Anti-lipopolysaccharide Antibodies Research Articles

Separation of Immunoglobulin and Transferrin from Blood Serum and Plasma by Metal Chelate Interaction Chromatography

Metal chelate interaction chromatography was used to separate Ig, transferrin, and albumin from blood serum and blood plasma. A column was packed with iminodiacetic acid: 1,4-butanediol diglycidyl Sepharose 6B or Sephacryl S-300 and loaded with copper, zinc, nickel, or cobalt ion. Radial immunodiffusion assay indicated that Ig-rich fractions of blood serum obtained from Zn-, Mi-, Co-, and Cu-loaded columns contained 23.2, 81.3, 79.4, and 98.1% active IgG, respectively. Transferrin was recovered from the second peak. When the same conditions of metal chelate interaction chromatography were used for blood plasma, hemoglobin tended to bind strongly to the Cu-loaded column and was eluted only with 50% ethanol. Modification of histidine residues in Ig and transferrin with diethyl pyrocarbonate almost completely destroyed their binding ability to the column. Immunoglobulin G separated showed antilipopolysaccharide antibody activity against Escherichia coli. Salmonella typhimurium, and Bordettella parapertussis.

Antibody inhibition of HeLa cell invasion by Yersinia enterocolitica.

Antisera were prepared in rabbits against formalized and heat-killed bacteria of Yersinia enterocolitica serotype O:5,27 and against formalized bacteria of serotype O:8. Both strains used for immunization demonstrated adhesion to and invasion of HeLa cells. Coating of the bacteria with antibody did not greatly alter adhesion (i.e., extracellular attachment) to HeLa cells; however, antibody against formalized bacteria of both serotypes inhibited HeLa cell invasion by the homologous and heterologous strains. The Fab fragments from purified immunoglobulins also demonstrated cross-reacting inhibition of HeLa cell invasion. Antibody against heat-killed bacteria of serotype O:5,27 had no inhibitory activity. Adsorption of the antiserum against formalized bacteria of serotype O:5,27 with lipopolysaccharide from the homologous strain removed anti-lipopolysaccharide antibody but did not remove the inhibitory activity. The antiserum against formalized bacteria of serotype O:8 showed no antibody against lipopolysaccharide from serotype O:5,27 and no agglutinins against heat-killed bacteria of this strain. From these results, it is tentatively suggested that protein structures are important in mediating epithelial cell invasion by Y. enterocolitica.

ELISA for human IgG and IgM anti-lipopolysaccharide antibodies with indirect standardization.

A new attempt to standardize ELISAs for the quantitation of human IgM and IgG anti-lipopolysaccharide antibodies (anti-LPS), without the use of a specific standard material, is described. Sandwich ELISAs for total IgG and IgM were combined with indirect ELISAs for anti-LPS IgG and IgM antibodies on a 96-well microtest plate using identical assay conditions. The concentration of specific IgG or IgM anti-LPS was read on the respective standard curve for total IgG or IgM. The results were corrected for residual immunoreactivity remaining unbound in the wells after one sample incubation in the combined assays. The quantitative results of IgG anti-LPS correlated well with results obtained using an ELISA with direct standardization (r = 0.969). 28 mg/l of IgM anti-LPS was found as median value among 121 blood donors using the described ELISA principle. Binding studies demonstrated a lower apparent affinity of donor anti-LPS IgM than anti-IgG.

Absence of complement fixing antibodies against lipopolysaccharides from Escherichia coli in a subgroup of patients with Crohn's disease.

Complement fixing antibodies against different Escherichia coli lipopolysaccharides were determined in patients with Crohn's disease and in healthy individuals and compared with antitetanus toxoid antibodies. All healthy individuals had antilipopolysaccharide antibodies, 10 of 27 patients with Crohn's disease had no antibodies and six had rapidly changing antibody titres. These abnormalities were found in patients with disease in the colon, with arthropathy and fistula. Antilipid A was found at lower titres in Crohn's disease. Neither antitetanus toxoid antibodies, nor immunoglobulin concentrations were different in patients with or without antilipopolysaccharide antibodies. There was no evidence for circulating immune complexes in patients lacking antilipopolysaccharide antibodies. Certain subgroups of patients with Crohn's disease have altered antibody levels to typical enteral antigens which most likely can be explained by local antibody binding to lipopolysaccharides at inflammatory sites, or by changes in immunoregulation in this disease.

Immunoglobulins in bile and serum of the rabbit associated with protection after Vibrio cholerae infection and vaccination

Previous studies have shown that cholera, as well as protective immunity against infection with Vibrio cholerae, can be induced in the rabbit. This protection is long-lasting (up to 30 months) and is characterized on challenge by rapid, symptom-free disappearance of V. cholerae from the intestine; we therefore believe this to be vibriocidal protection. In this study, we analysed the humoral and secretory immune response against various subcellular V. cholerae components in vibriocidally protected, non-vibriocidally protected, and unprotected animals. Only vibriocidal protection was found to be associated with high levels of biliary IgA directed against lipopolysaccharide O antigen. We did not find such a correlation between either type ot protection and response in serum. Therefore, anti-lipopolysaccharide antibodies are essential in protection against experimental infection with V. cholerae.

Protective immunity induced by outer membrane proteins of Salmonella typhimurium in mice.

Outer membrane proteins (OMP) extracted from both smooth (C5) and rough (Rb2) strains of Salmonella typhimurium were able to induce protective immunity to salmonellosis. The OMP-induced protection lasted for at least 6 months. The antibody level was estimated by passive hemagglutination. In the C5 OMP-immunized mice, antibodies to both proteins and lipopolysaccharide were detected. On the other hand, in the Rb2 OMP-immunized mice, antiprotein but not antilipopolysaccharide antibodies were detected. Delayed-type hypersensitivity appeared as early as the second week after immunization with OMP and persisted through the fourth week.

Lack of protection against ascending Escherichia coli pyelonephritis in diabetic rats following immunization with purified lipopolysaccharide.

Purified lipopolysaccharide of Escherichia coli produced specific antibody when injected intraperitoneally or given to rats orally. Either route of immunization did not prevent ascending pyelonephritis in a diabetic rat model. The use of purified LPS excludes the potential contribution of other virulence factors of E. coli as protective antigens in the prevention of ascending pyelonephritis and confirms that anti-lipopolysaccharide antibody is not protective for ascending pyelonephritis.

Strong biotype and serotype cross-protective antibacterial and antitoxic immunity in rabbits after cholera infection

We studied whether immunity evoked by infection with classical or El Tor V. cholerae 01 organisms in rabbits (the RITARD model) also gives protection against cholera caused by V. cholerae of heterologous biotype as well as serotype, and whether such protection is antibacterial and/or antitoxic. A primary infection with a classical Ogawa or El Tor Inaba strain resulted in intestinal colonization and diarrheal disease in a dose-related manner though the El Tor strain was more virulent. As few as 10 3 El Tor organisms gave disease in more than 90% of the rabbits as compared to 10 9 classical organisms (ED 90); the El Tor strain also gave rise to diarrhea with earlier onset and of greater severity and longer duration. The primary infection induced strong protective immunity against later challenge with either the homologous or the heterologous strain in doses that corresponded to 1000 × ED 90. Protection was associated with marked inhibition of colonization, and when rabbits convalescing from cholera infection were challenged with graded doses of bacteria or purified toxin in ligated intestinal loops significant antibacterial as well as antitoxic immunity was evident. Titer rises in serum vibriocidal and anti-lipopolysaccharide antibodies were similar after infection with either strain, whilst antitoxin titer rises were more marked after El Tor infection. During infection V. cholerae 01 organisms seem to express protective antigens that stimulate immunity which extends across both biotype and serotype barriers.

Purification and partial characterization of outer membrane proteins P5 and P6 from Haemophilus influenzae type b

The major outer membrane proteins of Haemophilus influenzae type b (Hib), designated P5 and P6 (R.S. Munson, Jr., J.L. Shenep, S.J. Barenkamp, and D.M. Granoff, J. Clin. Invest. 72:677-684, 1983), were purified to homogeneity and partially characterized. P5 was insoluble in octylglucoside-NaCl and could be extracted with 1% sodium dodecyl sulfate (SDS) in 20 mM phosphate (pH 7.5). Solubilized P5 was further purified on hydroxylapatite in 0.1% SDS. The purified protein had an apparent molecular weight of 27,000 as determined by SDS-polyacrylamide gel electrophoresis after sample preparation at room temperature. The protein migrated with an apparent molecular weight of 35,000 after heating for 30 min at 100 degrees C in the presence of 10% beta-mercaptoethanol (beta ME). Rabbit antisera prepared against the purified preparation immunoprecipitated solubilized protein P5 but had no protective activity in the infant rat bacteremic model. The SDS-insoluble residue was further extracted with 1% SDS-0.5 M NaCl-0.1% beta ME at 37 degrees C. A single outer membrane protein, designated P6, with an apparent molecular weight of 16,000, remained insoluble under these conditions. Antiserum prepared against this insoluble fraction contained antibodies which, after removal of anti-lipopolysaccharide antibody, immunoprecipitated P6 and protected infant rats challenged with Hib. Protein P6 could be released from the insoluble cell wall in the presence of SDS-NaCl-beta ME at 60 degrees C. Thus, proteins P5 and P6 could be purified from the cell envelope of Hib. Based on the results from infant rat passive protection experiments, antigens in the P6-cell wall fraction merit further investigation as possible vaccine components. In contrast, epitopes on protein P5 did not appear to elicit protective antibody.

Properties of Human Anti‐Lipopolysaccharide Gamma Globulin: Specificity and Protective Effects

Blood donated to the Natal Blood Transfusion Service was screened by an enzyme-linked immunosorbent assay (ELISA) for anti-lipopolysaccharide (anti-LPS) antibodies. Plasma units with high concentrations (greater than 40 micrograms/ml) of anti-LPS IgG were pooled and fractionated to obtain a gamma globulin (lot LG-1). The binding of LG-1 antibodies to LPS prepared from 14 bacterial species and strains was found to be the highest to LPS from Shigella flexneri, Salmonella abortus equi and Salmonella typhimurium and intermediate with Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis and Escherichia coli 026:B6. Differential absorption experiments showed that LG-1 contained a mixture of specific and cross-reacting antibodies. A large proportion of antibodies binding to Sh. flexneri LPS were mainly specific, while those binding to S. typhimurium and the other Salmonella species tested were largely cross-reactive. There was little correlation between the spectrum of activity of the LG-1 antibodies and the incidence of gram-negative bacteria in blood cultures taken from hospital patients in an area covered by the Transfusion Service. Mice treated with LG-1 prior to inoculation with P. aeruginosa were significantly protected against morbidity and mortality compared to controls.

Prophylaxis of superior mesenteric artery occlusion shock in rabbits by antilipopolysaccharide (anti-LPS) antibodies

Occlusion of the superior mesenteric artery (SMA) is known to produce lethal shock in experimental animals, probably due to the presence of endotoxins released from the gut. A 60-min SMA occlusion shock was produced in 20 rabbits. Eight were pretreated with equine antilipopolysaccharide (anti-LPS) hyperimmune plasma, and seven survived (87.5%) for 10 days. However, only 2 out of 12 (16.6% χ 2 = 9.73, P < 0.001) control rabbits survived. These results support the concept that endotoxins are involved in the etiology of SMA occlusion shock and that anti-LPS therapy may therefore be useful in the prevention of endotoxic shock.

Degree of antibody-independent activation of the classical complement pathway by K1 Escherichia coli differs with O antigen type and correlates with virulence of meningitis in newborns

A total of 95 K1 Escherichia coli strains of the O (lipopolysaccharide) serotypes O1, O7, or O18 had been analyzed previously for the ability to cause bacteremia after colonizing the gut of newborn rats. In this study, these strains were tested for their resistance to the bactericidal activity of rat serum. All strains that had caused bacteremia in a high percentage of the inoculated rats were able to survive for several hours in 90% adult rat serum. With only a few exceptions, O7:K1 and O18:K1 strains were serum resistant and virulent, whereas O1:K1 strains were serum sensitive and avirulent. Serum sensitivity was due to the classical complement pathway. K1 strains of all three O serotypes were resistant to the alternative complement pathway. O7:K1 and O18:K1 cells were killed efficiently after the classical pathway was triggered by specific antilipopolysaccharide antibodies. However, killing of O1:K1 bacteria by the classical pathway system did not require antibodies. Isolated O1-lipopolysaccharide fixed complement more efficiently than did isolated O7- or O18-lipopolysaccharide, suggesting that the differences in the chemical structure of the O antigens are responsible for the observed differences in complement sensitivity. In combination with epidemiological data, the results indicate that antibody-independent classical pathway activation provides an important defense mechanism for newborns against certain gram-negative infections.

Enzyme-linked immunoassays for antibodies against Vibrio cholerae.

Glutaraldehyde-treated V. cholerae organisms bind firmly to the surfaces of plastic microELISA plates, thus providing a stable immobilized antigen for use in enzyme-linked immunosorbent assays (ELISA). Serum absorption and ELISA-inhibition experiments indicate that, in addition to detecting natural antibodies in normal rat serum, the immobilized antigen may be used to quantitate specific anti-V. cholerae antibodies induced in rats by injection of live organisms. Apart from serotypically specific anti-lipopolysaccharide (LPS) antibodies, the reaction with immobilized organisms seems to involve antibodies common to Inaba and Ogawa serotypes; it is suggested that these antibodies are primarily directed against antigens of the outer membrane protein complex of V. cholerae cells. These findings have led to the development of an indirect ELISA method which quantitates levels of antibodies that react with heat-sensitive surface antigens of V. cholerae without involvement of anti-LPS antibodies. When used in conjunction with the indirect LPS-ELISA, the test has been found to provide a more detailed description of the serum antibody responses of rats to parenteral injections of live V. cholerae than has been reported previously.

Further studies of the role of noncapsular antibody in protection against experimental Haemophilus influenzae type b bacteremia.

Serum antibody against polyribosylribitol phosphate, the capsular antigen of Haemophilus influenzae type b, confers protection against experimental Haemophilus infection. Antibodies against noncapsular antigens are also protective, but the antigenic specificity of the protective antibodies remains unknown. Antilipopolysaccharide antibody was prepared by immunization of rabbits with boiled H. influenzae type b cells. Antilipopolysaccharide antibodies present in these sera did not protect against experimental Haemophilus bacteremia in infant rats. Antisera were also prepared by immunization of rabbits with live H. influenzae type b bacteria. After absorption of anticapsular and antilipopolysaccharide antibodies, these sera contained antibody to several outer membrane proteins which were accessible on the intact bacterial surface as detected by radioimmune precipitation. These absorbed sera prevented experimental Haemophilus bacteremia in infant rats. Thus, antibodies against noncapsular, non-lipopolysaccharide determinants, possibly against one or more outer membrane proteins, confer protection against experimental H. influenzae type b disease. In contrast, antibodies against lipopolysaccharide are ineffective.

Role of heat labile antigens of Shigella flexneri in HeLa cell invasion.

In studies of the role of surface antigens of Shigella flexneri in HeLa cell invasion, three antisera were employed to block the invasion. Antisera against live (ALS) and boiled (ABS) S. flexneri blocked invasion very effectively. Reduction in the numbers of intracellular shigellae was always accompanied by reduction in the number adherent to the cells, indicating the importance of adhesion in the invasive process. Anti-live absorbed antiserum (ALAS) prepared by exhaustive absorption of ALS with boiled S. flexneri blocked adhesion and invasion at dilutions of 20 or 50; the efficiency of the absorption was indicated by absence of agglutinating and anti-lipopolysaccharide (LPS) antibodies. S. flexneri LPS did not block adhesion and invasion even at a concentration of 1.0 mg/ml. Hence it was concluded that heat-labile surface antigens are important in adhesion and invasion of HeLa cells by S. flexneri. Antiserum against heat stable antigen (ABS) probably blocks adhesion by steric hindrance.

Isolation, purification, and partial characterization of Brucella abortus matrix protein.

Peptidoglycan sacculi with peptidoglycan-associated proteins were prepared from cell envelopes of Brucella abortus by extraction with sodium dodecyl sulfate (SDS) at 50 degrees C. On extraction of these preparations with SDS at 100 degrees C, a protein was obtained whose removal from peptidoglycan was confirmed by electron microscopy. Incubation of the 50 degrees C SDS-extracted cell envelopes with 50 mM MgCl2 in SDS-2-beta-mercaptoethanol at 37 degrees C also extracted the protein, along with lipopolysaccharide. At temperatures below 60 degrees C, the protein did not bind SDS strongly and had an apparent molecular weight greater than 92,000 in SDS-polyacrylamide gel electrophoresis. At higher temperatures, SDS bound strongly, and the apparent molecular weight was 38,000. Urea at 5 M did not alter the electrophoretic mobility of this 38,000-molecular-weight form. Immunoelectrophoresis in detergents with antisera to cell envelopes, carbohydrate staining of SDS-polyacrylamide gels, and production of anti-lipopolysaccharide antibodies by mice immunized with the purified protein indicated that lipopolysaccharide was present in free and protein-bound forms. Sequential gel filtration in SDS-EDTA and SDS-NaCl removed most lipopolysaccharide. After further purification by preparative SDS-polyacrylamide gel electrophoresis, a gas-liquid chromatographic analysis showed residual lipid tightly associated with the protein. The results suggested that the interactions between matrix proteins and other outer membrane components are stronger in B. abortus than in Escherichia coli, which was used as a control throughout.

Demonstration of antibodies against Yersinia enterocolitica lipopolysaccharide in human sera by enzyme-linked immunosorbent assay.

Antibodies against Yersinia enterocolitica serotype O:3 lipopolysaccharide present in sera from patients with Yersinia infection were studied by using an enzyme-linked immunosorbent assay. Of the sera with significant bacterial agglutination titers against Y. enterocolitica type O:3, 86% contained anti-lipopolysaccharide antibodies of the immunoglobulin G class. With the sera of some patients, we demonstrated increasing anti-lipopolysaccharide antibody levels of immunoglobulin G class in spite of decreasing bacterial agglutination titers. The assay was specific for lipopolysaccharide from Y. enterocolitica type O:3, and in inhibition experiments lipopolysaccharide could be detected in amounts of greater than or equal to 0.5 micrograms/ml.

Antibody-Dependent Cell-Mediated Cytotoxicity

Lymphoid cells from nonsensitized donors can be cytotoxic in vitro for target cells coated with antibody. The antibody can be directed either against antigens which are intrinsic to or experimentally attached to the target cell surface. Killing of target cells by this method has been termed antibody-dependent cell-mediated cytotoxicity (ADCC). In these investigations we compared the ability of Peyer's patch cells and spleen cells from nonsensitized mice to lyse lipoplysaccharide-coated and uncoated target cells by an ADCC mechanism. The results demonstrate that spleen cells, but not Peyer's patch cells, from identical mice can be cytotoxic for lipopolysaccharide-coated chicken erythrocytes (CRBC) in the presence of anti-lipopolysaccharide antibody and uncoated CRBC in the presence of anti-CRBC antibody. Thus, Peyer's patches in mice appear to be deficient in the effector cell type(s) required to kill antibody-coated erythrocyte target cells by an ADCC mechanism as well as the macrophage-like accessory adherent cell type(s) required for the induction of humoral antibody synthesis and cell-mediated cytotoxic allograft reactions in vitro.

A new method for strain identification of Rhizobium trifolii in nodules.

An indirect haemagglutination test has been developed for the detection of strains of Rhizobium trifolii in nodules of subterranean clover plants. Preserved sheep red blood cells, coated with isolated specific rhizobial lipopolysaccharide, were used as the indicator of agglutination; these cells were agglutinated by specific antilipopolysaccharide antibody. Detection of lipopolysaccharide antigen in a suspension of nodular tissue was carried out by reacting the suspension with antilipopolysaccharide antibody prior to the addition of coated red blood cells. The presence of antigen in the suspension was indicated by an inhibition of agglutination. The test was more sensitive than agglutination and immunodiffusion in the detection of rhizobial lipopolysaccharide antigens, and could be used for the rapid screening of large numbers of nodules.