THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136, NO.1. JULY 1977 © 1977 by the University of Chicago. All rights reserved.

Detection of IgG Antibodies to Type-Specific Pseudomonas aeruginosa Lipopolysaccharides by Solid-Phase Radioimmunoassay From the Veterans Administration Hospital and the Infectious Diseases Division, Department of Internal Medicine, Indiana University Medical Center, Indianapolis, Indiana

Richard Kohler and Arthur \Vhite

Pseudomonas aeruginosa organisms can be serotyped according to their type-specific cell wall Iipopolysaccharides [I]. In vitro opsonophagocytosis of P. aeruginosa by polymorphonuclear leukocytes requires the presence of type-specific antibodies [2-4]. Although both IgG and IgM antibodies can function as specific opsonins in vitro, an animal model suggests that IgG antibodies may provide better protection in vivo [5]. Sadoff and Artenstein reported that antibodies directed against the cell wall membrane of P. aeruginosa can be detected in rabbit antisera by solid-phase radioimmunoassay [6]. In the present study we evaluated solid-phase radioimmunoassay with use of polystyrene tubes coated with lipopolysaccharide for detection of type-speci-

fic IgG antibodies in human sera after either vaccination or natural infection. Materials and Methods

Human sera. Sera were obtained from 15 normal adult male volunteers before and 10-145 days after initiation of immunization with an investigational heptavalent pseudomonas vaccine [7]. Each injection of vaccine contained 17 p.g of lipopolysaccharide/kg from each of seven serotypes of P. aeruginosa; analysis of the vaccine components has been reported [7, 8]. Each volunteer received eight im injections of vaccine over a six-week period. In addition, four sera from drug addicts with endocarditis due to P. aeruginosa were donated by Drs. A. Martin Lerner and Milagros Reyes (Wayne State University, Detroit, Mich.). Radioactive label. Goat antiserum to human IgG Fe fragment was purchased from Cappel Laboratories (Dowingtown, Pa.) and iodinated with 1251 by New England Nuclear Corp., Boston, Mass.; the iodine monochloride technique [9J was used. After iodination the antiserum was found by immunoelectrophoresis to produce a precipitin arc only to IgG when tested against whole human serum. The specific activity of the labeled material was approximately 0.015 mCi/ mg of protein (1.18 mCi/ml). Solid-phase radioimmunoassay. Lipopolysaccharide or sonicated antigen was coated onto

Received for publication August 23, 1976, and in revised form February 27,1977. This study was supported by grants from Parke-Davis and Company, Detroit, Michigan, and Eli Lilly and Company, Indianapolis, Indiana. This work was presented in part at the 33rd Annual Meeting of the American Federation for Clinical Research, Atlantic City, New jersey, May 2, 1976. We thank Drs. Mike Fisher and Henry Devlin (ParkeDavis and Company) for supplying the sera of vaccinees and for performing the passive hemagglutination tests, Ms. jennifer Smith for providing technical assistance, Dr. james Norton for providing help with the statistics, and Ms. Jean Rutter for typing the manuscript. Please address requests for reprints to Dr. Richard Kohler, Department of Internal Medicine, Infectious Diseases Division. OP 317, Wishard Memorial Hospital, Indianapolis, Indiana 46202.

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Previous studies have suggested that IgG serotype-specific antibodies are protective against infections with Pseudomonas aeruginosa. In the present study, type-specific IgG antibodies to P. aeruginosa were detected by solid-phase radioimmunoassay in sera from 15 volunteers before and after vaccination with lipopolysaccharides from P. aeruginosa and from four patients with endocarditis due to P. aeruginosa. Significant type-specific increases in IgG antibody occurred after both vaccination and infection. The correlation coefficients comparing net counts per minute by solidphase radioimmunoassay with hemagglutination titers in the 15 vaccinees were 0.940,0.874,0.792,0.903,0.882,0.869, and 0.704 for serotypes 1-7, respectively.

Antibodies to P. aeruginosa LPS

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to-tube variability, the first two coating steps were done on a single day one week before initiation of the assays. Each antigen was from a single lot. The tubes were then stored dry at 4 C until the day of the assays. All sera were tested within a five-day period. Each serum was tested in simultaneously assayed duplicates against each antigen. Controls consisted of tubes coated with bovine serum albumin alone. Each control was tested in quadruplicate. Results are expressed as net cpm (mean cpm of antigen-coated tubes - mean cpm of control tubes). Passive hemagglutination. Each serum from P. aeruginosa-vaccinated or -infected patients was tested for HA antibodies as previously described [1 OJ. Statistical methods. For detection of any aberrant readings, the distribution of absolute differences between duplicate tubes was examined. For testing for potential outliers, the upper 99% limit of tolerance for the right-half normal distribution was calculated. This value may be obtained as the upper end of a two-tailed 99% tolerance

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Figure 1. Correlation between results of duplicate radioimrnunoassays of 30 sera from 15 persons vaccinated with lipopolysaccharides of Pseudomonas aeruginosa. All sera were tested against each of seven antigens (from P. aeruginosa serotypes 1-7). rI = intraclass correlation coefficient.

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the inside walls of 12- X 75-mm polystyrene tubes (Falcon Plastics, Oxnard, Calif.) by allowing 0.2 ml of the antigen solution to remain in the tube at 37 C for I hr. The Iipopolysaccharides were used at a concentration of 50 /Lg/ml; sonicates of other bacteria were used in a I: 100 dilution in 0.85% NaCt U nadsorbed antigen was then aspirated, the tubes were rinsed three times with 0.85% N aCl, and 0.2 ml of I % bovine serum albumin in phosphate-buffered saline, pH 7.4, was added; this mixture was incubated for 1 hr, aspirated, and rinsed. This step was performed to coat remaining binding sites on the walls of the tubes. Next, 0.2 ml of the test serum was added, incubated for 1 hr at 37 C, and aspirated. All sera were used at a 1: 10 dilution in I % bovine serum albumin in phosphate-buffered saline, pH 7.4, except for the four sera from patients with endocarditis which were diluted 1:30 because of the small quantities available. Finally, 0.2 ml of a 1: 100 dilution of the 125I-Iabe1ed goat antiserum to IgG Fe fragment was added, incubated for 1 hr, and rinsed as before. Each tube was then counted in a y-counter. To diminish tube-

Kohler and White

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Results

Agreement between duplicate tubes. The results of testing of duplicate sera from each of the 15 volunteers vaccinated against each of the sevSEROTYPE

SEROTYPE

SEROTYPE

1

2

3

en antigens of P. aeruginosa are shown in figure 1. The intraclass correlation between duplicate readings of cpm, excluding the four outliers represented by open circles, was 0.988. Detection of response to vaccination. Response to each antigen was analyzed first by passive HA testing of 2-mercaptoethanol-treated and untreated sera. Both 2-mercaptoethanol-treated and untreated sera from all 15 vaccinees demonstrated a fourfold rise in passive HA titer after vaccination against at least one of the seven antigens in the vaccine mixture. Eleven vaccinees responded to at least five antigens, and 10 demonstrated a significant rise in HA titer of antibody to one or two antigens in either the 2-mercaptoethanol-treated or untreated serum (but not both). For comparison with the results of solid-phase radioimmunoassay, these responses were defined as equivocal. By use of the Iipopolysaccharides from serotypes 1, 2, 4, 5, and 6 in the radioimmunoassay, appropriate rises (or lack thereof) were detected (figure 2). A similar correlation was seen for serotype 3, although the increases in cpm after vaccination were less marked than for the aforementioned serotypes. Responses to the lipopolysaccharide of serotype SEROTYPE

SEROTYPE

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Detection of IgG antibodies to type-specific Pseudomonas aeruginosa lipopolysaccharides by solid-phase radioimmunoassay.

THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136, NO.1. JULY 1977 © 1977 by the University of Chicago. All rights reserved. Detection of IgG Antibodies t...
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