Normally occurring human anti-GM1 immunoglobulin M antibodies and the immune response to bacteria

Abstract

Anti-GM(1) antibodies of the immunoglobulin M (IgM) isotype are normal components of the antibody repertoire of adult human serum. Using a sensitive high-performance thin-layer chromatography (HPTLC) immunostaining assay, we found that these antibodies were absent in the umbilical vein and children <1 month of age but could be detected after 1 month of age. Although most of the children older than 6 months of age were positive, there were still a few negative children. The appearance of anti-GM(1) IgM antibodies showed a perfect concordance with two well-characterized antibacterial antibodies, anti-Forssman and anti-blood group A, which indicates a similar origin. We also studied IgM reactivity with lipopolysaccharides (LPSs) from gram-negative bacteria isolated from stool samples from healthy babies and from Escherichia coli HB101 in serum from individuals of different ages. We found a positive reaction with both LPSs in all the children more than 1 month of age analyzed, even in those that were negative for anti-GM(1) antibodies. Anti-GM(1) IgM antibodies were purified from adult serum by affinity chromatography and tested for the ability to bind LPSs from different bacteria. This highly specific preparation showed reactivity only with LPS from a strain of Campylobacter jejuni isolated from a patient with diarrhea. We conclude that normally occurring IgM antibodies are generated after birth, probably during the immune defense against specific bacterial strains.

FIG. 1.

Antiglycan IgM antibodies in normal human serum. Forssman and blood group A glycolipids (lane 1, upper and lower bands, respectively), and gangliosides GA₁, GM₁, GD_1a, and GD_1b (lane 2, top to bottom) were separated by HPTLC as described in Materials and Methods. Plates were incubated with the stated human serum, and after being washed, IgM antibody binding was detected with peroxidase-labeled anti-human IgM antibodies. One plate was stained with orcinol reagent for chemical detection of glycolipids.

FIG. 2.

Normal human serum IgM antibody binding to bacterial LPSs. LPSs from gram-negative bacteria isolated from stool samples from healthy babies (lane 1) and from Escherichia coli HB101 (lane 2) were prepared by the miniphenol-water method (16). Both preparations and GM₁ (lane 3) were separated by HPTLC and assayed for IgM antibody binding as described in Materials and Methods. Serum samples from an umbilical vein, an adult, and two children were used. Notice the absence of anti-GM₁ IgM antibody binding in the serum sample from the 9-month-old child. Arrows indicate a nonspecific spot originating from the culture medium.

FIG. 3.

Cross-reactivity of anti-GM₁ antibodies with LPS from E. O127-B8 (lane 1), gram-negative bacteria isolated from stool samples from healthy babies (lane 2), Pseudomonas aeruginosa serotype 10 (lane 3), Shigella flexneri (lane 4), Salmonella enteritidis (lane 5), Campylobacter jejuni isolate 1 and C. jejuni isolate 2 (lane 6), were chromatographed together with GM₁ (lane 8) and assayed for IgM antibody binding as described in Materials and Methods. HPTLC plates were incubated either with total adult serum samples (lane 7) or (B) their anti-GM₁ IgM antibodies purified by affinity chromatography (C). One plate was stained with orcinol reagent (A). The asterisk indicates a nonspecific spot originating from the culture medium.