Journal of Immunological Methods, 154 (1992) 217-223
217
© 1992 ElsevierSciencePublishers B.V. All rights reserved0022-1759/92/$05.00
JIM 06436
A quantitative method for measuring the adherence of group B streptococci to murine peritoneal exudate macrophages A n n e R. Sloan and T h o m a s G. Pistole Department of Microbiology, Universityof New Hampshire, Durham, Nil, USA
(Received 12 February 1992,revisedreceived30 April 1992,accepted 1 May 1992)
We have developed a solid phase, direct binding, enzyme-linked immunosorbent assay (ELISA) to detect and quantify the adherence of group B streptococci to murine macrophages. The assay correlated well with direct microscopic quantification of adherence. As few as 3.8 x 104 bacteria/assay well or less than one bacterium per macrophage could be detected. This assay is both quantitative and selective, and is readily adaptable for multiple sample analysis. It provides a valuable alternative to visual detection of bacterial adherence. Key words: Group B streptococcus; ELISA; Macrophage;Adherence
Introduction The group B streptococcus (GBS) is the single most common agent associated with bacteremia and meningitis during the neonatal period (Rubin and McDonald, 1991). Polymorphonuclear neutrophils are known to phagocytose and destroy these bacteria, but this requires opsonization with either specific antibody or complement (Smith et al., 1990). We have been examining the possibility that in individuals such as neonates, for whom serum opsonins may not yet be fully functional, macrophages serve to recognize and destroy 13oCorrespondence to: T.G. Pistole,Department of Microbiology, Universityof New Hampshire, Durham, NH 03824-3544, USA. Abbreviations: DMSO, dimethylsulfoxide; DPBS, Dulbecco's phosphate-bufferedsaline; ELISA, enzyme-linkedimmunosorbent assay; GBS, group B streptococci; M199, Medium 199 with Earle's salts; MEM, minimal essential medium; PBS, phosphate-buffered saline; pM~b, peritoneal macrophage; TMB, 3,3',5,5'-tetramethylbenzidine.
tential pathogens in the absence of serum factors. As part of that study we require an in vitro binding assay for measuring the adherence of GBS to host defense cells. Our initial studies used the binding assay developed by Glass et al. (1981), which involves the direct microscopic examination of stained cells. Although a valuable assay, it suffers from several limitations. The procedure is labor-intensive, the data are subjectively derived, ann ~ne assay lacks good reproducibility. Other protocols for measuring bacterial attachment to eukaryotic cells have been developed, including ones using fluorescence-activated cell sorting (Ogle et al., 1988), fluorescence microscopy (Oben and Foreman, 1988), radiolabeled bacteria (Verhoeff et al., 1977), or electronic particle coL:nters (Gorman et al., 1986), but none provides the composite attributes of repeatability, versatility, and low cost we sought. Ofek et al. (1986) reported on an ELISA-based system for determining the adherence of bacteria
218
to emerocytes or oral epithelial cells. Subsequently Athamna and Ofek (1988) applied this approach to quantify the adherence of Klebsiella pneumon/ae to mouse peritoneal macrophages. Here we report on an adaptation of this technique for quantifying adherence of a gram-positive bacterium to phagocytic cells.
Materials and methods
Bacteria Type III group B streptococci, strain 18RS21, kindly provided by Dr. Dennis Kasper, Harvard Medical School, Boston, MA, were grown in Todd-Hewitt broth (Difco Laboratories, Detroit, MI) at 37°C to mid-log phase under static conditions. The bacteria were harvested by centrifugation and were washed in Dulbecco's phosphatebuffered saline (DPBS). Stock suspensions of GBS were stored at -70°C in DPBS containing 8% dimethylsulfoxide (DMSO; Sigma Chemical Co., St. Louis, Me). Before use in each assay frozen aliquots were thawed, washed with DPBS, and adjusted to the appropriate density. The concentration of bacteria in the suspension was determined by direct counts in a Petroff-Hansser chamber (C.A. Hausser and Son, Philadelphia, PA). Macrophages Peritoneal exudate macrophages (pM~b) from 8-12-week-old female BALB/c mice (originally obtained from Charles River Laboratories, Wilmington, MA and subsequently bred at our institution) were elicited by intraperitoneai injections of 2 ml of Brewer thioglycollate (Difco Laboratories). After 3 days the pM~ were harvested by lavage using 10 ml of cold Medium 199 with Earle's salts (M199; Gibco Laboratories, Grand Island, NY). The p M 0 were washed twice and resuspended in M199 to a concentration of 106 cells/ml. Ant/body Rabbit antiserum against the cell wall polysaccharide of type Ill GBS was kindly provided by Dr. Dennis Kasper.
Cell lines The WISH (ATCC CCL 25, Rockville, MD) and L929 (ATCC CCL1) cell lines were used as controls for bacterial attachment. Both cell lines were maintained in log phase using minimal essential medium (MEM) with Earle's salts containing 15% fetal bovine serum (Hyclone Laboratories, Logan, UT), 2 mM L-glutamine (Gibco), and 100 /~M non-essential amino acids (Gibco) at 37°C with 5% CO 2, 95% air. The cell lines were harvested using 0.05% trypsin-EDTA (Gibco) and resuspended in M199 at a concentration of 106 cells/mi. Immobilization of cells onto the microtitration plate 200 /tl of pM0 or of control cell lines (106 cells/ml) were distributed to the wells of a 16chamber glass slide (Lab-Tek, Nunc, Naperville, IL). The cells were sedimented to the bottom of the plate by centrifugation at 100 × g for 5 min, then allowed to attach for 3 h at 37°C. The cell monolayers were washed twice with DPBS to remove unattached cells. Microscopic assay for measuring GBS binding to pM~ We developed a modification of the original procedure (Glass et al., 1981). Suspensions of GBS in 100/zl of DPBS, at concentrations of 109, 2 × l0 s, and 4 x 107 bacteria/ml, were added to the pM~ and control cells in duplicate. After incubation at 37°(2 for 1 h the wells were washed four times with DPBS to remove non-adherent bacteria. The chambers were then removed and the slide air-dried, fixed in methanol, and stained with a modified Wright's stain (Leukostat, Fisher Scientific, Pittsburgh, PA). Approximately 200 pM~ were examined per well for bacterial adherence. Those with two or more (Glass et al., 1981) or five or more bacteria attached were scored as positive. ELISA Suspensions of GBS in 100 ~I of DPBS, at concentrations of 109, 2 × l0 s, and 4 × 107 bacteria/ml were added to the macrophage and control cells in quadruplicate. After incubation at 37°C for 30 min the wells were washed four times with DPBS to remove non-adherent bacteria. The
219 plates were air-dried and fixed with methanol for 10 min. Bacteria extracellularly attached to macrophages or control cells were quantified by ELISA. The choice of a control for bacterial adherence is crucial. We attempted to use empty plastic wells or wells coated with protein blocking agents such as bovine serum albumin, gelatin, or hemoglobin (Ofek et al., 1986) as negative controls. In all cases we found a high degree of bacterial adherence to the empty wells or protein-coated wells when compared with bacterial attachment to pM~5. For this reason we chose to compare bacterial attachment to two types of easily obtained, non-phagocytic cell lines with that of the macrophage. By using either the epithelial-like WISH cell line or the fibroblast-like L929 cell line as controls for non-specific attachment of GBS to the wells of the assay chamber, we obtained low background ELISA readings in these wells, compared with ELISA values for GBS adhering to pM~. The procedure of At.hamna and Ofek (1988) was modified for use with our system. To each well, containing methanol-fixed cells, was added 200/tl of 20 mM phosphate, 0.15 M NaCI (PBS), pH 7.2, containing 1% gelatin (Bio-Rad Laboratories, Richmond, CA), to block non-specific binding of antibody, and 10 /~g/ml of goat immunoglobulin G (Organon Teknika, Durham, NC), to block the Fc receptors on the macrophages. After incubation for 1 h at 37°C, the monolayers were washed three times with PBS containing 0.05% Tween 20 (Bio-Rad), followed by the addition of 100/~i/well of specific antiGBS serum diluted 1/500 in PBS containing 1% gelatin and 0.1% Tween 20 for 1 h at 37°C. The plates were washed three times with PBSge!atln-Twee~ 20~ followed by the addition of 100 /~l/well of horseradish peroxidase-labeled, affinity-purified anti-rabbit immunoglobulin G (Organon Teknika), diluted 1/5000 in PBS-gelatin-Tween 20 for 1 h at 37°C. After three washes with PBS-gelatin-Tween 20, 200 /~1 of the substrate 3,3',5,5'-tetramethylbenzidine (TMB; Sigma) in acetate buffer was added to each well. Since TMB is poorly soluble in aqueous solution, it was first dissolved in DMSO to a final concentration of 42 mM and 1 ml of this TMB-DMSO
solution was added dropwise with gentle shaking to 100 ml of 0.1 M sodium acetate-citric acid buffer, pH 4.9. Just before use, 14.7/zi of 30% hydrogen peroxide was added to the acetate buffer containing TMB for a final concentration of 1.3 mM H20 2. The blue color was allowed to develop for 15 min at room temperature and the enzyme reaction was stopped with the addition of 50 /~1 of 2.0 M H2SO4 to each well. The absorbance was read at 450 nm with an ELISA plate reader (Whittaker M.A. Bioproducts, Walkerviile, MD). The following control systems were included: (1) blank wells (no bacteria or eukaryotic cells), (2) wells with macrophages only (no bacteria), and (3) wells with control cells c~,'y (no bacteria). These controls were included t~ ensure that the eukaryotic cells did not react with the first antibody and to detect any non-specific binding of the antibodies.
Determination of the number of bacteria per monolayer A standard curve made for each test served to derive the number of bacteria per monolayer. For this purpose GBS of known concentration in 100 /~! of distilled water were allowed to dry overnight in the wells of a microtitration plate, followed by fixation with methanol for 10 min. An ELISA was performed on the immobilized bacteria as described above. The ELISA values in OD4s0 units (i.e., optical density at 450 nm) were plotted as a function of the number of bacteria in each well. The curve obtained was used to calculate the number of bacteria attached to the pM~ or cell line monolayer from the ELLS#. values obtained in the test experiment. The standard curve was adjusted for loss of dried bacteria due to washi~ig following the procedure of Athamna and Ofek (1988). GBS were grown in tryptic soy broth (Dffco) containing 1.5 /tCi of 5-[125I]iodo-2'-deoxyuridine (NEN-Dupont, Boston, MA) per ml to mid-log phase at 37°C under static conditions. The radiolabeled bacteria were harvested by centrifugation and washed free of excess radioactivity with DPBS. The bacteria were adjusted as above to the desired concentration and their radioactivity was determined using a gamma counter (Gamma 5500, Beckman Instruments, Fullerton, CA). The radio-
22o labeled bacteria contained 3800 cpm/107 bacteria. The bacterial suspension was diluted in distilled water, dried, and fixed onto two sets of flat-bottomed E I A / R I A Strip-Plate-8 (Costar, Cambridge, MA). One of the sets was washed nine times and the radioactivity of the individual wells of the two sets was determined.
]7
O.2
Determination of the number of pMdp per well This determination was based on the selective staining of the p M ~ nuclei with methylene blue, followed by extraction of the stain (Bracha and Mirelman, 1984). Various concentrations of pM~b in 100/LI of M199 were sedimented in the wells of a microtitration plate, air-dried, and fixed with m e t h a n o l These monolayers were stained with 100/~i of 1% methylene blue solution per well for 10 min, followed by washing with boric acid buffer (pH 8.6). The stain was extracted by adding 0.1 N HCI and reading at 620 nm in the ELISA plate reader. The OD620 values of the extracted stain were plotted as a function of the number of p M ~ in each well to obtain a standard curve. This curve was used to estimate the number of pM~b in each experimental well after extracting and reading the methylene blue stain from each test monolayer. A standard curve made for each test served to derive the number of p M ~ per monolayer. After determination of the number of bacteria in the experimental assays was completed, the same monolayers were washed and stained to quantify the number of p M ~ , using the standard curve previously derived.
Results Standard curves for adherence of GBS and pM~b In order to quantify bacterial adherence using the ELISA system it was first necessary to construct standard curves to determine the number of bacteria and of macrophages in each system. Serially diluted suspensions of GBS, dried and fixed on microtitration plates, were reacted with the ELISA reagents. As shown in Fig. 1, a linear relationship between the number of immobilized bacteria and the ELISA values was obtained over
o olo 4
lO5
lO6
1~7
Bacteria/well
Fig. 1. Standard curve for the determination of numbers of bacteria per well. ELISA values (A450) as a function of increasingnumbers of dried, immobilizedstreptococci the range of 6 x 104 to 6 x 106 bacteria/well. To determine whether significant numbers of bacteria were lost during washing surface-labeled (t25 I) bacteria were dried and fixed to the bottom of microtitration plate wells. One series was washed nine times while the others remained unwashed. The amount of remaining radioactivity was determined for each sample. The data, depicted in Table I, indicate there was negligible loss of radioactivity, and hence of bacteria, due to washing over the concentration range of 2 × 10 3 to 2 x 108 bacteria added. The number of adherent pM~b was estimated by staining with methylene blue and quantifying the extracted dye. As shown in Fig. 2, the OD620 values of the extracted methylene blue were linTABLE ! EFFECT OF WASHING ON THE ADHERENCE OF BACTERIA Use of 1251-labeledGBS. No. of bacteria added
cpm for Non-washeda
2 x IOs 36,403+353 4 × 1 0 7 6,990_+!16 8 ×106 i,372+ 15 1.6×106 301_+ 13 3.2x 105 106_+ II 6.4x 104 57± 8 1.3x 104 43_+ 7 2.6× 10 3 49± 5 a Mean+SE (n a4).
Washeda
Percentage remaining after washing
35,687+ 477 6,848+ 65 1,353-+ 8 323± 12 104+ 19 58+ 13 50_+ 16 54_+ 3
98 98 99 107 98 102 116 110
221 1.2 1.o ]
TABLE II ATTACHMENT OF GROUP B STREPTOCOCCI TO MACROPHAGES AND CONTROL CELL LINES
o.s
Number of bacteria per well is based on ELISA values. o.s 0.4
.
.
.
.
. . MKmphageNwell
1'07
Fig. 2. Standard curve for the determination of numbers of
Cells
GBS added/ well
Ratio of added
Mean no. ( _+SE) of
pM~b
lxl0 s 2x 107 4X 106 1 × l0 s 2×107 4× 106 1 × 10s 2× 107 4× 106
1,000:1 200:~! 40:1 i,000:1 200:1 40:1 1.000:1 200:1 40:1
per Mc.bb 8.5 _+!.7 t,.5 _+0.4 0.384-0.03 0.67_+0.10 0.27_+0.06 0.13_+0.04 0.53_+0.06 0.22 :~0.04 0.15_+0.01
L929
macrophages per well. Values of extracted methylene blue
stain (A620) as a function of number of sedimented and dried peritoneal macrophages. e a r over t h e r a n g e o f l 0 s to 106 p M ~ / w e l l . T h e s e s t a n d a r d curves w e r e m a d e in e a c h experim e n t a n d u s e d to d e t e r m i n e t h e n u m b e r o f adh e r e n t bacteria p e r p M ~ , w h i c h w a s calculated by dividing t h e total n u m b e r o f bacteria by t h e t o t a l n u m b e r o f p M ~ p e r well.
Adherence assays measured by ELISA T h e n u m b e r o f pM~b o r control cells a d d e d to e a c h well of t h e assay plate w a s 2 x l 0 s. Approxim a t e l y 1 × l 0 s pM4) w e r e immobilized p e r well, as d e t e r m i n e d f r o m t h e pM~b s t a n d a r d curve. T h i s n u m b e r o f cells f o r m e d a c o m p l e t e m o n o layer covering t h e s u r f a c e a r e a o f t h e well. By visual inspection t h e W I S H a n d L929 cells also c o m p l e t e l y covered t h e s u r f a c e a r e a o f t h e well. T h u s , a n y d i f f e r e n c e s in E L I S A v a l u e s b e t w e e n wells c o n t a i n i n g immobilized pM~b a n d t h o s e with control cells is a reflection o f specific bacterial a d h e r e n c e to t h e p M ~ . T h e a d h e r e n c e o f G B S to p M ~ was d o s e - d e p e n d e n t over t h e c o n c e n t r a t i o n r a n g e o f bacteria a d d e d , n a m e l y 4 x 106 to 1 x 1 0 8 / m l . A d h e r e n c e o f less t h a n o n e b a c t e r i u m / c e l l w a s d e t e c t e d by t h e E L I S A t e c h n i q u e (Table II). T o c o n f i r m that E L I S A r e a d i n g s c o r r e s p o n d e d to bacterial a d h e r e n c e to p M ~ , t h e b o t t o m s o f t h e wells were c u t off a n d m o u n t e d o n t o a glass microscope slide, a n d t h e n u m b e r o f a d h e r e n t bacteria p e r p M ~ or control cell w a s c o u n t e d microscopically. C o m p a r a b l e s t u d i e s p e r f o r m e d with n o n phagocytic cell lines revealed significantly lower v a l u e s for microbial a d h e r e n c e . E v e n at t h e high-
WISH
~ l0 s M0/weii.
bn=12. est d o s a g e tested (1 x 10 a bacteria added), t h e b i n d i n g ratios were at least ten-fold lower t h a n t h o s e for p M ~ .
Visual assay for detecting microbial attachment S t a n d a r d a d h e r e n c e assays were p e r f o r m e d by a modification of t h e t e c h n i q u e d e v e l o p e d by G l a s s et ai. (1931). F e r e a c h assay s y s t e m two v a l u e s were d e t e r m i n e d : t h e n u m b e r o f p M O
TABLE Iil ATTACHMENT OF GROUP B STREPTOCOCCI TO MACROPHAGES AND CONTROL CELL LINES Numbers are based on visual assessmentby direct microscopic counting. Cells
GBS added
pM~b
1 × 10s 2×i07 4X106 1×108 2× 107 4×106 1×108 2x107 4×10 ~
L929 WISH
a Mean_+SE (n = 6).
Percentage with >i two bacteria/ cell a 96 + 1.2 82 _+1.7 10 _+2.0 7.6 _+1.1 1.9 _ + 0 . 0 2 0.40_+0.04 6.0 _+0.57 1.6 _ + 0 . 2 6 0.34_+0.05
>/five bacteria/ cell a 33 _+3.5 9.7 _+2.1 !.9 -+0.8 2.7 -+0.42 0.13-+0.02 0.05_+0.02 2.3 _+0.4 0.09-+0.02 0.05_+0.03
222
j'°I ~.,
'
'
"
....
',
'
"
.....
,'o
ELISA Fig. 3. CompaO.~nof bacterial bindingobtained from the direct visualassayaml f~omELISA.Plot with open squares relates results~romvisualassay(M~ binding:g two bacteria) with ELISAdata (numberof bacteria bound9er M~). Plot with closed squares depicts a comparable relationshipbetween visualasse,y (M~bbinding ~ fivebacteria)and ELISA data. with ~ two bacteria attached and the number of pM~ with ~ five bacteria attached. The former is the value used in the original description of this protocol, while the latter is a more rigorous criterion used in our laboratory. The results of these assays are summarized in Table IlL As expected, there was a positive correlation between the number of bacteria added and the percentage of pM~ exhibiting adherence. This was true regardless of which binding criterion was used. At the higher level of GBS added (1 × 10s) virtually all the pM~ bound at least two bacteria; however, only one-third of them were scored positive when the criterion was > five bacteria bound per cell. Comparable studies with nonphagocytic cell lines revealed adherence values ten times lower for each concentration of GBS tested, confirming that the adherence was not a general eukaryotic cell function. AS shown in Fig. 3, there was good correlation between the ELISA readings for adherence and the values obtained by direct microscopic counts. Discussion Phagocytosis of microorganisms is an important host defense among members of the animal kingdom. In order for phagocytosis to be initiated there must be contact between the engulfing cell and the potential pathogen. Assuming that these are not chance encounters there must be some form of recognition between these two participants. In the case of non-opsonin-mediated
phagocytosis the ability to phagocytose microorganisms must be due to inherent properties of the host cell, most likely structures located on the plasma membrane. As part of our studies to identify the receptor on the mouse pM0 responsible for recognition of GBS we sought a binding assay that could be readily performed, gave reproducible results, and was relatively inexpensive to perform. An enzyme-linked assay has the potential to provide objective data and to allow simultaneous testing of multiple variables. Athamna and Ofek (1988) developed an ELISA-based adherence assay using K. pneumoniae and mouse pM~. Our studies, reported here, indicate that this assay can he modified to detect binding of GBS to pM~. In this system bacteria are allowed to adhere to mot~olayers of pM~ and their presence is both detected and quantified by an ELISA, using specific av:ibody to the bacteria as the primary antibody. To calculate binding ratios it was first necessary to verify the numbers of each cell type in our assay chambers. Standard curves plotting the numbers of bacteria per well vs. absorbance at 450 nm (to detect the product formed by horseradish peroxidase in the antibody-enzyme conjugate; Fig. 1) and the number of pM~ per well vs. absorbance at 620 nm (to detect the methylene blue eluted from stained pM~; Fig. 2) each yielded sigmoidal dose-response curves with a linear mid-section. Using this approach we determined the binding ratios for GBS and murine pM~. As shown in Table II, the data obtained are highly replicable. At high ratios of added bacteria to pM0 (1000:1) our system detected 8.5 bacteria per pM~, whereas at a lower ratio (200:1) there were 1.5 bacteria detected per pM4,. Under comparable conditions the control eukaryotic cells consistently exhibited bacterial binding ratios of < 1 per cell. An important difference from other ELISAbased systems for detecting bacterial adherence to M~ was our use of non-phagocytic cell lines as controls. Commonly used blocking agents, including bovine serum albumin, hemoglobin, and gelatin (Ofek et al., 1986) were ineffective, leading to high background levels of bacterial binding in wells devoid of M~. In using the WISH and
223 L929 cells we c r e a t e d conditions in which approximately t h e s a m e a m o u n t o f surface covered by t h e Md~ in t h e e x p e r i m e n t a l s y s t e m s was covered by t h e s e non-phagocytic cells in t h e control wells. Nonspecific b i n d i n g of streptococci to t h e polys t y r e n e plate was t h u s both r e d u c e d a n d m a d e c o m p a r a b l e in all wells. D a t a p r e s e n t e d in T a b l e Ill provides information o b t a i n e d from bacterial b i n d i n g s t u d i e s using direct microscopic counting. A s with t h e E L I S A b a s e d system, results f r o m t h e direct c o u n t i n g assays also yielded a positive d o s e - r e s p o n s e effect. Similarly, t h e control cells gave consistently low b i n d i n g values. A l t h o u g h t h e E L I S A - b a s e d system for q u a n t i ~ i n g bacterial b i n d i n g to eukaryotic cells provides s o m e obvious a d v a n t a g e s over direct visual assay, e a c h s y s t e m is, in fact, assessing a s o m e w h a t different aspect o f a d h e r e n c e . U s i n g E L I S A we c a n obtain i n f o r m a t i o n on a p o p u l a t i o n of M~b, b u t we c a n n o t d e t e r m i n e t h e distribution o f t h e b o u n d a n d d e t e c t e d bacteria over this p o p u lation. T h a t is, we c a n calculate an average binding ratio, e.g., 8.5 bacteria p e r pM~b (Table I1, at high ratio o f a d d e d G B S to MdD, b u t we c a n n o t a s c e r t a i n t h e b i n d i n g p r o p e r t i e s o f individual M~b; for this t h e visual assay s y s t e m r e m a i n s t h e definitive p r o c e d u r e . N o n e t h e l e s s t h e u s e of an E L I S A - b a s e d app r o a c h to m e a s u r e bacterial a d h e r e n c e provides a valuable tool for s c r e e n i n g v a r i o u s assay conditions o r t r e a t m e n t s to d e t e r m i n e their effect on binding, since m a n y variables c a n be s i m u l t a n e ously tested. T h e ability o f this s y s t e m to d e t e c t as few as 3.8 × 10 4 G B S c o m p a r e s favorably with t h e d a t a o f A t h a m n a a n d O f e k (1988) in which t h e y d e t e c t e d 5 × 104 K. pneumoniae. T h e s e studies indicate that a n E L I S A - b a s e d system h a s b r o a d application in s t u d i e s o n microbial p h a g o cytosis.
Acknowledgements W e t h a n k D. K a s p e r for providing streptococcal strain a n d specific a n t i s e r a a n d I. O f e k for valuable discussion.
T h i s r e s e a r c h was s u p p o r t e d in part by Public H e a l t h Service G r a n t A127930 from t h e National Institutes o f H e a l t h a n d Biomedical R e s e a r c h S u p p o r t G r a n t s 2 S07 RR07108-15 a n d 2 S07 RR07108-17.
References Athamna. A. and !. Ofek. (1988) Enzyme-linked immunosorbent assay for quantitation of attachment and ingestion stages of bacterial phagocytosis. J. Clin. Microbiol. 26, 62. Braeha, R. and Mirelman, D. (1984) Virulence of Entamoeba histolytica trophosoites, Effect of bacteria, microaerobic conditions and metronidazole. J. Exp. Med. 160, 353. Glass, E.. Stewart, J. and Weir, D.M. (1981) Presence of bacterial binding qectin-like" receptors on phagocytes. Immunology 44, 529. Gorman, S.P., McCafferty. D.F. and Anderson, L. (1986) Application of an electronic particle counter to the quantification of bacterial and Candida adherence to mucosal epithelial cell~::L~,. Appl. Microbiol. 2, 97. Kasper, D.L.. :Baker, C.L., Galdes, B., Katzenellenbogen, E. and Jen~ngs, H.J. (1983) Immunochemical analysis and immuncgenicity of the type I1 group B streptococcal capsular polysaccharide. J. Clin. Invest. 72, 260. Oben, J.A. and Foreman, J.C. (1988) A simple quantitative fluorimetric assay of in vitro phagooytosis in human neutrophils. J. lmmunol. Methods 112, 99. Ofek, I., Courtney, H.S., Schifferli, D.M. and Beachey, E.H. (1986) Enzyme-linked assay for adherence of bacteria to animal cells. J. Clin. Microbiol. 24, 512. Ogle, J.D., Noel, J.G., Sramkoski, R.M., Ogle, C.K. and Alexander, J.W. (1988) Phagocytosis of opsonized fluorescent mierospheres by human neutrophils. A two-color cytometric method for the determination of attachment and ingestion. J. immunol. Methods 115, 17. Rubin, E.E. and McDonald, LC. (1991) Group B streptococcal disease in twins: Failure of empiric therapy to prevent late onset disease in the second twin. Pediatr. Infect. Dis. J. 10, 621. Smith, C.L., Baker, C.J., Anderson, D.C. and Edwards, M.S. (1990) Role of complement receptors in opsonophagocytosis of group B streptococci by adult and neonatal neutrophils. J. Infect. Dis. 162, 489. Verhoeff, J., Peterson, P.K. and Quie, P.G. (1977)Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: A quantative assay using [3H]thymidine labeled bacteria. J. Immunol. Methods 14, 303.
217
© 1992 ElsevierSciencePublishers B.V. All rights reserved0022-1759/92/$05.00
JIM 06436
A quantitative method for measuring the adherence of group B streptococci to murine peritoneal exudate macrophages A n n e R. Sloan and T h o m a s G. Pistole Department of Microbiology, Universityof New Hampshire, Durham, Nil, USA
(Received 12 February 1992,revisedreceived30 April 1992,accepted 1 May 1992)
We have developed a solid phase, direct binding, enzyme-linked immunosorbent assay (ELISA) to detect and quantify the adherence of group B streptococci to murine macrophages. The assay correlated well with direct microscopic quantification of adherence. As few as 3.8 x 104 bacteria/assay well or less than one bacterium per macrophage could be detected. This assay is both quantitative and selective, and is readily adaptable for multiple sample analysis. It provides a valuable alternative to visual detection of bacterial adherence. Key words: Group B streptococcus; ELISA; Macrophage;Adherence
Introduction The group B streptococcus (GBS) is the single most common agent associated with bacteremia and meningitis during the neonatal period (Rubin and McDonald, 1991). Polymorphonuclear neutrophils are known to phagocytose and destroy these bacteria, but this requires opsonization with either specific antibody or complement (Smith et al., 1990). We have been examining the possibility that in individuals such as neonates, for whom serum opsonins may not yet be fully functional, macrophages serve to recognize and destroy 13oCorrespondence to: T.G. Pistole,Department of Microbiology, Universityof New Hampshire, Durham, NH 03824-3544, USA. Abbreviations: DMSO, dimethylsulfoxide; DPBS, Dulbecco's phosphate-bufferedsaline; ELISA, enzyme-linkedimmunosorbent assay; GBS, group B streptococci; M199, Medium 199 with Earle's salts; MEM, minimal essential medium; PBS, phosphate-buffered saline; pM~b, peritoneal macrophage; TMB, 3,3',5,5'-tetramethylbenzidine.
tential pathogens in the absence of serum factors. As part of that study we require an in vitro binding assay for measuring the adherence of GBS to host defense cells. Our initial studies used the binding assay developed by Glass et al. (1981), which involves the direct microscopic examination of stained cells. Although a valuable assay, it suffers from several limitations. The procedure is labor-intensive, the data are subjectively derived, ann ~ne assay lacks good reproducibility. Other protocols for measuring bacterial attachment to eukaryotic cells have been developed, including ones using fluorescence-activated cell sorting (Ogle et al., 1988), fluorescence microscopy (Oben and Foreman, 1988), radiolabeled bacteria (Verhoeff et al., 1977), or electronic particle coL:nters (Gorman et al., 1986), but none provides the composite attributes of repeatability, versatility, and low cost we sought. Ofek et al. (1986) reported on an ELISA-based system for determining the adherence of bacteria
218
to emerocytes or oral epithelial cells. Subsequently Athamna and Ofek (1988) applied this approach to quantify the adherence of Klebsiella pneumon/ae to mouse peritoneal macrophages. Here we report on an adaptation of this technique for quantifying adherence of a gram-positive bacterium to phagocytic cells.
Materials and methods
Bacteria Type III group B streptococci, strain 18RS21, kindly provided by Dr. Dennis Kasper, Harvard Medical School, Boston, MA, were grown in Todd-Hewitt broth (Difco Laboratories, Detroit, MI) at 37°C to mid-log phase under static conditions. The bacteria were harvested by centrifugation and were washed in Dulbecco's phosphatebuffered saline (DPBS). Stock suspensions of GBS were stored at -70°C in DPBS containing 8% dimethylsulfoxide (DMSO; Sigma Chemical Co., St. Louis, Me). Before use in each assay frozen aliquots were thawed, washed with DPBS, and adjusted to the appropriate density. The concentration of bacteria in the suspension was determined by direct counts in a Petroff-Hansser chamber (C.A. Hausser and Son, Philadelphia, PA). Macrophages Peritoneal exudate macrophages (pM~b) from 8-12-week-old female BALB/c mice (originally obtained from Charles River Laboratories, Wilmington, MA and subsequently bred at our institution) were elicited by intraperitoneai injections of 2 ml of Brewer thioglycollate (Difco Laboratories). After 3 days the pM~ were harvested by lavage using 10 ml of cold Medium 199 with Earle's salts (M199; Gibco Laboratories, Grand Island, NY). The p M 0 were washed twice and resuspended in M199 to a concentration of 106 cells/ml. Ant/body Rabbit antiserum against the cell wall polysaccharide of type Ill GBS was kindly provided by Dr. Dennis Kasper.
Cell lines The WISH (ATCC CCL 25, Rockville, MD) and L929 (ATCC CCL1) cell lines were used as controls for bacterial attachment. Both cell lines were maintained in log phase using minimal essential medium (MEM) with Earle's salts containing 15% fetal bovine serum (Hyclone Laboratories, Logan, UT), 2 mM L-glutamine (Gibco), and 100 /~M non-essential amino acids (Gibco) at 37°C with 5% CO 2, 95% air. The cell lines were harvested using 0.05% trypsin-EDTA (Gibco) and resuspended in M199 at a concentration of 106 cells/mi. Immobilization of cells onto the microtitration plate 200 /tl of pM0 or of control cell lines (106 cells/ml) were distributed to the wells of a 16chamber glass slide (Lab-Tek, Nunc, Naperville, IL). The cells were sedimented to the bottom of the plate by centrifugation at 100 × g for 5 min, then allowed to attach for 3 h at 37°C. The cell monolayers were washed twice with DPBS to remove unattached cells. Microscopic assay for measuring GBS binding to pM~ We developed a modification of the original procedure (Glass et al., 1981). Suspensions of GBS in 100/zl of DPBS, at concentrations of 109, 2 × l0 s, and 4 x 107 bacteria/ml, were added to the pM~ and control cells in duplicate. After incubation at 37°(2 for 1 h the wells were washed four times with DPBS to remove non-adherent bacteria. The chambers were then removed and the slide air-dried, fixed in methanol, and stained with a modified Wright's stain (Leukostat, Fisher Scientific, Pittsburgh, PA). Approximately 200 pM~ were examined per well for bacterial adherence. Those with two or more (Glass et al., 1981) or five or more bacteria attached were scored as positive. ELISA Suspensions of GBS in 100 ~I of DPBS, at concentrations of 109, 2 × l0 s, and 4 × 107 bacteria/ml were added to the macrophage and control cells in quadruplicate. After incubation at 37°C for 30 min the wells were washed four times with DPBS to remove non-adherent bacteria. The
219 plates were air-dried and fixed with methanol for 10 min. Bacteria extracellularly attached to macrophages or control cells were quantified by ELISA. The choice of a control for bacterial adherence is crucial. We attempted to use empty plastic wells or wells coated with protein blocking agents such as bovine serum albumin, gelatin, or hemoglobin (Ofek et al., 1986) as negative controls. In all cases we found a high degree of bacterial adherence to the empty wells or protein-coated wells when compared with bacterial attachment to pM~5. For this reason we chose to compare bacterial attachment to two types of easily obtained, non-phagocytic cell lines with that of the macrophage. By using either the epithelial-like WISH cell line or the fibroblast-like L929 cell line as controls for non-specific attachment of GBS to the wells of the assay chamber, we obtained low background ELISA readings in these wells, compared with ELISA values for GBS adhering to pM~. The procedure of At.hamna and Ofek (1988) was modified for use with our system. To each well, containing methanol-fixed cells, was added 200/tl of 20 mM phosphate, 0.15 M NaCI (PBS), pH 7.2, containing 1% gelatin (Bio-Rad Laboratories, Richmond, CA), to block non-specific binding of antibody, and 10 /~g/ml of goat immunoglobulin G (Organon Teknika, Durham, NC), to block the Fc receptors on the macrophages. After incubation for 1 h at 37°C, the monolayers were washed three times with PBS containing 0.05% Tween 20 (Bio-Rad), followed by the addition of 100/~i/well of specific antiGBS serum diluted 1/500 in PBS containing 1% gelatin and 0.1% Tween 20 for 1 h at 37°C. The plates were washed three times with PBSge!atln-Twee~ 20~ followed by the addition of 100 /~l/well of horseradish peroxidase-labeled, affinity-purified anti-rabbit immunoglobulin G (Organon Teknika), diluted 1/5000 in PBS-gelatin-Tween 20 for 1 h at 37°C. After three washes with PBS-gelatin-Tween 20, 200 /~1 of the substrate 3,3',5,5'-tetramethylbenzidine (TMB; Sigma) in acetate buffer was added to each well. Since TMB is poorly soluble in aqueous solution, it was first dissolved in DMSO to a final concentration of 42 mM and 1 ml of this TMB-DMSO
solution was added dropwise with gentle shaking to 100 ml of 0.1 M sodium acetate-citric acid buffer, pH 4.9. Just before use, 14.7/zi of 30% hydrogen peroxide was added to the acetate buffer containing TMB for a final concentration of 1.3 mM H20 2. The blue color was allowed to develop for 15 min at room temperature and the enzyme reaction was stopped with the addition of 50 /~1 of 2.0 M H2SO4 to each well. The absorbance was read at 450 nm with an ELISA plate reader (Whittaker M.A. Bioproducts, Walkerviile, MD). The following control systems were included: (1) blank wells (no bacteria or eukaryotic cells), (2) wells with macrophages only (no bacteria), and (3) wells with control cells c~,'y (no bacteria). These controls were included t~ ensure that the eukaryotic cells did not react with the first antibody and to detect any non-specific binding of the antibodies.
Determination of the number of bacteria per monolayer A standard curve made for each test served to derive the number of bacteria per monolayer. For this purpose GBS of known concentration in 100 /~! of distilled water were allowed to dry overnight in the wells of a microtitration plate, followed by fixation with methanol for 10 min. An ELISA was performed on the immobilized bacteria as described above. The ELISA values in OD4s0 units (i.e., optical density at 450 nm) were plotted as a function of the number of bacteria in each well. The curve obtained was used to calculate the number of bacteria attached to the pM~ or cell line monolayer from the ELLS#. values obtained in the test experiment. The standard curve was adjusted for loss of dried bacteria due to washi~ig following the procedure of Athamna and Ofek (1988). GBS were grown in tryptic soy broth (Dffco) containing 1.5 /tCi of 5-[125I]iodo-2'-deoxyuridine (NEN-Dupont, Boston, MA) per ml to mid-log phase at 37°C under static conditions. The radiolabeled bacteria were harvested by centrifugation and washed free of excess radioactivity with DPBS. The bacteria were adjusted as above to the desired concentration and their radioactivity was determined using a gamma counter (Gamma 5500, Beckman Instruments, Fullerton, CA). The radio-
22o labeled bacteria contained 3800 cpm/107 bacteria. The bacterial suspension was diluted in distilled water, dried, and fixed onto two sets of flat-bottomed E I A / R I A Strip-Plate-8 (Costar, Cambridge, MA). One of the sets was washed nine times and the radioactivity of the individual wells of the two sets was determined.
]7
O.2
Determination of the number of pMdp per well This determination was based on the selective staining of the p M ~ nuclei with methylene blue, followed by extraction of the stain (Bracha and Mirelman, 1984). Various concentrations of pM~b in 100/LI of M199 were sedimented in the wells of a microtitration plate, air-dried, and fixed with m e t h a n o l These monolayers were stained with 100/~i of 1% methylene blue solution per well for 10 min, followed by washing with boric acid buffer (pH 8.6). The stain was extracted by adding 0.1 N HCI and reading at 620 nm in the ELISA plate reader. The OD620 values of the extracted stain were plotted as a function of the number of p M ~ in each well to obtain a standard curve. This curve was used to estimate the number of pM~b in each experimental well after extracting and reading the methylene blue stain from each test monolayer. A standard curve made for each test served to derive the number of p M ~ per monolayer. After determination of the number of bacteria in the experimental assays was completed, the same monolayers were washed and stained to quantify the number of p M ~ , using the standard curve previously derived.
Results Standard curves for adherence of GBS and pM~b In order to quantify bacterial adherence using the ELISA system it was first necessary to construct standard curves to determine the number of bacteria and of macrophages in each system. Serially diluted suspensions of GBS, dried and fixed on microtitration plates, were reacted with the ELISA reagents. As shown in Fig. 1, a linear relationship between the number of immobilized bacteria and the ELISA values was obtained over
o olo 4
lO5
lO6
1~7
Bacteria/well
Fig. 1. Standard curve for the determination of numbers of bacteria per well. ELISA values (A450) as a function of increasingnumbers of dried, immobilizedstreptococci the range of 6 x 104 to 6 x 106 bacteria/well. To determine whether significant numbers of bacteria were lost during washing surface-labeled (t25 I) bacteria were dried and fixed to the bottom of microtitration plate wells. One series was washed nine times while the others remained unwashed. The amount of remaining radioactivity was determined for each sample. The data, depicted in Table I, indicate there was negligible loss of radioactivity, and hence of bacteria, due to washing over the concentration range of 2 × 10 3 to 2 x 108 bacteria added. The number of adherent pM~b was estimated by staining with methylene blue and quantifying the extracted dye. As shown in Fig. 2, the OD620 values of the extracted methylene blue were linTABLE ! EFFECT OF WASHING ON THE ADHERENCE OF BACTERIA Use of 1251-labeledGBS. No. of bacteria added
cpm for Non-washeda
2 x IOs 36,403+353 4 × 1 0 7 6,990_+!16 8 ×106 i,372+ 15 1.6×106 301_+ 13 3.2x 105 106_+ II 6.4x 104 57± 8 1.3x 104 43_+ 7 2.6× 10 3 49± 5 a Mean+SE (n a4).
Washeda
Percentage remaining after washing
35,687+ 477 6,848+ 65 1,353-+ 8 323± 12 104+ 19 58+ 13 50_+ 16 54_+ 3
98 98 99 107 98 102 116 110
221 1.2 1.o ]
TABLE II ATTACHMENT OF GROUP B STREPTOCOCCI TO MACROPHAGES AND CONTROL CELL LINES
o.s
Number of bacteria per well is based on ELISA values. o.s 0.4
.
.
.
.
. . MKmphageNwell
1'07
Fig. 2. Standard curve for the determination of numbers of
Cells
GBS added/ well
Ratio of added
Mean no. ( _+SE) of
pM~b
lxl0 s 2x 107 4X 106 1 × l0 s 2×107 4× 106 1 × 10s 2× 107 4× 106
1,000:1 200:~! 40:1 i,000:1 200:1 40:1 1.000:1 200:1 40:1
per Mc.bb 8.5 _+!.7 t,.5 _+0.4 0.384-0.03 0.67_+0.10 0.27_+0.06 0.13_+0.04 0.53_+0.06 0.22 :~0.04 0.15_+0.01
L929
macrophages per well. Values of extracted methylene blue
stain (A620) as a function of number of sedimented and dried peritoneal macrophages. e a r over t h e r a n g e o f l 0 s to 106 p M ~ / w e l l . T h e s e s t a n d a r d curves w e r e m a d e in e a c h experim e n t a n d u s e d to d e t e r m i n e t h e n u m b e r o f adh e r e n t bacteria p e r p M ~ , w h i c h w a s calculated by dividing t h e total n u m b e r o f bacteria by t h e t o t a l n u m b e r o f p M ~ p e r well.
Adherence assays measured by ELISA T h e n u m b e r o f pM~b o r control cells a d d e d to e a c h well of t h e assay plate w a s 2 x l 0 s. Approxim a t e l y 1 × l 0 s pM4) w e r e immobilized p e r well, as d e t e r m i n e d f r o m t h e pM~b s t a n d a r d curve. T h i s n u m b e r o f cells f o r m e d a c o m p l e t e m o n o layer covering t h e s u r f a c e a r e a o f t h e well. By visual inspection t h e W I S H a n d L929 cells also c o m p l e t e l y covered t h e s u r f a c e a r e a o f t h e well. T h u s , a n y d i f f e r e n c e s in E L I S A v a l u e s b e t w e e n wells c o n t a i n i n g immobilized pM~b a n d t h o s e with control cells is a reflection o f specific bacterial a d h e r e n c e to t h e p M ~ . T h e a d h e r e n c e o f G B S to p M ~ was d o s e - d e p e n d e n t over t h e c o n c e n t r a t i o n r a n g e o f bacteria a d d e d , n a m e l y 4 x 106 to 1 x 1 0 8 / m l . A d h e r e n c e o f less t h a n o n e b a c t e r i u m / c e l l w a s d e t e c t e d by t h e E L I S A t e c h n i q u e (Table II). T o c o n f i r m that E L I S A r e a d i n g s c o r r e s p o n d e d to bacterial a d h e r e n c e to p M ~ , t h e b o t t o m s o f t h e wells were c u t off a n d m o u n t e d o n t o a glass microscope slide, a n d t h e n u m b e r o f a d h e r e n t bacteria p e r p M ~ or control cell w a s c o u n t e d microscopically. C o m p a r a b l e s t u d i e s p e r f o r m e d with n o n phagocytic cell lines revealed significantly lower v a l u e s for microbial a d h e r e n c e . E v e n at t h e high-
WISH
~ l0 s M0/weii.
bn=12. est d o s a g e tested (1 x 10 a bacteria added), t h e b i n d i n g ratios were at least ten-fold lower t h a n t h o s e for p M ~ .
Visual assay for detecting microbial attachment S t a n d a r d a d h e r e n c e assays were p e r f o r m e d by a modification of t h e t e c h n i q u e d e v e l o p e d by G l a s s et ai. (1931). F e r e a c h assay s y s t e m two v a l u e s were d e t e r m i n e d : t h e n u m b e r o f p M O
TABLE Iil ATTACHMENT OF GROUP B STREPTOCOCCI TO MACROPHAGES AND CONTROL CELL LINES Numbers are based on visual assessmentby direct microscopic counting. Cells
GBS added
pM~b
1 × 10s 2×i07 4X106 1×108 2× 107 4×106 1×108 2x107 4×10 ~
L929 WISH
a Mean_+SE (n = 6).
Percentage with >i two bacteria/ cell a 96 + 1.2 82 _+1.7 10 _+2.0 7.6 _+1.1 1.9 _ + 0 . 0 2 0.40_+0.04 6.0 _+0.57 1.6 _ + 0 . 2 6 0.34_+0.05
>/five bacteria/ cell a 33 _+3.5 9.7 _+2.1 !.9 -+0.8 2.7 -+0.42 0.13-+0.02 0.05_+0.02 2.3 _+0.4 0.09-+0.02 0.05_+0.03
222
j'°I ~.,
'
'
"
....
',
'
"
.....
,'o
ELISA Fig. 3. CompaO.~nof bacterial bindingobtained from the direct visualassayaml f~omELISA.Plot with open squares relates results~romvisualassay(M~ binding:g two bacteria) with ELISAdata (numberof bacteria bound9er M~). Plot with closed squares depicts a comparable relationshipbetween visualasse,y (M~bbinding ~ fivebacteria)and ELISA data. with ~ two bacteria attached and the number of pM~ with ~ five bacteria attached. The former is the value used in the original description of this protocol, while the latter is a more rigorous criterion used in our laboratory. The results of these assays are summarized in Table IlL As expected, there was a positive correlation between the number of bacteria added and the percentage of pM~ exhibiting adherence. This was true regardless of which binding criterion was used. At the higher level of GBS added (1 × 10s) virtually all the pM~ bound at least two bacteria; however, only one-third of them were scored positive when the criterion was > five bacteria bound per cell. Comparable studies with nonphagocytic cell lines revealed adherence values ten times lower for each concentration of GBS tested, confirming that the adherence was not a general eukaryotic cell function. AS shown in Fig. 3, there was good correlation between the ELISA readings for adherence and the values obtained by direct microscopic counts. Discussion Phagocytosis of microorganisms is an important host defense among members of the animal kingdom. In order for phagocytosis to be initiated there must be contact between the engulfing cell and the potential pathogen. Assuming that these are not chance encounters there must be some form of recognition between these two participants. In the case of non-opsonin-mediated
phagocytosis the ability to phagocytose microorganisms must be due to inherent properties of the host cell, most likely structures located on the plasma membrane. As part of our studies to identify the receptor on the mouse pM0 responsible for recognition of GBS we sought a binding assay that could be readily performed, gave reproducible results, and was relatively inexpensive to perform. An enzyme-linked assay has the potential to provide objective data and to allow simultaneous testing of multiple variables. Athamna and Ofek (1988) developed an ELISA-based adherence assay using K. pneumoniae and mouse pM~. Our studies, reported here, indicate that this assay can he modified to detect binding of GBS to pM~. In this system bacteria are allowed to adhere to mot~olayers of pM~ and their presence is both detected and quantified by an ELISA, using specific av:ibody to the bacteria as the primary antibody. To calculate binding ratios it was first necessary to verify the numbers of each cell type in our assay chambers. Standard curves plotting the numbers of bacteria per well vs. absorbance at 450 nm (to detect the product formed by horseradish peroxidase in the antibody-enzyme conjugate; Fig. 1) and the number of pM~ per well vs. absorbance at 620 nm (to detect the methylene blue eluted from stained pM~; Fig. 2) each yielded sigmoidal dose-response curves with a linear mid-section. Using this approach we determined the binding ratios for GBS and murine pM~. As shown in Table II, the data obtained are highly replicable. At high ratios of added bacteria to pM0 (1000:1) our system detected 8.5 bacteria per pM~, whereas at a lower ratio (200:1) there were 1.5 bacteria detected per pM4,. Under comparable conditions the control eukaryotic cells consistently exhibited bacterial binding ratios of < 1 per cell. An important difference from other ELISAbased systems for detecting bacterial adherence to M~ was our use of non-phagocytic cell lines as controls. Commonly used blocking agents, including bovine serum albumin, hemoglobin, and gelatin (Ofek et al., 1986) were ineffective, leading to high background levels of bacterial binding in wells devoid of M~. In using the WISH and
223 L929 cells we c r e a t e d conditions in which approximately t h e s a m e a m o u n t o f surface covered by t h e Md~ in t h e e x p e r i m e n t a l s y s t e m s was covered by t h e s e non-phagocytic cells in t h e control wells. Nonspecific b i n d i n g of streptococci to t h e polys t y r e n e plate was t h u s both r e d u c e d a n d m a d e c o m p a r a b l e in all wells. D a t a p r e s e n t e d in T a b l e Ill provides information o b t a i n e d from bacterial b i n d i n g s t u d i e s using direct microscopic counting. A s with t h e E L I S A b a s e d system, results f r o m t h e direct c o u n t i n g assays also yielded a positive d o s e - r e s p o n s e effect. Similarly, t h e control cells gave consistently low b i n d i n g values. A l t h o u g h t h e E L I S A - b a s e d system for q u a n t i ~ i n g bacterial b i n d i n g to eukaryotic cells provides s o m e obvious a d v a n t a g e s over direct visual assay, e a c h s y s t e m is, in fact, assessing a s o m e w h a t different aspect o f a d h e r e n c e . U s i n g E L I S A we c a n obtain i n f o r m a t i o n on a p o p u l a t i o n of M~b, b u t we c a n n o t d e t e r m i n e t h e distribution o f t h e b o u n d a n d d e t e c t e d bacteria over this p o p u lation. T h a t is, we c a n calculate an average binding ratio, e.g., 8.5 bacteria p e r pM~b (Table I1, at high ratio o f a d d e d G B S to MdD, b u t we c a n n o t a s c e r t a i n t h e b i n d i n g p r o p e r t i e s o f individual M~b; for this t h e visual assay s y s t e m r e m a i n s t h e definitive p r o c e d u r e . N o n e t h e l e s s t h e u s e of an E L I S A - b a s e d app r o a c h to m e a s u r e bacterial a d h e r e n c e provides a valuable tool for s c r e e n i n g v a r i o u s assay conditions o r t r e a t m e n t s to d e t e r m i n e their effect on binding, since m a n y variables c a n be s i m u l t a n e ously tested. T h e ability o f this s y s t e m to d e t e c t as few as 3.8 × 10 4 G B S c o m p a r e s favorably with t h e d a t a o f A t h a m n a a n d O f e k (1988) in which t h e y d e t e c t e d 5 × 104 K. pneumoniae. T h e s e studies indicate that a n E L I S A - b a s e d system h a s b r o a d application in s t u d i e s o n microbial p h a g o cytosis.
Acknowledgements W e t h a n k D. K a s p e r for providing streptococcal strain a n d specific a n t i s e r a a n d I. O f e k for valuable discussion.
T h i s r e s e a r c h was s u p p o r t e d in part by Public H e a l t h Service G r a n t A127930 from t h e National Institutes o f H e a l t h a n d Biomedical R e s e a r c h S u p p o r t G r a n t s 2 S07 RR07108-15 a n d 2 S07 RR07108-17.
References Athamna. A. and !. Ofek. (1988) Enzyme-linked immunosorbent assay for quantitation of attachment and ingestion stages of bacterial phagocytosis. J. Clin. Microbiol. 26, 62. Braeha, R. and Mirelman, D. (1984) Virulence of Entamoeba histolytica trophosoites, Effect of bacteria, microaerobic conditions and metronidazole. J. Exp. Med. 160, 353. Glass, E.. Stewart, J. and Weir, D.M. (1981) Presence of bacterial binding qectin-like" receptors on phagocytes. Immunology 44, 529. Gorman, S.P., McCafferty. D.F. and Anderson, L. (1986) Application of an electronic particle counter to the quantification of bacterial and Candida adherence to mucosal epithelial cell~::L~,. Appl. Microbiol. 2, 97. Kasper, D.L.. :Baker, C.L., Galdes, B., Katzenellenbogen, E. and Jen~ngs, H.J. (1983) Immunochemical analysis and immuncgenicity of the type I1 group B streptococcal capsular polysaccharide. J. Clin. Invest. 72, 260. Oben, J.A. and Foreman, J.C. (1988) A simple quantitative fluorimetric assay of in vitro phagooytosis in human neutrophils. J. lmmunol. Methods 112, 99. Ofek, I., Courtney, H.S., Schifferli, D.M. and Beachey, E.H. (1986) Enzyme-linked assay for adherence of bacteria to animal cells. J. Clin. Microbiol. 24, 512. Ogle, J.D., Noel, J.G., Sramkoski, R.M., Ogle, C.K. and Alexander, J.W. (1988) Phagocytosis of opsonized fluorescent mierospheres by human neutrophils. A two-color cytometric method for the determination of attachment and ingestion. J. immunol. Methods 115, 17. Rubin, E.E. and McDonald, LC. (1991) Group B streptococcal disease in twins: Failure of empiric therapy to prevent late onset disease in the second twin. Pediatr. Infect. Dis. J. 10, 621. Smith, C.L., Baker, C.J., Anderson, D.C. and Edwards, M.S. (1990) Role of complement receptors in opsonophagocytosis of group B streptococci by adult and neonatal neutrophils. J. Infect. Dis. 162, 489. Verhoeff, J., Peterson, P.K. and Quie, P.G. (1977)Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: A quantative assay using [3H]thymidine labeled bacteria. J. Immunol. Methods 14, 303.
