Vol. 9, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1979, p. 38-48 0095-1137/79/01-0038/11$02.00/0
Sensitive Microplate Enzyme-Linked Immunosorbent Assay for Detection of Antibodies Against the Scrub Typhus Rickettsia, Rickettsia tsutsugamushi GREGORY A. DASCH,* SIDNEY HALLE, AND A. LOUIS BOURGEOIS Department of Microbiology, Naval Medical Research Institute, Bethesda, Maryland 20014 Received for publication 30 October 1978
A microtiter enzyme-linked immunosorbent assay (ELISA) has been developed for the titration of antibodies against scrub typhus in human and animal sera. Scrub typhus rickettsiae were grown in monolayers of irradiated mouse LM3 cells and separated from host cell materials by differential centrifugation, filtration through a glass filter (AP-20, Millipore Corp.), and isopycnic banding in Renografin density gradients. The scrub typhus ELISA antigens were obtained from the purified viable rickettsiae by French pressure cell disruption and addition of 0.2% Formalin to the soluble extract. Antisera prepared in rabbits against the prototype Karp, the Kato, and the Gilliam strains of scrub typhus were used to standardize the ELISA and to compare its sensitivity and specificity to that of the indirect fluorescent antibody test (IFA). ELISA titers were measured as the greatest serum dilution showing an optical density 0.25 above controls or by the optical density achieved at a fixed serum dilution. The IFA and ELISA end point titers were quite similar, and all three measures of titer had comparable specificity for the strains of scrub typhus. No cross-reactions between the typhus and scrub typhus sera were observed by ELISA. Both the immunoglobulin M (IgM) and IgG antibody titers of 12 sequential sera from four patients with scrub typhus were obtained by IFA and ELISA. The IFA and ELISA end point titers for IgM and IgG had correlation coefficients of 0.91 and 0.97, respectively, whereas the ELISA optical density values at a serum dilution of 1:100 had slightly lower correlations with the IFA titers (0.80 and 0.94). Early rising IgM titers followed by rising IgG titers were demonstrated by ELISA in three patients with primary scrub typhus infections, whereas the IgG response predominated in a patient with a reinfection. It is concluded that the ELISA for scrub typhus is a very satisfactory alternative to the IFA test.
The serological diagnosis of infection with the scrub typhus rickettsia, Rickettsia tsutsugamushi, is complicated by the antigenic diversity of its strains (2, 3, 18, 20, 25) and the difficulty of preparing suitable antigens (14, 26, 31). Although the simplest procedure, the nonspecific Weil-Felix reaction in which agglutinins for the OXK strain of Proteus mirabilis are detected, has been widely used to diagnose primary scrub typhus infections, it is neither specific nor very sensitive. A positive Weil-Felix test can be observed with louse-borne relapsing fever (41), leptospirosis (8, 11), or other febrile illnesses (4), as well as with Proteus urinary tract infections (8). Furthermore, fewer than 50% of patients with well-documented scrub typhus infections have rising or strongly positive Weil-Felix titers (1, 4, 10, 15), and reinfection with scrub typhus does not provoke a second rise in OXK agglutinins (33). Finally, experimentally infected animals
have a weak and variable Weil-Felix response (6, 23), which makes the test unsuitable for seroepidemiological studies. Although specific rickettsial microagglutination tests have been widely used for murine and epidemic typhus (19), they have not been applied to the scrub typhus rickettsiae, possibly because of their instability. Specific antigens for use in the complement fixation (CF) test have been available for many years (2, 26, 31). Their chief drawback has been their specificity: a significant CF titer, particularly with acute-phase sera, is obtained only with the antigen homologous to the strain of scrub typhus causing the infection (17, 31). Consequently, all strains endemic to a region must be used to ensure the detection of every positive serum. Although a soluble group-specific antigen has been obtained from the scrub typhus rickettsiae (26, 32), it is difficult to prepare and has 38
VOL. 9, 1979
ELISA FOR ANTIBODIES AGAINST SCRUB TYPHUS
not been used routinely. Because the CF antigens require considerable time for preparation, are often difficult to standardize, and preclude the use of anticomplementary sera, the CF test has seen little use in recent years since the advent of the indirect fluorescent antibody assay (IFA) for scrub typhus. The use of CF antigens in an indirect hemagglutination assay has also been reported (24), but it presents the same difficulties as the CF test and has not been used clinically. The IFA for scrub typhus uses as antigen acetone-fixed smears of rickettsiae grown in the yolk sacs of embryonated chicken eggs (8, 9). Both the antigen preparation and method of assay are simple. Because the IFA is considerably more sensitive than the CF test, the common group antigen of scrub typhus rickettsiae is generally detected much more readily even though the serum titer against the homologous strain is considerably higher than against the heterologous strains (16, 18). The microimmunofluorescent antibody test modification has greatly increased the utility of this test in screening sera against a large number of strains, either in routine clinical serology or in epidemiological surveys of animals for the incidence of scrub typhus in endemic foci (10, 35, 36). However, the IFA method requires a fluorescence microscope and the capacity to store antigens at -20 or -70°C and is therefore cumbersome for field applications. The enzyme-linked immunosorbent assay (ELISA) procedure has been adapted to a great variety of viral, bacterial, and parasitic diseases (30). It is suitable for use either as a fully automated and objective quantitative test in sophisticated clinical laboratories or as a qualitative but sensitive visual test appropriate for the limited facilities of field situations. Consequently, we have investigated the ELISA as a flexible alternative to the IFA technique. We report here a new method for preparing antigen fractions from viable purified scrub typhus rickettsiae and their use in a microplate ELISA for the detection of antibodies present in tsutsugamushi disease. The scrub typhus ELISA was evaluated for its sensitivity and strain specificity vis-a-vis the IFA test with both rabbit and human antisera. MATERLALS AND METHODS Preparation of scrub typhus antigens for the ELISA. The Gilliam, Karp, and Kato strains of R. tsutsugamushi (egg passages 181, 50 to 53, and 128, respectively) were obtained from B. L. Elisberg and F. M. Bozeman (Bureau of Biologics, Food and Drug Administration, Bethesda, Md.). All seed preparations were produced from the yolk sacs of embryonated chicken eggs, processed through the bovine plasma
39
albumin (BPA) purification step, and stored at -700C (39). Monolayers of mouse LM3 cells (40 to 45 16ounce [480-ml] flasks per preparation), which had been irradiated 6 or 7 days previously with 3,000 rads with 'Co, were inoculated with the rickettsial seed preparations diluted in brain heart infusion (106 to 107 rickettsial plaque-forming units/16-ounce flask set with 3 x 106 irradiated cells) in a manner similar to that described previously for R. typhi (39). Five to 7 days after inoculation, the L-cell monolayers were removed by brief trypsinization with 0.25% trypsin (Difco) in Dulbecco phosphate-buffered saline (PBS). The flasks were then rinsed with PBS, and the cells were collected by centrifugation at 12,000 rpm for 10 min in a Sorvall SS-34 rotor. The L cells were resuspended in 40 ml of PBS containing 1% BPA and homogenized by passage twice each through 22- and 26-gauge needles. The homogenate was centrifuged at 1,000 rpm for 10 min to remove the nuclei and crude cell debris, and the supernatant fluid was saved. The nuclear pellet was suspended in 40 ml of PBS-1% BPA, again passed through the syringe needles, and centrifuged. The two supernatant fluids were combined and filtered through a glass depth filter (AP-20, Millipore Corp.), and the rickettsiae in the filtrate were collected by centrifugation at 12,000 rpm for 15 min. The crude rickettsial pellet was resuspended in 15 ml of PBS-1% BPA, and 2.5 ml was layered over each of six 20 to 45% Renografin gradients (34 ml each) made in the PBS-BPA diluent. These gradients were centrifuged in an SW27 Spinco rotor for 1 h at 25,000 rpm. The rickettsiae band dispersed in this gradient so that two fractions were routinely collected: a very sharp lower band of 2 to 3 mm in thickness at a position comparable to the single band obtained with typhus group rickettsiae (39), and an upper 10- to 15mm-thick, disperse band between the lower band and the cellular debris which did not penetrate the gradient. The lower band consisted of aggregates of highly purified scrub typhus rickettsiae, and the top band material contained scattered rickettsiae in varying amounts of cellular debris. Each band was washed twice by centrifugation in PBS. The washed material was either treated with bee venom phospholipase A2 (10-,tg/ml final concentration; Sigma or Calbiochem) or trypsin (0.25%) in PBS for 30 min at 25°C and then washed once in PBS or was not treated and collected directly by centrifugation at 12,000 rpm for 15 min. The final pellets were resuspended in 5 ml of 0.04 M KPO4 buffer (pH 7.3), disrupted by passage through an Aminco French pressure cell twice at 20,000 lb/in2, and centrifuged at 8,000 rpm for 10 min to remove crude debris. Formalin was added to a 0.2% final concentration, and the antigens were stored at 4°C. Protein concentrations were determined by the method of Lowry et al. (27). Preparation of animal antisera. The preparation of typhus, yolk sac, and L-cell antisera has been described (13, 39). New Zealand white rabbits (6 to 8 pounds [ca. 2.7 to 3.6 kg]) were inoculated intraperitoneally with suspensions of R. tsutsugamushi grown in chicken yolk sacs and purified as follows. The Gilliam inoculum was purified through the BPA step, and the yield from two heavily infected yolk sacs in PBS was inoculated into each rabbit. The Gilliam
40
DASCH, HALLE, AND BOURGEOIS
immune sera were obtained by intracardiac puncture 27 days later. BPA-treated Karp and Kato preparations were further purified by a single cycle of Renografin density gradient centrifugation (as described above, but in the diluent K36 [39]), and the yield from five to seven yolk sacs suspended in PBS was inoculated into each rabbit. The Kato immune sera were obtained 22 days after infection. Karp and Kato hyperimmune sera were obtained after booster immunization of the rabbits with similar freshly prepared inocula 1 and 4 months, respectively, after initial immunization and were collected 8 and 11 days after the second inoculation. Some of the scrub typhus sera had antibody titers detectable by ELISA against yolk sac antigens. However, the titers against yolk sac antigens were much lower than those against scrub typhus antigens, and no cross-reactions with control L-cell antigens were observed in the ELISA with these sera. IFA test. The Wilmington strain of R. typhi and the Karp, Gilliam, and Kato strains of R. tsutsugamushi were grown in the yolk sacs of embryonated chicken eggs and purified through the BPA step (39): Dulbecco PBS was substituted for K36 in the final BPA step and for washing the rickettsial suspension free of BPA. The final rickettsial pellet was diluted in PBS to make a stock 20% (wt/vol) suspension, which was stored in 0.1-ml volumes at -70°C. The appropriate working dilution of each antigen was determined by making twofold dilutions of the stock antigen and examining them by the microimmunofluorescent antibody technique of Wang (38), as adapted recently for use with spotted fever, typhus, and scrub typhus rickettsiae (28, 29). Antigen dilutions in PBS were applied to each slide with a dip pen-point (AdCom, Silver Spring, Md.) in 15 groups of 4 antigen spots each. The slides were air dried for 30 min and then fixed in acetone for 10 min at room temperature. After the slides were allowed to air dry again, they were stored at -20°C. Fresh slides were prepared biweekly. Slides were warmed to room temperature, and condensation was removed with forced air at room temperature before the application of 5-,ul drops of diluted antiserum to each group of four antigen spots. Serum was diluted twofold serially in PBS from an initial 1: 40 dilution. Known positive and negative control sera were included on each slide. After applying the serum dilutions, the slides were incubated for 30 min at 37°C in a moist chamber, washed twice with PBS (5 min each time), and air dried. Drops of appropriate dilutions (standardized by the method of Beutner et al. [5]) of fluorescein-conjugated goat anti-human immunoglobulin M (IgM; ,u-chain specific), goat antihuman IgG (-y-chain specific), or goat anti-rabbit immunoglobulin (Cappel Laboratories, Inc., Cochranville, Pa.) were then placed on each group of antigen spots and the slides were incubated for 30 min at 37°C. The slides were rinsed twice in PBS (5 min each time) and then in distilled water, air dried, and mounted in buffered glycerol (90% glycerol with 10% 0.1 M PBS, pH 9.4) for examination. Slides were examined at x500 magnification, using a Zeiss Photomicroscope III with vertical illumination provided by a III RS reflected light fluorescent illuminator equipped with an HBO
J. CLIN. MICROBIOL. 50-W mercury vapor lamp and a Zeiss filter set no. 10 for fluorescein isothiocyanate blue excitation. Antigens and antibodies were both coded, and the slides were read independently by three observers. Slides were stored at 4°C until read by all observers (generally within 3 days). Each IFA titer reported here is the highest dilution of serum at which definite fluorescence (1+) with typical rickettsial morphology could be detected, although the last serum dilution at which strong fluorescence (3+) could be detected was also determined. Geometric means of antiserum titers obtained by all three observers were calculated. Deviations in titers, between day, between observer, and between repeat titrations, were rarely greater than fourfold and generally averaged about twofold for either 1+ or 3+ end points. Background fluorescence, due to low levels of antibodies to yolk sac present in the rabbit antisera prepared against the scrub typhus rickettsiae, was generally minimal except at the initial serum dilutions and was easily distinguished from the specific rickettsial fluorescence. Source of human sera. The acute- and convalescent-phase human sera used in these studies were obtained from patients participating in clinical and immunological studies of scrub typhus conducted by the U.S. Naval Medical Research Unit No. 2 in the Pescadores Islands of Taiwan. These patients all had typical signs and symptoms of scrub typhus including eschar, and R. tsutsugamushi had been isolated from each of these patients by the inoculation of acutephase blood specimens into laboratory mice (7). Microplate ELISA procedure. The tube ELISA described previously (22) was modified in the microplate ELISA as follows. Antigens diluted in coating buffer (0.1 M Na2CO3, 0.02% NaN3, pH 9.6) were applied to disposable "U"-bottom microtiter plates (Immulon, no. 18-440, Microbiological Associates, Bethesda, Md.) in 100 pl/well. The plates were stacked and incubated overnight in a humidified incubator at 37°C. Serum, conjugate dilutions, and substrate were added as described previously in 100-pl volumes per well. All intermediate wash steps of the wells were done five times with 200 to 300 t,l of working buffer per well with a 24-channel multiple automated sample harvester (MASH-II, no. 18-902, Microbiological Associates). The final alkaline phosphatase reaction was terminated after 60 min at 37°C with 100 tl of 2 N NaOH per well. The total well contents (200 tl) were then transferred into tubes of transfer frames (no. 77851-00 Flow Laboratories, Inc., Rockville, Md.) each containing 0.8 ml of 0.1 N NaOH for a final dilution of 1:10. Additions of antigens, conjugates, and enzyme substrate and transfers of well contents were made most conveniently with a 100- or 200-lI, eight-channel Titertek pipette (no. 78-211-05, Flow Laboratories). Absorbance was read at 400 nm in a Stasar II spectrophotometer equipped with a rapid sampling cuvette and automatic thermal printer (Gilford Instruments,
Oberlin, Ohio).
RESULTS Characterization by ELISA of antigens prepared from partially purified scrub ty-
ELISA FOR ANTIBODIES AGAINST SCRUB TYPHUS
VOL. 9, 1979
phus rickettsiae. All rickettsiae of the typhus group can be completely freed of host cell contaminants by Renografin density gradient centrifugation (12, 39, 40). In marked contrast, the scrub typhus rickettsiae avidly retain some host cell materials under a variety of modifications of the Renografin procedure (14; G. A. Dasch and E. Weiss, manuscript in preparation). With the modified Renografin procedure described here, about 10% of the viable scrub typhus rickettsiae present in the initial crude cytoplasmic fractions of infected L cells were recovered from the final bottom band formed in the Renografin density gradients. The scrub typhus rickettsiae obtained from L cells were more highly purified than were those obtained from infected chicken yolk sacs (14). Since neither yolk sac- nor L-cell-grown scrub typhus rickettsiae could be freed completely of host cell materials, the mixed system using antisera obtained from animals infected with yolk sac-grown rickettsiae and antigens prepared from infected L cells was used to ensure the rickettsial specificity of the ELISA. Each of the partially purified scrub typhus antigens was tested for its capacity to bind to polystyrene microtiter plates, using a fixed dilution of rabbit antiserum (CF titers of 128 to 512 with particulate antigens) prepared against the homologous strain (Fig. 1, Table 1). In every case, the more highly purified antigens in the
1.5
I ANTIGENS --- P -BOTTOM
41
bottom band bound more specific antibodies, resulting in significantly higher ELISA optical density (OD) values, than did the less satisfactory antigens obtained from the top band fraction (Table 1). These results suggested that the residual contaminating host L-cell materials inhibited binding of the rickettsial antigens present in the top band fractions, much as had been observed previously with typhus group antigens which contained significant yolk sac contamination (22). Similarly, a French pressure cell extract of the nuclear pellet of the infected L cells did not bind any rickettsial antibody in the ELISA even though numerous rickettsiae were present in the fraction. The ratio of L-cell materials to rickettsial antigen appears critical since excellent ELISA antigens were occasionally obtained from both the top and bottom Renografin gradient bands (Gilliam no. 2 and 3, Kato no. 1). In these cases the L-cell monolayers were exceptionally well infected, and the top fractions appeared quite similar to the bottom band fractions in Giemsa-stained smears. Some of the host cell materials adhering to the scrub typhus rickettsiae can be released by treating the suspensions with either phospholipase A2 or proteases (14). When top and bottom band rickettsial fractions were treated with bee venom phospholipase A2 before the preparation of French pressure cell antigen extracts, a small
r
KARP *
GILLIAM BOTTOM ANTIGEN
@10
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L~~~~~~~~~~~~~~~~~b
0 FIG. 1. sILLIAn antigelb TitrationAof U 0
KARP
Lb
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PROW0Ah CON .01
.1
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50 .1
NO Ab 10
mg PROTEIN/mi FIG. 1. Titration of antigens from scrub typhus rickettsiae by microplate ELISA. Goat antirabbit immunoglobulin-alkaline phosphatase conjugate at 1:400 dilution. (A) Karp no. 2 antigens titrated with rabbit antiserum no. 20 against Karp strain at 1:1,000 dilution. Symbols: Untreated top fraction (U); phospholipasetreated (PL) top fraction (O); untreated bottom fraction (0); phospholipase-treated (PL) bottom fraction (0). Control titrations without antiserum gave similar results for treated or untreated top (A) and bottom (A) fractions. (B) Gilliam bottom fraction antigen no. 1 titrated with rabbit antiserum no. 10 against R. prowazekii (0), antiserum no. 20 against Karp strain (0), antiserum no. 13 against Kato strain (U), antiserum no. 1 against Gilliam strain (A), all at 1:1,000 dilution, and without serum (A).
42
DASCH, HALLE, AND BOURGEOIS
J. CLIN. MICROBIOL.
TABLE 1. Characterization of scrub typhus antigens by microplate ELISA ELISA antigen dilution
Antigen prepn Strain
Karp no. 1
Karp no. 2
bNo. of
Protein
Fraction
Combined Bands
Top Top Bottom Bottom Top Bottom Top
Treatment
None Trypsin Phospholipase None Phospholipase None Phospholipase None None None None None None None None None None
0roteinODb
30 7.2 2.5 25 3.5
0.63 0.44 0.80 0.44 0.51 1.33 1.54 0.37 1.16 0.14 1.26 0.74 1.26 0.71 1.07 0.80 1.39
wells/prepnc
500
1,600 1,600
160 500 20.5 (2.1)d (0.92) 5,000 10 (1.0) (1.12) 5,000 61 500 Karp no. 3 4.9 (1.6) (0.92) 5,000 Gilliam no. 1 210 160 Bottom 15.6 (1.6) (0.97) 1,600 Gilliam no. 2 0.8 Top 16,000 Bottom 16,000 Gilliam no. 3 4.4 Top 500 Bottom 0.5 5,000 Kato no. 1 1.4 Top 1,600 Bottom 1.2 (0.4) (1.34) 5,000 (0.08) (1.00) 25,000 a Antigen concentration giving the greatest OD in the ELISA with a standard rabbit serum against the homologous antigen (anti-Gilliam serum no. 1, anti-Karp serum no. 20, or hyperimmune serum anti-Kato no. 13, all at 1:1,000 dilution, using a goat immunoglobulin-alkaline phosphatase conjugate at 1:400 dilution). b OD at 400 nm after 60 min of incubation with the optimum or "usable" (in parentheses) dilution of antigen. 'Number of ELISA microplate wells that could be coated using the whole antigen fraction from 40 to 45 16ounce tissue culture flasks at the most useful ELISA antigen dilution. d Best antigen concentration giving an OD in the ELISA over 0.80 OD, the "usable" dilution.
but significant improvement of the antigens in the ELISA was observed (Fig. 1A; Table 1, Karp no. 1 and 2). In contrast, trypsin-treated antigens were less satisfactory (Table 1, Karp no. 1), even though this treatment, as well as the phospholipase treatment, reduced the protein content of the antigens significantly (Table 1; 14). Since the phospholipase treatment did not have a pronounced effect on the OD values and its effect on antigen stability was unknown (14, 26), only the untreated bottom band antigen fraction was used in further studies. Antigen titration curves were obtained with homologous and heterologous rabbit antisera against scrub typhus strains and with several types of control sera, including antisera to murine and epidemic typhus. The optimal coating dilution of antigen was arbitrarily defined as that antigen dilution in a geometric series of half-log dilution steps giving the greatest OD with a high-titered homologous antiserum (Table 1). However, the optimal concentration often required either prohibitive amounts of antigen or produced excessive background OD levels with control sera. Consequently, the "usable" antigen dilution was chosen as the lowest concentration, of the ones used, that gave a reading above 0.8 OD. As seen in Table 1 (Kato no. 1 antigen), moderate reductions in required OD
readings resulted in considerable saving of antigen. The resultant protein loadings (0.1 to 2.0 ,ug of protein per ml) of antigens in bottom fractions were quite similar to those required with antigens from typhus rickettsiae (22). Antigens of each scrub typhus strain also reacted with rabbit antisera prepared against heterologous strains of R. tsutsugamushi (Fig. 1B). Although the absolute OD reached depended on the particular heterologous strain and the strength of the antiserum, the heterologous antigen titration curves were quite similar to those obtained with the homologous antiserum. In contrast, OD values obtained with antisera against R. typhi (not shown) or R. prowazekii (Fig. 1B) were not significantly different from those with normal control or anti-yolk sac antisera (not shown) and only slightly higher than the ODs attained in the absence of antiserum (Fig. 1B). Comparison of titers of rabbit antisera against scrub typhus rickettsiae by IFA and ELISA. Sixteen antisera were obtained from three groups of four rabbits infected with the Gilliam (immune sera), Karp (hyperimmune sera), and Kato (both immune and hyperimmune sera) strains of scrub typhus. These sera were not obtained with the same immunization schedules and represent a random set of possible
ELISA FOR ANTIBODIES AGAINST SCRUB TYPHUS
VOL. 9, 1979
sera. These sera were titrated against each strain, using intact rickettsiae in the IFA or the French pressure cell-extracted, bottom-band antigens in the ELISA. The ELISA end point titer (greatest antiserum dilution calculated by interpolation resulting in an OD 0.25 above background controls [without antiserum or with normal rabbit serum]) (Fig. 2A) or the ELISA OD value (after subtracting the background control) obtained at serum dilutions of 1:1,000 (Fig. 2B) and 1:3,200 (not shown) were plotted against the corresponding IFA titer obtained with antigen from the same strain. Linear regressions of the IFA and ELISA titers were obtained for the 16 sera against each antigen and for the combined data, but only the latter are shown. The IFA and ELISA end point titers (Fig. 2A) correlated better than the IFA titers and the ELISA OD 1/1,000 values (Fig. 2B), probably because the very high-titered sera had reached the maximum ELISA OD obtainable with these antigens (particularly the Gilliam antigen) even at a 1:1,000 dilution. When the regressions of the individual antigens are compared by using Fisher's z statistic (34), only the Gilliam and Karp regressions with the ELISA OD 1/1,000 end point have differences approaching significance (0.05 < P < 0.1). This discrepancy in regressions is reduced when the lines for the IFA titer and the ELISA OD at a 1:3,200 antiserum dilution are compared (P > 0.3). Although the resultant correlation
43
coefficient obtained with the combined data from the ELISA OD 1/3,200 end point was much better (0.85 versus 0.77), this dilution was quite unsatisfactory because some weakly reacting sera were not significantly above background with heterologous antigens. Consequently, the ELISA end point titer method is the more accurate measure of antiserum titer when compared to the IFA titer. On the other hand, the ELISA OD method is highly satisfactory (especially within specific OD limits) for surveys of large numbers of sera requiring less precision but considerable antigen economy. The sensitivity of the ELISA titer with respect to the IFA titer may also be seen in Fig. 2A. Remarkably similar results were generally attained with these two techniques. Those points or portions of the solid regression line (medium and strong sera) to the lower right of the dotted equivalence line indicate sera in which the ELISA titers were higher than those attained by IFA. Conversely, the individual points or the portion of the regression line (low serum titers) falling to the left of the equivalence line have greater IFA titers than ELISA titers. The strain specificity of the titers of each group of antisera against the Karp, Kato, and Gilliam antigens was determined by IFA and ELISA. As expected from the high correlation between the IFA and ELISA titers (Fig. 2), both the sensitivity and strain specificity of the two
A
A M T ICE N1>
S *
,&CIL L IA N 4 _-S' "
(3
/
t
/
r .92
A
/ 2
/
2
4 3 LOG ELISA TITER
5
0
.25
.50
.75
1.00 1.25 1.50
OD(400om)/60.in FIG. 2. Correlation of IFA and ELISA titers of rabbit antisera against the Karp, Kato, and Gilliam strains of R. tsutsugamushi. ELISA titers were obtained with the Karp no. 2 bottom untreated antigen (0, at 2.1 pg ofprotein per ml), Gilliam no. 1 bottom antigen (A, at 1.6 pg ofprotein per ml), and Kato no. 1 bottom antigen (U, at 0.08 pg of protein per ml) and a goat anti-rabbit immunoglobulin-alkaline phosphatase enzyme conjugate at 1:400 dilution. The solid line is the linear regression of log IFA titer and ELISA titers; r is the correlation coefficient for each respective linear regression. (A) Log IFA titer and log ELISA end point titer (reciprocal of greatest serum dilution having an OD 0.25 above controls) of each antiserum against each antigen. Dotted line represents locus of points, log IFA titer = log ELISA end point titer. (B) Log IFA titer and ELISA OD titer (OD above controls at serum dilution of 1:1,000) of each antiserum against each antigen.
44
DASCH, HALLE, AND BOURGEOIS
J. CLIN. MICROBIOL.
techniques were very similar (Fig. 3). The geo- antigens obtained by IFA and those obtained by metric mean antibody end point titers by ELISA ELISA end point had a correlation coefficient of were greater than the IFA mean titers with all 0.97, whereas the regression of differences obantigens except the weak Kato immune sera (cf. tained by IFA and ELISA OD 1/1,000 had a Fig. 2). By either assay, with few exceptions, the correlation coefficient of 0.86. In conclusion, differences in titers against the three antigens these results suggest that none of the major were highly significant only when the homoloantigen determinants detected in the intact rickgous titer and a heterologous titer were com- ettsial antigen by IFA has been lost during our pared, whereas the titers were generally similar method of purification of the rickettsiae and when two heterologous antigens were used. preparation of the antigen extracts for the Whereas the specificity of the sera could be ELISA. determined by all three measures of antibody Comparison of titers of human antisera titer, the levels of significance of titer differences against scrub typhus rickettsiae by IFA were generally greater by the IFA and ELISA and ELISA. Twelve sera were obtained from end point titer methods (Fig. 3). The linear four cases of naturally occurring scrub typhus regression of all the differences in titers between infection among Chinese military personnel stationed in the Pescadores Islands, Republic of China. In addition, two control sera were obtained from two fever patients admitted to the same hospital with a self-resolving fever of unknown origin (M-109) and influenza (A-Victoria strain) (M-210), respectively. All cases of scrub typhus were confirmed by presence of eschar and rickettsial isolation and were treated with o tetracycline or doxycycline commencing on the date of admission (date of the first serum taken), at J whereas the controls were negative for rickettsial isolation. 5 IEach antiserum was titrated against antigens of the Karp, Gilliam, and Kato strains of scrub typhus by IFA and ELISA, using specific anti-u O IgM and anti-IgG conjugates (Table 2). Linear 3 regressions for both IgM and IgG titers were gen is given above the respective heterologous anti- 0.2, Fisher's z statistic) (34). A comparison of gen. The difference in titers between the two heterol- the calculated regression line of the IFA and ogous antigens was generally not significant (P > 0.1, ELISA end point titers with a line through the not shown). points of equivalent titers indicated that the two 0
-J
Ia
-
-J
VOL. 9, 1979
45
ELISA FOR ANTIBODIES AGAINST SCRUB TYPHUS
TABLE 2. Titers of human scrub typhus antiseraa Antigen used Antiserum
Patient
Kato
Gilliam
Karp
IFF ISA ti- EOD 1:A tiEL ti- Or past Conju- IFAtitebELISA Days LS ELSAtOD 1: IFA titer ELISA Dy titer ter i ter ter' gate IFA titerb EIAt onsetatCnu oo
10
ELISA
OD 1: 0
2,870 1,150 0.45 5,000 0.11 9,130 43 254 7 IgM M-101 75 0.19 180 190 527
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1979, p. 38-48 0095-1137/79/01-0038/11$02.00/0
Sensitive Microplate Enzyme-Linked Immunosorbent Assay for Detection of Antibodies Against the Scrub Typhus Rickettsia, Rickettsia tsutsugamushi GREGORY A. DASCH,* SIDNEY HALLE, AND A. LOUIS BOURGEOIS Department of Microbiology, Naval Medical Research Institute, Bethesda, Maryland 20014 Received for publication 30 October 1978
A microtiter enzyme-linked immunosorbent assay (ELISA) has been developed for the titration of antibodies against scrub typhus in human and animal sera. Scrub typhus rickettsiae were grown in monolayers of irradiated mouse LM3 cells and separated from host cell materials by differential centrifugation, filtration through a glass filter (AP-20, Millipore Corp.), and isopycnic banding in Renografin density gradients. The scrub typhus ELISA antigens were obtained from the purified viable rickettsiae by French pressure cell disruption and addition of 0.2% Formalin to the soluble extract. Antisera prepared in rabbits against the prototype Karp, the Kato, and the Gilliam strains of scrub typhus were used to standardize the ELISA and to compare its sensitivity and specificity to that of the indirect fluorescent antibody test (IFA). ELISA titers were measured as the greatest serum dilution showing an optical density 0.25 above controls or by the optical density achieved at a fixed serum dilution. The IFA and ELISA end point titers were quite similar, and all three measures of titer had comparable specificity for the strains of scrub typhus. No cross-reactions between the typhus and scrub typhus sera were observed by ELISA. Both the immunoglobulin M (IgM) and IgG antibody titers of 12 sequential sera from four patients with scrub typhus were obtained by IFA and ELISA. The IFA and ELISA end point titers for IgM and IgG had correlation coefficients of 0.91 and 0.97, respectively, whereas the ELISA optical density values at a serum dilution of 1:100 had slightly lower correlations with the IFA titers (0.80 and 0.94). Early rising IgM titers followed by rising IgG titers were demonstrated by ELISA in three patients with primary scrub typhus infections, whereas the IgG response predominated in a patient with a reinfection. It is concluded that the ELISA for scrub typhus is a very satisfactory alternative to the IFA test.
The serological diagnosis of infection with the scrub typhus rickettsia, Rickettsia tsutsugamushi, is complicated by the antigenic diversity of its strains (2, 3, 18, 20, 25) and the difficulty of preparing suitable antigens (14, 26, 31). Although the simplest procedure, the nonspecific Weil-Felix reaction in which agglutinins for the OXK strain of Proteus mirabilis are detected, has been widely used to diagnose primary scrub typhus infections, it is neither specific nor very sensitive. A positive Weil-Felix test can be observed with louse-borne relapsing fever (41), leptospirosis (8, 11), or other febrile illnesses (4), as well as with Proteus urinary tract infections (8). Furthermore, fewer than 50% of patients with well-documented scrub typhus infections have rising or strongly positive Weil-Felix titers (1, 4, 10, 15), and reinfection with scrub typhus does not provoke a second rise in OXK agglutinins (33). Finally, experimentally infected animals
have a weak and variable Weil-Felix response (6, 23), which makes the test unsuitable for seroepidemiological studies. Although specific rickettsial microagglutination tests have been widely used for murine and epidemic typhus (19), they have not been applied to the scrub typhus rickettsiae, possibly because of their instability. Specific antigens for use in the complement fixation (CF) test have been available for many years (2, 26, 31). Their chief drawback has been their specificity: a significant CF titer, particularly with acute-phase sera, is obtained only with the antigen homologous to the strain of scrub typhus causing the infection (17, 31). Consequently, all strains endemic to a region must be used to ensure the detection of every positive serum. Although a soluble group-specific antigen has been obtained from the scrub typhus rickettsiae (26, 32), it is difficult to prepare and has 38
VOL. 9, 1979
ELISA FOR ANTIBODIES AGAINST SCRUB TYPHUS
not been used routinely. Because the CF antigens require considerable time for preparation, are often difficult to standardize, and preclude the use of anticomplementary sera, the CF test has seen little use in recent years since the advent of the indirect fluorescent antibody assay (IFA) for scrub typhus. The use of CF antigens in an indirect hemagglutination assay has also been reported (24), but it presents the same difficulties as the CF test and has not been used clinically. The IFA for scrub typhus uses as antigen acetone-fixed smears of rickettsiae grown in the yolk sacs of embryonated chicken eggs (8, 9). Both the antigen preparation and method of assay are simple. Because the IFA is considerably more sensitive than the CF test, the common group antigen of scrub typhus rickettsiae is generally detected much more readily even though the serum titer against the homologous strain is considerably higher than against the heterologous strains (16, 18). The microimmunofluorescent antibody test modification has greatly increased the utility of this test in screening sera against a large number of strains, either in routine clinical serology or in epidemiological surveys of animals for the incidence of scrub typhus in endemic foci (10, 35, 36). However, the IFA method requires a fluorescence microscope and the capacity to store antigens at -20 or -70°C and is therefore cumbersome for field applications. The enzyme-linked immunosorbent assay (ELISA) procedure has been adapted to a great variety of viral, bacterial, and parasitic diseases (30). It is suitable for use either as a fully automated and objective quantitative test in sophisticated clinical laboratories or as a qualitative but sensitive visual test appropriate for the limited facilities of field situations. Consequently, we have investigated the ELISA as a flexible alternative to the IFA technique. We report here a new method for preparing antigen fractions from viable purified scrub typhus rickettsiae and their use in a microplate ELISA for the detection of antibodies present in tsutsugamushi disease. The scrub typhus ELISA was evaluated for its sensitivity and strain specificity vis-a-vis the IFA test with both rabbit and human antisera. MATERLALS AND METHODS Preparation of scrub typhus antigens for the ELISA. The Gilliam, Karp, and Kato strains of R. tsutsugamushi (egg passages 181, 50 to 53, and 128, respectively) were obtained from B. L. Elisberg and F. M. Bozeman (Bureau of Biologics, Food and Drug Administration, Bethesda, Md.). All seed preparations were produced from the yolk sacs of embryonated chicken eggs, processed through the bovine plasma
39
albumin (BPA) purification step, and stored at -700C (39). Monolayers of mouse LM3 cells (40 to 45 16ounce [480-ml] flasks per preparation), which had been irradiated 6 or 7 days previously with 3,000 rads with 'Co, were inoculated with the rickettsial seed preparations diluted in brain heart infusion (106 to 107 rickettsial plaque-forming units/16-ounce flask set with 3 x 106 irradiated cells) in a manner similar to that described previously for R. typhi (39). Five to 7 days after inoculation, the L-cell monolayers were removed by brief trypsinization with 0.25% trypsin (Difco) in Dulbecco phosphate-buffered saline (PBS). The flasks were then rinsed with PBS, and the cells were collected by centrifugation at 12,000 rpm for 10 min in a Sorvall SS-34 rotor. The L cells were resuspended in 40 ml of PBS containing 1% BPA and homogenized by passage twice each through 22- and 26-gauge needles. The homogenate was centrifuged at 1,000 rpm for 10 min to remove the nuclei and crude cell debris, and the supernatant fluid was saved. The nuclear pellet was suspended in 40 ml of PBS-1% BPA, again passed through the syringe needles, and centrifuged. The two supernatant fluids were combined and filtered through a glass depth filter (AP-20, Millipore Corp.), and the rickettsiae in the filtrate were collected by centrifugation at 12,000 rpm for 15 min. The crude rickettsial pellet was resuspended in 15 ml of PBS-1% BPA, and 2.5 ml was layered over each of six 20 to 45% Renografin gradients (34 ml each) made in the PBS-BPA diluent. These gradients were centrifuged in an SW27 Spinco rotor for 1 h at 25,000 rpm. The rickettsiae band dispersed in this gradient so that two fractions were routinely collected: a very sharp lower band of 2 to 3 mm in thickness at a position comparable to the single band obtained with typhus group rickettsiae (39), and an upper 10- to 15mm-thick, disperse band between the lower band and the cellular debris which did not penetrate the gradient. The lower band consisted of aggregates of highly purified scrub typhus rickettsiae, and the top band material contained scattered rickettsiae in varying amounts of cellular debris. Each band was washed twice by centrifugation in PBS. The washed material was either treated with bee venom phospholipase A2 (10-,tg/ml final concentration; Sigma or Calbiochem) or trypsin (0.25%) in PBS for 30 min at 25°C and then washed once in PBS or was not treated and collected directly by centrifugation at 12,000 rpm for 15 min. The final pellets were resuspended in 5 ml of 0.04 M KPO4 buffer (pH 7.3), disrupted by passage through an Aminco French pressure cell twice at 20,000 lb/in2, and centrifuged at 8,000 rpm for 10 min to remove crude debris. Formalin was added to a 0.2% final concentration, and the antigens were stored at 4°C. Protein concentrations were determined by the method of Lowry et al. (27). Preparation of animal antisera. The preparation of typhus, yolk sac, and L-cell antisera has been described (13, 39). New Zealand white rabbits (6 to 8 pounds [ca. 2.7 to 3.6 kg]) were inoculated intraperitoneally with suspensions of R. tsutsugamushi grown in chicken yolk sacs and purified as follows. The Gilliam inoculum was purified through the BPA step, and the yield from two heavily infected yolk sacs in PBS was inoculated into each rabbit. The Gilliam
40
DASCH, HALLE, AND BOURGEOIS
immune sera were obtained by intracardiac puncture 27 days later. BPA-treated Karp and Kato preparations were further purified by a single cycle of Renografin density gradient centrifugation (as described above, but in the diluent K36 [39]), and the yield from five to seven yolk sacs suspended in PBS was inoculated into each rabbit. The Kato immune sera were obtained 22 days after infection. Karp and Kato hyperimmune sera were obtained after booster immunization of the rabbits with similar freshly prepared inocula 1 and 4 months, respectively, after initial immunization and were collected 8 and 11 days after the second inoculation. Some of the scrub typhus sera had antibody titers detectable by ELISA against yolk sac antigens. However, the titers against yolk sac antigens were much lower than those against scrub typhus antigens, and no cross-reactions with control L-cell antigens were observed in the ELISA with these sera. IFA test. The Wilmington strain of R. typhi and the Karp, Gilliam, and Kato strains of R. tsutsugamushi were grown in the yolk sacs of embryonated chicken eggs and purified through the BPA step (39): Dulbecco PBS was substituted for K36 in the final BPA step and for washing the rickettsial suspension free of BPA. The final rickettsial pellet was diluted in PBS to make a stock 20% (wt/vol) suspension, which was stored in 0.1-ml volumes at -70°C. The appropriate working dilution of each antigen was determined by making twofold dilutions of the stock antigen and examining them by the microimmunofluorescent antibody technique of Wang (38), as adapted recently for use with spotted fever, typhus, and scrub typhus rickettsiae (28, 29). Antigen dilutions in PBS were applied to each slide with a dip pen-point (AdCom, Silver Spring, Md.) in 15 groups of 4 antigen spots each. The slides were air dried for 30 min and then fixed in acetone for 10 min at room temperature. After the slides were allowed to air dry again, they were stored at -20°C. Fresh slides were prepared biweekly. Slides were warmed to room temperature, and condensation was removed with forced air at room temperature before the application of 5-,ul drops of diluted antiserum to each group of four antigen spots. Serum was diluted twofold serially in PBS from an initial 1: 40 dilution. Known positive and negative control sera were included on each slide. After applying the serum dilutions, the slides were incubated for 30 min at 37°C in a moist chamber, washed twice with PBS (5 min each time), and air dried. Drops of appropriate dilutions (standardized by the method of Beutner et al. [5]) of fluorescein-conjugated goat anti-human immunoglobulin M (IgM; ,u-chain specific), goat antihuman IgG (-y-chain specific), or goat anti-rabbit immunoglobulin (Cappel Laboratories, Inc., Cochranville, Pa.) were then placed on each group of antigen spots and the slides were incubated for 30 min at 37°C. The slides were rinsed twice in PBS (5 min each time) and then in distilled water, air dried, and mounted in buffered glycerol (90% glycerol with 10% 0.1 M PBS, pH 9.4) for examination. Slides were examined at x500 magnification, using a Zeiss Photomicroscope III with vertical illumination provided by a III RS reflected light fluorescent illuminator equipped with an HBO
J. CLIN. MICROBIOL. 50-W mercury vapor lamp and a Zeiss filter set no. 10 for fluorescein isothiocyanate blue excitation. Antigens and antibodies were both coded, and the slides were read independently by three observers. Slides were stored at 4°C until read by all observers (generally within 3 days). Each IFA titer reported here is the highest dilution of serum at which definite fluorescence (1+) with typical rickettsial morphology could be detected, although the last serum dilution at which strong fluorescence (3+) could be detected was also determined. Geometric means of antiserum titers obtained by all three observers were calculated. Deviations in titers, between day, between observer, and between repeat titrations, were rarely greater than fourfold and generally averaged about twofold for either 1+ or 3+ end points. Background fluorescence, due to low levels of antibodies to yolk sac present in the rabbit antisera prepared against the scrub typhus rickettsiae, was generally minimal except at the initial serum dilutions and was easily distinguished from the specific rickettsial fluorescence. Source of human sera. The acute- and convalescent-phase human sera used in these studies were obtained from patients participating in clinical and immunological studies of scrub typhus conducted by the U.S. Naval Medical Research Unit No. 2 in the Pescadores Islands of Taiwan. These patients all had typical signs and symptoms of scrub typhus including eschar, and R. tsutsugamushi had been isolated from each of these patients by the inoculation of acutephase blood specimens into laboratory mice (7). Microplate ELISA procedure. The tube ELISA described previously (22) was modified in the microplate ELISA as follows. Antigens diluted in coating buffer (0.1 M Na2CO3, 0.02% NaN3, pH 9.6) were applied to disposable "U"-bottom microtiter plates (Immulon, no. 18-440, Microbiological Associates, Bethesda, Md.) in 100 pl/well. The plates were stacked and incubated overnight in a humidified incubator at 37°C. Serum, conjugate dilutions, and substrate were added as described previously in 100-pl volumes per well. All intermediate wash steps of the wells were done five times with 200 to 300 t,l of working buffer per well with a 24-channel multiple automated sample harvester (MASH-II, no. 18-902, Microbiological Associates). The final alkaline phosphatase reaction was terminated after 60 min at 37°C with 100 tl of 2 N NaOH per well. The total well contents (200 tl) were then transferred into tubes of transfer frames (no. 77851-00 Flow Laboratories, Inc., Rockville, Md.) each containing 0.8 ml of 0.1 N NaOH for a final dilution of 1:10. Additions of antigens, conjugates, and enzyme substrate and transfers of well contents were made most conveniently with a 100- or 200-lI, eight-channel Titertek pipette (no. 78-211-05, Flow Laboratories). Absorbance was read at 400 nm in a Stasar II spectrophotometer equipped with a rapid sampling cuvette and automatic thermal printer (Gilford Instruments,
Oberlin, Ohio).
RESULTS Characterization by ELISA of antigens prepared from partially purified scrub ty-
ELISA FOR ANTIBODIES AGAINST SCRUB TYPHUS
VOL. 9, 1979
phus rickettsiae. All rickettsiae of the typhus group can be completely freed of host cell contaminants by Renografin density gradient centrifugation (12, 39, 40). In marked contrast, the scrub typhus rickettsiae avidly retain some host cell materials under a variety of modifications of the Renografin procedure (14; G. A. Dasch and E. Weiss, manuscript in preparation). With the modified Renografin procedure described here, about 10% of the viable scrub typhus rickettsiae present in the initial crude cytoplasmic fractions of infected L cells were recovered from the final bottom band formed in the Renografin density gradients. The scrub typhus rickettsiae obtained from L cells were more highly purified than were those obtained from infected chicken yolk sacs (14). Since neither yolk sac- nor L-cell-grown scrub typhus rickettsiae could be freed completely of host cell materials, the mixed system using antisera obtained from animals infected with yolk sac-grown rickettsiae and antigens prepared from infected L cells was used to ensure the rickettsial specificity of the ELISA. Each of the partially purified scrub typhus antigens was tested for its capacity to bind to polystyrene microtiter plates, using a fixed dilution of rabbit antiserum (CF titers of 128 to 512 with particulate antigens) prepared against the homologous strain (Fig. 1, Table 1). In every case, the more highly purified antigens in the
1.5
I ANTIGENS --- P -BOTTOM
41
bottom band bound more specific antibodies, resulting in significantly higher ELISA optical density (OD) values, than did the less satisfactory antigens obtained from the top band fraction (Table 1). These results suggested that the residual contaminating host L-cell materials inhibited binding of the rickettsial antigens present in the top band fractions, much as had been observed previously with typhus group antigens which contained significant yolk sac contamination (22). Similarly, a French pressure cell extract of the nuclear pellet of the infected L cells did not bind any rickettsial antibody in the ELISA even though numerous rickettsiae were present in the fraction. The ratio of L-cell materials to rickettsial antigen appears critical since excellent ELISA antigens were occasionally obtained from both the top and bottom Renografin gradient bands (Gilliam no. 2 and 3, Kato no. 1). In these cases the L-cell monolayers were exceptionally well infected, and the top fractions appeared quite similar to the bottom band fractions in Giemsa-stained smears. Some of the host cell materials adhering to the scrub typhus rickettsiae can be released by treating the suspensions with either phospholipase A2 or proteases (14). When top and bottom band rickettsial fractions were treated with bee venom phospholipase A2 before the preparation of French pressure cell antigen extracts, a small
r
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GILLIAM BOTTOM ANTIGEN
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0 FIG. 1. sILLIAn antigelb TitrationAof U 0
KARP
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mg PROTEIN/mi FIG. 1. Titration of antigens from scrub typhus rickettsiae by microplate ELISA. Goat antirabbit immunoglobulin-alkaline phosphatase conjugate at 1:400 dilution. (A) Karp no. 2 antigens titrated with rabbit antiserum no. 20 against Karp strain at 1:1,000 dilution. Symbols: Untreated top fraction (U); phospholipasetreated (PL) top fraction (O); untreated bottom fraction (0); phospholipase-treated (PL) bottom fraction (0). Control titrations without antiserum gave similar results for treated or untreated top (A) and bottom (A) fractions. (B) Gilliam bottom fraction antigen no. 1 titrated with rabbit antiserum no. 10 against R. prowazekii (0), antiserum no. 20 against Karp strain (0), antiserum no. 13 against Kato strain (U), antiserum no. 1 against Gilliam strain (A), all at 1:1,000 dilution, and without serum (A).
42
DASCH, HALLE, AND BOURGEOIS
J. CLIN. MICROBIOL.
TABLE 1. Characterization of scrub typhus antigens by microplate ELISA ELISA antigen dilution
Antigen prepn Strain
Karp no. 1
Karp no. 2
bNo. of
Protein
Fraction
Combined Bands
Top Top Bottom Bottom Top Bottom Top
Treatment
None Trypsin Phospholipase None Phospholipase None Phospholipase None None None None None None None None None None
0roteinODb
30 7.2 2.5 25 3.5
0.63 0.44 0.80 0.44 0.51 1.33 1.54 0.37 1.16 0.14 1.26 0.74 1.26 0.71 1.07 0.80 1.39
wells/prepnc
500
1,600 1,600
160 500 20.5 (2.1)d (0.92) 5,000 10 (1.0) (1.12) 5,000 61 500 Karp no. 3 4.9 (1.6) (0.92) 5,000 Gilliam no. 1 210 160 Bottom 15.6 (1.6) (0.97) 1,600 Gilliam no. 2 0.8 Top 16,000 Bottom 16,000 Gilliam no. 3 4.4 Top 500 Bottom 0.5 5,000 Kato no. 1 1.4 Top 1,600 Bottom 1.2 (0.4) (1.34) 5,000 (0.08) (1.00) 25,000 a Antigen concentration giving the greatest OD in the ELISA with a standard rabbit serum against the homologous antigen (anti-Gilliam serum no. 1, anti-Karp serum no. 20, or hyperimmune serum anti-Kato no. 13, all at 1:1,000 dilution, using a goat immunoglobulin-alkaline phosphatase conjugate at 1:400 dilution). b OD at 400 nm after 60 min of incubation with the optimum or "usable" (in parentheses) dilution of antigen. 'Number of ELISA microplate wells that could be coated using the whole antigen fraction from 40 to 45 16ounce tissue culture flasks at the most useful ELISA antigen dilution. d Best antigen concentration giving an OD in the ELISA over 0.80 OD, the "usable" dilution.
but significant improvement of the antigens in the ELISA was observed (Fig. 1A; Table 1, Karp no. 1 and 2). In contrast, trypsin-treated antigens were less satisfactory (Table 1, Karp no. 1), even though this treatment, as well as the phospholipase treatment, reduced the protein content of the antigens significantly (Table 1; 14). Since the phospholipase treatment did not have a pronounced effect on the OD values and its effect on antigen stability was unknown (14, 26), only the untreated bottom band antigen fraction was used in further studies. Antigen titration curves were obtained with homologous and heterologous rabbit antisera against scrub typhus strains and with several types of control sera, including antisera to murine and epidemic typhus. The optimal coating dilution of antigen was arbitrarily defined as that antigen dilution in a geometric series of half-log dilution steps giving the greatest OD with a high-titered homologous antiserum (Table 1). However, the optimal concentration often required either prohibitive amounts of antigen or produced excessive background OD levels with control sera. Consequently, the "usable" antigen dilution was chosen as the lowest concentration, of the ones used, that gave a reading above 0.8 OD. As seen in Table 1 (Kato no. 1 antigen), moderate reductions in required OD
readings resulted in considerable saving of antigen. The resultant protein loadings (0.1 to 2.0 ,ug of protein per ml) of antigens in bottom fractions were quite similar to those required with antigens from typhus rickettsiae (22). Antigens of each scrub typhus strain also reacted with rabbit antisera prepared against heterologous strains of R. tsutsugamushi (Fig. 1B). Although the absolute OD reached depended on the particular heterologous strain and the strength of the antiserum, the heterologous antigen titration curves were quite similar to those obtained with the homologous antiserum. In contrast, OD values obtained with antisera against R. typhi (not shown) or R. prowazekii (Fig. 1B) were not significantly different from those with normal control or anti-yolk sac antisera (not shown) and only slightly higher than the ODs attained in the absence of antiserum (Fig. 1B). Comparison of titers of rabbit antisera against scrub typhus rickettsiae by IFA and ELISA. Sixteen antisera were obtained from three groups of four rabbits infected with the Gilliam (immune sera), Karp (hyperimmune sera), and Kato (both immune and hyperimmune sera) strains of scrub typhus. These sera were not obtained with the same immunization schedules and represent a random set of possible
ELISA FOR ANTIBODIES AGAINST SCRUB TYPHUS
VOL. 9, 1979
sera. These sera were titrated against each strain, using intact rickettsiae in the IFA or the French pressure cell-extracted, bottom-band antigens in the ELISA. The ELISA end point titer (greatest antiserum dilution calculated by interpolation resulting in an OD 0.25 above background controls [without antiserum or with normal rabbit serum]) (Fig. 2A) or the ELISA OD value (after subtracting the background control) obtained at serum dilutions of 1:1,000 (Fig. 2B) and 1:3,200 (not shown) were plotted against the corresponding IFA titer obtained with antigen from the same strain. Linear regressions of the IFA and ELISA titers were obtained for the 16 sera against each antigen and for the combined data, but only the latter are shown. The IFA and ELISA end point titers (Fig. 2A) correlated better than the IFA titers and the ELISA OD 1/1,000 values (Fig. 2B), probably because the very high-titered sera had reached the maximum ELISA OD obtainable with these antigens (particularly the Gilliam antigen) even at a 1:1,000 dilution. When the regressions of the individual antigens are compared by using Fisher's z statistic (34), only the Gilliam and Karp regressions with the ELISA OD 1/1,000 end point have differences approaching significance (0.05 < P < 0.1). This discrepancy in regressions is reduced when the lines for the IFA titer and the ELISA OD at a 1:3,200 antiserum dilution are compared (P > 0.3). Although the resultant correlation
43
coefficient obtained with the combined data from the ELISA OD 1/3,200 end point was much better (0.85 versus 0.77), this dilution was quite unsatisfactory because some weakly reacting sera were not significantly above background with heterologous antigens. Consequently, the ELISA end point titer method is the more accurate measure of antiserum titer when compared to the IFA titer. On the other hand, the ELISA OD method is highly satisfactory (especially within specific OD limits) for surveys of large numbers of sera requiring less precision but considerable antigen economy. The sensitivity of the ELISA titer with respect to the IFA titer may also be seen in Fig. 2A. Remarkably similar results were generally attained with these two techniques. Those points or portions of the solid regression line (medium and strong sera) to the lower right of the dotted equivalence line indicate sera in which the ELISA titers were higher than those attained by IFA. Conversely, the individual points or the portion of the regression line (low serum titers) falling to the left of the equivalence line have greater IFA titers than ELISA titers. The strain specificity of the titers of each group of antisera against the Karp, Kato, and Gilliam antigens was determined by IFA and ELISA. As expected from the high correlation between the IFA and ELISA titers (Fig. 2), both the sensitivity and strain specificity of the two
A
A M T ICE N1>
S *
,&CIL L IA N 4 _-S' "
(3
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/
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5
0
.25
.50
.75
1.00 1.25 1.50
OD(400om)/60.in FIG. 2. Correlation of IFA and ELISA titers of rabbit antisera against the Karp, Kato, and Gilliam strains of R. tsutsugamushi. ELISA titers were obtained with the Karp no. 2 bottom untreated antigen (0, at 2.1 pg ofprotein per ml), Gilliam no. 1 bottom antigen (A, at 1.6 pg ofprotein per ml), and Kato no. 1 bottom antigen (U, at 0.08 pg of protein per ml) and a goat anti-rabbit immunoglobulin-alkaline phosphatase enzyme conjugate at 1:400 dilution. The solid line is the linear regression of log IFA titer and ELISA titers; r is the correlation coefficient for each respective linear regression. (A) Log IFA titer and log ELISA end point titer (reciprocal of greatest serum dilution having an OD 0.25 above controls) of each antiserum against each antigen. Dotted line represents locus of points, log IFA titer = log ELISA end point titer. (B) Log IFA titer and ELISA OD titer (OD above controls at serum dilution of 1:1,000) of each antiserum against each antigen.
44
DASCH, HALLE, AND BOURGEOIS
J. CLIN. MICROBIOL.
techniques were very similar (Fig. 3). The geo- antigens obtained by IFA and those obtained by metric mean antibody end point titers by ELISA ELISA end point had a correlation coefficient of were greater than the IFA mean titers with all 0.97, whereas the regression of differences obantigens except the weak Kato immune sera (cf. tained by IFA and ELISA OD 1/1,000 had a Fig. 2). By either assay, with few exceptions, the correlation coefficient of 0.86. In conclusion, differences in titers against the three antigens these results suggest that none of the major were highly significant only when the homoloantigen determinants detected in the intact rickgous titer and a heterologous titer were com- ettsial antigen by IFA has been lost during our pared, whereas the titers were generally similar method of purification of the rickettsiae and when two heterologous antigens were used. preparation of the antigen extracts for the Whereas the specificity of the sera could be ELISA. determined by all three measures of antibody Comparison of titers of human antisera titer, the levels of significance of titer differences against scrub typhus rickettsiae by IFA were generally greater by the IFA and ELISA and ELISA. Twelve sera were obtained from end point titer methods (Fig. 3). The linear four cases of naturally occurring scrub typhus regression of all the differences in titers between infection among Chinese military personnel stationed in the Pescadores Islands, Republic of China. In addition, two control sera were obtained from two fever patients admitted to the same hospital with a self-resolving fever of unknown origin (M-109) and influenza (A-Victoria strain) (M-210), respectively. All cases of scrub typhus were confirmed by presence of eschar and rickettsial isolation and were treated with o tetracycline or doxycycline commencing on the date of admission (date of the first serum taken), at J whereas the controls were negative for rickettsial isolation. 5 IEach antiserum was titrated against antigens of the Karp, Gilliam, and Kato strains of scrub typhus by IFA and ELISA, using specific anti-u O IgM and anti-IgG conjugates (Table 2). Linear 3 regressions for both IgM and IgG titers were gen is given above the respective heterologous anti- 0.2, Fisher's z statistic) (34). A comparison of gen. The difference in titers between the two heterol- the calculated regression line of the IFA and ogous antigens was generally not significant (P > 0.1, ELISA end point titers with a line through the not shown). points of equivalent titers indicated that the two 0
-J
Ia
-
-J
VOL. 9, 1979
45
ELISA FOR ANTIBODIES AGAINST SCRUB TYPHUS
TABLE 2. Titers of human scrub typhus antiseraa Antigen used Antiserum
Patient
Kato
Gilliam
Karp
IFF ISA ti- EOD 1:A tiEL ti- Or past Conju- IFAtitebELISA Days LS ELSAtOD 1: IFA titer ELISA Dy titer ter i ter ter' gate IFA titerb EIAt onsetatCnu oo
10
ELISA
OD 1: 0
2,870 1,150 0.45 5,000 0.11 9,130 43 254 7 IgM M-101 75 0.19 180 190 527
