 0 0 0 d i l u t i o n o f a h u m a n s e r u m in 1% B S A a n d 1% B S A o n l y ; b o t h were r e a c t e d w i t h E. coli sensit i z e d b e a d s . R e s u l t s are given in t a b l e 4. T h e c o e f f i c i e n t of v a r i a t i o n was 5% w i t h h u m a n s e r u m a n d 6% w i t h t h e 1% B S A d i l u e n t . In p r e v i o u s e x p e r i m e n t s t h e c o e f f i c i e n t o f v a r i a t i o n has r e a c h e d 15%.
321 'FABLE 4 Results of replicate RIA tests. Test number
E. coli sensitized beads reacted with 1:1000 dilution of a human serum
1% BSA
1 2 3 4 5 6 7 8 9 10
12,671 12,555 12,833 11,803 13,411 11,626 12,798 12,991 13,520 13,9.16
1,740 1,671 1,641 1,585 1,457 1,856 1,668 1,730 1,671 1,763
mean
12,815
1,678
Standard deviation Coefficient of variation (%)
721.85 5%
107.80 6%
Proof of specificity of the RIA reaction In evaluating the usefulness o f a n y i m m u n o l o g i c a l p r o c e d u r e , especially one w h i c h shows a high degree o f sensitivity, it is necessary to d e m o n s t r a t e specificity o f the test system. We p r o p o s e d to do this by using 2 antigenically dissimilar organisms, E. eoli and F. polymorphum, as test antigens. A series o f a b s o r p t i o n e x p e r i m e n t s were d o n e using either E. coli a b s o r b e n t or F. p o l y m o r p h u m a b s o r b e n t . An h y p e r i m m u n e serum against E. coli was used as the test serum. T w o sets o f 3-fold serial dilutions o f s e r u m , 1 : 3 0 0 - 1 : 6 5 6 , 1 0 0 , were m a d e in each o f the f o l l o w i n g : (1) 1% BSA, (2) ' c r u d e ' E. coli antigen (1.6 mg p r o t e i n / m l ) in 1% BSA, and (3) ' c r u d e ' F. p o l y m o r p h u m antigen (1.5 mg p r o t e i n / m l ) in 1% BSA. A f t e r i n c u b a t i o n at 25°C for 1 h, sensitized beads were a d d e d ; I set o f serum dilutions received beads sensitized with E. coli antigen, the s e c o n d set received beads sensitized with F. polymorphurn antigen. C o n t r o l s i n c l u d e d the f o l l o w i n g : 1) 1% BSA vs. E. coli antigen. 2) 1% BSA vs. F. p o l y m o r p h u m antigen. 3) ' c r u d e ' E. coli antigen in 1% BSA vs. E. coli antigen. 4) ' c r u d e ' E. coli antigen in 1% BSA vs. F. p o l y m o r p h u m antigen. 5) ' c r u d e ' F. polymorphum antigen in 1% BSA vs. E. coli antigen. 6) ' c r u d e ' F. p o l y m o r p h u m antigen in 1% BSA vs. F. polymorphum antigen. The R I A test was carried o u t as described. Results were p l o t t e d as serum e o u n t s / m i n minus the c o r r e s p o n d i n g c o n t r o l c o u n t s / m i n versus the recipro-
322
'41
\,
I 300
900
2,700
8/00
24,300
72,900
218,700
656,100
I/SERUM DILUTION
Fig. 3. E. coli antibody titration by RIA of a human serum in 1% BSA: unabsorbed (®); absorbed with E. coli antigen ("); absorbed with F. polyrnorphum antigen (A). Control counts/rain were 1,244 (o), 1,296 (m) and 1,902 (A). cal o f the serum dilution. Titers were read at the p o i n t w h e r e the curve d r o p p e d b e l o w 1 , 0 0 0 c o u n t s / m i n . Results with E. eoli antigen-sensitized beads (fig. 3) indicate t h a t using E. eoli a b s o r b e n t r e d u c e d the serum titer 76-fold f r o m a titer o f 9 0 , 0 0 0 to a titer o f 1,190 c o m p a r e d to o n l y a 2-fold decrease in titer w h e n F. p o l y r n o r p h u r n was used as a b s o r b e n t . When F. p o l y r n o r p h u r n sensitized beads were tested, the data (fig. 4) s h o w e d t h a t the serum had a low level o f a n t i b o d y to F. p o l y r n o r p h u m which was a b s o r b e d o u t with F. p o l y m o r p h u r n a b s o r b e n t b u t was n o t a f f e c t e d by E. coli absorbent. The test serum was s h o w n to have a high titer of a n t i b o d y against E. eoli, b u t a low titer o f a n t i b o d y against a n o t h e r gram-negative bacillus, F. polyrnorphurn. In addition, a n t i b o d y titers were significantly r e d u c e d by absorbing the serum with h o m o l o g o u s b u t n o t h e t e r o l o g o u s bacterial antigens, indicating the i m m u n o l o g i c a l specificity o f the R I A .
Z
x
rrZ w 0 coo
I
~ D .,,..,.~ l
300
'~
A--
9Go
2,7~o
8,,bo
I/SERUM DILUTION
Fig. 4. F. p o l y m o r p h u m a n t i b o d y t i t r a t i o n b y R I A o f a h u m a n serum in 1% B S A : unabsorbed (o); absorbed with F. polymorphum antigen (eJ); absorbed with E. coli antigen (/,).
Control counts/rain were 2,256 (o), 2,028 (~1), and 1,453 (/~).
323
Prevalence o f a n t i b o d i e s to E. coli in h u m a n sera T h e R I A p r o c e d u r e was u s e d t o s c r e e n sera f o r t h e p r e s e n c e o f a n t i b o d i e s t o E. coll. T w e n t y - f o u r h u m a n sera w e r e t e s t e d s i m u l t a n e o u s l y a n d in d u p l i c a t e at a d i l u t i o n o f 1 : 2 0 0 f o r a n t i b o d i e s t o E. coli; c o n t r o l s i n c l u d e d b e a d s e x p o s e d t o d i l u e n t o n l y (1% B S A in PBS). T i t e r s w e r e d e t e r m i n e d on e a c h o f t h e 24 sera, using t h e s t a n d a r d curve p r i n c i p l e d e s c r i b e d . T h e serial d i l u t i o n m e t h o d was used to t i t r a t e a s e r u m f o r a n t i b o d i e s t o E. coli. R e s u l t s w e r e p l o t t e d as c o u n t s / m i n versus t h e r e c i p r o c a l o f t h e s e r u m d i l u t i o n a n d this c u r v e was u s e d as a s t a n d a r d c u r v e . T i t e r s w e r e r e a d as t h e r e c i p r o c a l o f t h e d i l u t i o n giving 6 0 1 2 c o u n t s / r a i n w h i c h was t w i c e t h e b a c k g r o u n d c o u n t s o f t h e d i l u e n t c o n t r o l . L o w t i t e r s o f a n t i b o d i e s to E. coli w e r e p r e v a l e n t in t h e s e n o r m a l sera; t h e t i t e r s r a n g e d f r o m < 9 0 t o 1400. T h e s e r e s u l t s are c o n s i s t e n t w i t h o t h e r p u b l i s h e d r e p o r t s c o n f i r m i n g t h e p r e s e n c e of l o w levels o f E. coli a n t i b o d y in n o r m a l h u m a n sera ( N e t e r et al., 1 9 5 2 ; W i n b e r g e t al., 1 9 6 3 ; T h o m a s et al., 1 9 7 4 ) . ACKNOWLEDGEMENT S u p p o r t e d in p a r t b y G r a n t R 0 1 NS 1 0 3 0 9 - 0 2 f r o m t h e N e u r o l o g i c Diseases a n d S t r o k e I n s t i t u t e , N . I . H . REFERENCES Daugharty, H., D.T. Warfield and M.L. Davis, 1972, Appl. Mierobiol. 23, 360. Hebert, G.A., P.L. Pelham and B. Pittman, 1973, Appl. Microbiol. 25,26. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194, 495. Hutchinson, H.D. and D.W. Ziegler, 1972, Appl. Microbiol. 24, 742. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Nassau, E., E.R. Parsons and G.D. Johnson, 1975, J. Immunol. Methods 6, 261. Neter, E., L. Bertram, D. Zak, M. Murdock and C. Arbesman, 1952, J. Exp. Med. 96, 1. Patterson, W.R. and K.O. Smith, 1975, J. Clin. Microbiol. 2, 130. Smith, K.O., D. Gehle and A.W. McCracken, 1974, J. Immunol. Methods 5, 337. Thomas, V., A. Shelokov and M. Forland, 1974, New England J. Med. 290, 588. Winberg, J., H.J. Andersen, L.A. Hanson and K. Lincoln, 1963, Brit. Med. J. 2, 524.
