Int. Archs Allergy appl. Immun. 48: 485-494 (1975)

Quantitation of Salm onella O-Antibodies in Human Sera by Enzyme-Linked Immunosorbent Assay (ELISA) H ans E. C arlsson, A lf A. Lindberg , Sten H ammarstrom and A ke L junggren Department of Immunology, Wenner Gren Institute. University of Stockholm, and Department of Bacteriology, National Bacteriological Laboratory, Stockholm

Abstract. The enzyme-linked immunosorbent assay (ELISA) has been applied to the detection of antibodies against Salmonella O-antigens in human sera. Phenol-water ex­ tracted lipopolysaccharides (LPS) from serogroups A (O-antigens 2,12), B (4, 5,12) and D (9,12) were used as antigens. When compared to the tube agglutination method according to Widal employing sera from patients with verified or suspected typhoid - or paratyphoid fever and from healthy controls it was found that ELISA (i) correlated significantly with the Widal reaction, (ii) was up to 100-fold more sensitive, and (iii) showed a greater re­ producibility.

Introduction

Received: October 9, 1974.

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The immunological diagnosis of typhoid and paratyphoid fever is classi­ cally done by the tube agglutination method (Widal’s reaction [13]) or by passive hemagglutination [10]. Although both tests are often indispensable in the diagnosis they suffer from certain disadvantages. The Widal reaction often gives false-positive reactions and it is also relatively insensitive. Passive hemagglutination is more sensitive but detects preferentially antibodies of the IgM class, as does the Widal test. Therefore, it has for a long time been desired to increase both the sensitivity and the specificity of the tests in order to improve the immunological diagnosis of these and related infectious diseases. In a previous paper we described the use of an enzyme-linked immunosorbent assay (ELISA) employing phenol-water extracted lipopoly­ saccharide (LPS) from Salmonella as the antigen and rabbit O-antibodies [2], ELISA was found to be specific and sensitive; 10-100 times more sensitive

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than the Widal and hemagglutination reactions [2], In addition, antibodies of the IgG and IgM classes were detected with approximately the same efficiency. This paper describes the use of ELISA to estimate the titer of antiSalmonella O-antibodies in human sera. We found that ELISA correlated well with the Widal reaction, and was a more sensitive test with an improved reproducibility as compared to the Widal reaction.

Material and Methods Bacterial Strains For preparation of antigen the following strains were used : S. paratyphi A var. durazzo (O-antigen 2, 12i, 123) S. typhimurium LT2 (O-antigen 4, 5, 122) and S. typhi T2 (Oantigen 9, 12i, 123). The bacteria were cultured in complex broth medium as described earlier [6]. The yield was 4-6 g bacteria/1 culture. Isolation o f LPS The bacteria were harvested by centrifugation, killed by y-irradiation and washed twice in phosphate-buffered saline (PBS) pH 7.3. Bacterial cell-walls were prepared as described earlier [9], and the LPS was extracted by the hot phenol-water method [12]. All LPS preparations were spun in the ultracentrifuge (105,000 g for 4 h) until they were essen­ tially free from material absorbing at 260 nm. If necessary, remaining nucleic acid was removed by treatment with ribonucléase (type XII-A, Sigma Chemical Co., St. Louis, Mo.). The chemical structure of these LPS species is shown in figure 1 [7, 8], Sera Sera positive in the Widal reaction (titer> 1/160) were collected from patients with suspected or verified typhoid or paratyphoid fever. Sera from 41 randomly selected patients positive in serogroup D (O-antigen 9,12) and from 12 randomly selected patients positive in serogroup B (O-antigen 4, 5, 12) were chosen. Sera negative in the Widal reaction (titer< 1/80) were obtained from 36 healthy blood donors. The sera were stored at -20 °C until used.

Widal Reaction Tube agglutination for titration of O-antigens was performed using the procedure according to F elix [5], To glass tubes containing 0.5 ml doubling dilutions of serum in PBS a 0.5 ml amount of a standardized suspension of the appropriate heat-killed bacterium was added. The agglutination was recorded after incubation for 18 h at 37 °C. Sera ag­ glutinating in dilutions of 1/160 or more were considered positive.

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Absorption 0.5-ml amounts of serum were absorbed twice with 109 - 1010 heat-killed bacteria/ml for 1 h at 25°C. Sera were cleared by centrifugation at 5,000 g for 20 min at 4°C and stored at -20°C.

Salmonella O-Antibodies in Human Sera R

Glc

R

.1

a4

(Man 1

487

4 or 6

«4 ,

4 Rha 1 £ 3 Gal 1 -)-* -2 Man 1 ¿ 4 Rha 1 ¿ 3 Gal 1

Fig. 1. Main structural features of the Salmonella O-polysaccharide chain. R in serogroup A is paratose, serogroup B 20Ac-abequose and scrogroup D tyvelose. Abbreviations: Man = Mannose; Rha=rhamnose; Gal = galactose, and Glc= glucose.

Enzyme Conjugated Anti-Immunoglobulin Antibodies against human immunoglobulin were obtained by immunospecific purifica­ tion of hyperimmune rabbit antiserum on insolubilized human IgG (Gamma KABI, AB KABI, Stockholm). A 0.5-mg amount of anti-human immunoglobulin was conjugated with 1.5 mg alkaline phosphatase (calf intestinal mucosa, type VII; Sigma Chemical Co., St. Louis, Mo.) by the addition of glutardialdehyde [3], Conjugated material was separated from unconjugated enzyme and immunoglobulin by gel-filtration on Sepharose 6B (Phar­ macia AB, Uppsala). The conjugate was stored at 4°C in 5% human serum albumin (AB KABI, Stockholm) in 0.05 m Tris-HCl buffer (pH 8.0) containing 0.02% NaNs. Enzyme-Linked Immunosorbent Assay ELISA was performed essentially as described by E ngvall and P erlmann [4]. Dis­ posable polystyrene tubes [11 x 55 mm, Heger Plastics AB, Stallarholmen, Sweden) were incubated with 1 ml of LPS solution containing 5 US LPS/ml in 0.05 m carbonate buffer (pH 9.6) containing 0.02 % N aN 3, for 3 h at 37 °C. The tubes were washed three times with 0.9% NaCl containing 0.05% Tween 20 (Kemi Intressen AB, Sundbyberg, Sweden) be­ fore incubating with serum. To the washed LPS-coated tubes were added 1 ml serum diluted in PBS with 0.05% Tween 20 and 0.02% NaNj (PBST) and the mixture was in­ cubated at room temperature for 5 h. The tubes were washed as before, and 1 ml of rabbit anti-human Ig-alkaline phosphatase conjugate diluted 1/500 in PBST was added. After incubation at room temperature overnight (18 h), the tubes were washed as above, and 1 ml of a solution of enzyme substrate, p-nitrophenylphosphate (Sigma Chemical Co., St. Louis, Mo.) 1 mg/ml in 0.05 m carbonate buffer (pH 9.8) containing 0.001 m MgCk was added. The enzyme reaction was performed ar room temperature for 100 min or until the absorbance at 400 nm reached approximately 0.8. The reaction was stopped by the addition of 0.1 ml of 1.1 m NaOH. In the present system, the titre was arbitrarily chosen as the dilution of serum that gives an absorbance of 0.5 at 400 nm/100 min.

Titration o f Sera in ELISA Sera, in duplicates, were tested in dilutions 10“2 - 10~5 using tenfold dilution steps. In all experiments sera positive in serogroup B and D (Widal

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Results

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titers >1/160) and negative control sera were run together. A reference human serum was always included. As a control for the LPS coating of the test tubes, a rabbit antiserum against the Salmonella O-factor specific for the LPS under study was also included. In eight experiments the mean ab­ sorbance per 100 min of the reference human serum at a dilution of 10-3 was 11.2 (SD = 0.90). Figure 2 shows the mean titration curves for the three groups of sera (41 positive in serogroup D, 12 positive in serogroup B and 36 blood donor sera) tested against tubes coated with LPS from S. typhi T2 (serogroup D). The sera positive in serogroup D and B (mean titers, expressed as the dilu­ tion of serum giving an absorbance of 0.5/100 min at 400 nm, were 1/12,000 and 1/5,000, respectively) could be diluted approximately 10-20 times more than control sera (mean titer 1/600) giving the same absorbance at 400 nm/100 min. There was a statistically significant difference between the mean titration curves of patient sera positive in serogroup D and the sera from healthy blood donors (p0.05). The mean titration curves for the three groups of sera (each group containing 12 sera randomly selected from the larger groups given above)

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Fig. 2. Titration of human sera in ELISA against LPS from S. typhi T2. The diagram shows mean titration curves of 41 sera from patients Widal positive in serogroup D (•), 12 patients positive in serogroup B (A), and 36 healthy blood donors (■) negative in Widal agglutination. The horizontal bars show the 95% confidence limits.

Salmonella O-Antibodies in Human Sera

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Fig. 3. Titration of human sera in ELISA against LPS from S. typhimurium LT2. The diagram shows mean titration curves of 12 sera from each of the groups: patients Widal positive in serogroup D (•) , patients positive in serogroup B (A), and healthy blood donors (■) negative in Widal agglutination. The horizontal bars show the 95% confidence limits.

Comparison between Titer in ELISA and Widal Agglutination The degree of association between results obtained in ELISA and the Widal reaction was estimated in Spearman’s rank correlation. All titers

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tested against tubes coated with LPS from S. typhimurium LT2 (serogroup B) and S. paratyphi A var. durazzo (serogroup A), respectively, are shown in figures 3 and 4. The mean titer of sera positive in serogroup B was 1/25,000 or about 25 times higher than that of blood donors (mean titer 1/1,000) when tested against S. typhimurium LPS (serogroup B), (fig. 3). The mean titer of sera positive in serogroup D (1/3,000) was only 3-4 times higher than the mean titer of the control group (1/1,000). The difference between mean titration curves of all the three groups tested is statistically significant at least with serum diluted 1/1,000 and 1/10,000 (p0.05).

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Fig. 4. Titration of human sera in ELISA against LPS from S. paratyphi A. The dia­ gram shows mean titration curves of 12 sera from each of the groups: patients Widal positive in serogroup D (•), patients positive in serogroup B (A), and healthy blood donors (■), negative in Widal agglutination. The horizontal bars show the 95 % confidence limits.

obtained in the two systems were used for calculation, giving a correlation coefficient, rs = 0.62; N = 161; (p

Quantitation of Salmonella O-antibodies in human sera by enzyme-linked immunosorbent assay (ELISA).

Int. Archs Allergy appl. Immun. 48: 485-494 (1975) Quantitation of Salm onella O-Antibodies in Human Sera by Enzyme-Linked Immunosorbent Assay (ELISA...
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