A comparative analysis of antibody repertoire against Staphylococcus aureus antigens in patients with deep-seated versus superficial staphylococcal infections

Abstract

Immunoblot and an enzyme-linked immunosorbent assays were used to evaluate and compare IgG antibodies against S. aureus whole cell lysate, cell wall peptidoglycan and lipoteichoic acid to discriminate between deep-seated and superficial S. aureus infection. Serum samples were examined from patients with deep-seated (n = 25) and superficial (n = 25) S. aureus infections and 15 healthy controls. Patients with deep-seated infections exhibited a large number of immuno-reactive bands in their IgG immunoblot profile as compared to those with superficial infections and healthy controls. Anti-staphylococcal IgG antibodies that reacted with two antigens of apparent molecular weight 110 and 98 kDa were specifically present in 96% (24/25) of patients with deep-seated infections, and were absent in, superficial and control sera. Moreover other Gram-positive and Gram-negative bacteria did not share these two unique antigens. The ELISA assays revealed significantly elevated levels of IgG antibodies to peptidoglycan (PG) in 18 of 25 (72%) patients with deep infection and 15 of 25 (60%) patients with superficial staphylococcal infection. The elevated levels of IgG antibodies to teichoic acid (TA) antigen were detected in all (100%) deep-seated group patients and among 40% (10/25) patients with superficial infection. An increase in levels of antibodies to PG showed a positive correlation trend with levels of IgG antibodies to TA only in deep infection group. Thus immunoblot detection of these two unique antigens as well as detection of elevated antibodies against PG and TA may be useful for the discrimination of staphylococcal deep-seated and superficial infection in humans.

Figure 1

Whole-cell protein profile of representative S. aureus strains. Clinical isolates of S. aureus were taken from patients with deep-seated (A) and superficial staphylococcal infection (B). The crude protein extracts were separated by SDS–PAGE and stained with Coomassie blue. Lane M; molecular weight marker; Lane A; standard S. aureus strain (ATCC 12600); Lane 1-6; whole cell lysates of representative S. aureus isolates. Figures are representative of two independent experiments

Figure 2

Immunoblot profiles of patients with deep-seated and superficial S. aureus infection. Whole cell protein lysate of S. aureus (ATCC 12600) was separated by SDS–PAGE and transferred onto nitrocellulose membranes which were probed with serum (1:1000) from patients with deep-seated (A) and superficial (B) infection. Each lane represents serum from individual patients. Serum samples were drawn 2-3 day after detection of S. aureus in clinical specimens. Results are representative of two independent experiments

Figure 3

Western blot analysis of S. aureus immunodominant antigens. Whole cell protein lysate of S. epidermidis (lane 1), S. aureus (lane 2), clinical isolates of coagulase negative staphylococci (lane 3-5), E. coli (lane 5) and Klebsiella sp (lane 6) were separated by SDS–PAGE and transferred onto nitrocellulose membranes and probed with pooled serum (1:1000) from patients with deep-seated (A) and superficial (B) infection and healthy controls (C). Results are representative of three independent experiments

Figure 4

Levels of antistaphylococcal IgG antibodies. Antibodies against S. aureus cell wall antigen peptidoglycan (A) and teichoic acid (B) were measured ELISA in sera (1:1000) from helathy individuals and patients with superficial and deep-seated infection. Violin plots were constructed on the basis of OD 492 nm to show the distribution IgG antibody levels in each group. Data are the representative of three independent experiments

Figure 5

Correlation plots of antibodies to PG and TA. Antibody levels against peptidoglycan and teichoic acid in patients with superficial (A) and those with deep-seated (B) as measured by ELISA were plotted against each other to elucidate correlation. Coefficient of correlation was determined by r² values as shown. Results represent the values of experiment in duplicates