Measurement of anti-Haemophilus influenzae type b capsular polysaccharide antibodies by ELISA

Measurement of antibodies to the capsular polysaccharide polyribosylribitolphosphate (PRP) of Haemophilus influenzae type b by ELISA is made difficult by the poor binding of this antigen to the solid phase. Six coating conditions were compared using immune and non-immune human sera. Direct coating with PRP was inefficient. Precoating with protamine or poly-L-lysine (PLL) yielded irreproducible results and high background levels. Assays with PRP conjugated with PLL as coat were not sensitive enough. In addition, anti-PRP antibodies, especially those belonging to the IgM class, crossreacted with PLL. Coating with avidin or streptavidin followed by incubation with biotin-coupled PRP was not satisfactory either, due to binding of certain sera in the absence of PRP. Coating with PRP coupled to tyramine resulted in low backgrounds and acceptable specific binding levels. However, the finding that the binding of a few sera was only partially inhibited by soluble PRP led us to include an inhibition step in every experiment. Only optical densities inhibited by the antigen were taken into account. In view of the lack of parallelism of dilution curves from different sera, no attempt was made to express the results in weight units. They were expressed in arbitrary units calculated by comparison with internal standards. Under such conditions, the assay permitted a reproducible (interassay coefficients of variation around 10%) determination of PRP-Ab belonging to the various immunoglobulin classes and IgG subclasses and showed a good correlation with results obtained using the Farr assay.     

Some biological activities of rabbit anti-interferon serum.

After prolonged immunization of rabbits with a semipurified mouse interferon preparation in Freund's incomplete or Al-Span-Oil adjuvant, a specific interferon-neutralizing immunoglobulin was obtained from antiserum with a capacity of neutralizing about 49000 mouse interferon units per ml. The specific activity of the antiserum and immunoglobulin was confirmed in tests in which the interaction of antibodies with the cell surface was ruled out. The antiserum (and the immunoglobulin) neutralized both the antiviral and the cell-growth inhibitory activities of interferon. The "slow" and the "fast" fractions of purified interferon preparations were equally sensitive to the neutralizing effect of antibodies. On the other hand, the reaction of heat-inactivated interferon with the antiserum did not diminish the neutralizing activity of the latter, suggesting a destruction of interferon antigenic sites.     

Chemical composition and biological activities of a phenol-water extract from Haemophilus influenzae type a.

Ribonucleic acid was removed from a phenol-water extract of Haemophilus influenzae type a by streptomycin sulfate. This preparation was called purified preparation or PP. It contained neutral sugars (glucose, galactose, mannose, pentose), glucosamine, amino acids, and fatty acids. Heptose and 2-keto-3-deoxyoctonic acid were not present. The biological properties and immunogenicity were compared with the activities of lipopolysaccharide of Escherichia coli or Salmonella typhimurium. Higher doses were necessary to obtain lethality in mice and Sanarelli and Shwartzman reactions with our preparations than were necessary with lipopolysaccharide. The Limulus test and pyrogen assay in rabbits gave the same results with purified preparation and lipopolysaccharide, but pyrogenicity of purified preparation was not destroyed by NaOH treatment. Purified preparation was not as immunogenic at low doeses for rabbits as lipopolysaccharide. The results were different from those obtained with lipopolysaccharide but similar to those known from peptidoglycan studies. The contamination of purified preparation with peptidoglycan was negligible and cannot explain the biological activities of purified preparation. We suggest that the phenol-water extract from H. influenzae is not a classical endotoxin, but rather an endotoxin-like substance.     

Development of an immunoglobulin A-specific anti-Haemophilus influenzae antibody assay for detection of antibody in human mucosal secretions.

A micro-enzyme-linked immunosorbent assay for quantitation of immunoglobulin A-specific anti-Haemophilus influenzae antibody is described and characterized. It had a sensitivity of 27 ng/ml, which is appropriate to detect antibody levels in normal saliva. Specificity for H. influenzae was achieved with the H1H2 group of antigens. Absorption studies for a range of bacteria showed little cross-reactivity, with the exception of Pseudomonas aeruginosa. Absorption studies involving various antigen preparations obtained from H. influenzae indicated that the H1H2 antigen group included significant amounts of surface antigen. An analysis of saliva from normal subjects and patients with chronic obstructive lung disease showed significant differences in levels of antibody, highlighting the potential value of the assay.     

Complement-mediated serum activities against genetically defined capsular transformants of Haemophilus influenzae

Although there are six different capsular serotypes of Haemophilus influenzae (a-f), only type b strains commonly cause systemic infections in man. The present study was performed to determine whether the propensity of the type b organism to cause invasive infections is due to a unique ability to evade complement-mediated host defenses. The ability of genetically defined capsular transformants (a-f) of an unencapsulated H. influenzae to resist the bactericidal and opsonic activities of serum was examined. The unencapsulated organism and the type f transformants were relatively susceptible to serum bactericidal activity in both adult and infant serum pools, the type a and e transformants were relatively resistant, and the types b, c and d transformants were intermediate. With respect to serum opsonic activity in both adult and infant serum pools, the unencapsulated organism and the type f transformant were relatively susceptible, the type a, b and e transformants were relatively resistant and the type c and d transformants were intermediate. Thus, although the type b capsule endows the organism with the ability to resist the bactericidal and opsonic effects of complement, this property is not unique to type b.     

Serological grouping of meningococci and encapsulated Haemophilus influenzae strains by latex agglutination.

The latex agglutination method, utilizing antibody-coated latex particles, was adapted for serogrouping of Neisseria meningitidis and serotyping of encapsulated Haemophilus influenzae strains from agar plates. It was found to give more clear-cut results than conventional slide agglutination. A 100% agreement with the antiserum agar method was found for all strains isolated from blood or cerebrospinal fluid. Many meningococcal strains from nasopharyngeal carriers are autoagglutinable, but some of these gave a positive reaction with the group B latex reagent, although they were negative by the antiserum agar method. The latex agglutination method has several advantages over others: the lack of autoagglutination, easy performance, easy interpretation, and very low consumption of antisera.     

Serological, electrophoretic, and biological properties of Cryptococcus neoformans antigens.

We compared a cryptococcal culture filtrate antigen referred to as CneF with chemically defined cryptococcal antigen fractions isolated by Cherniak and co-workers by using double immunodiffusion gels, polyacrylamide gel electrophoresis, immunoblots, and footpad reactivity of immunized mice. The three previously described components of cryptococcal culture filtrates are a high-molecular-weight glucuronoxylo-mannan (GXM), which is the major constituent, a galactoxylomannan (GaIXM), and a mannoprotein (MP). In this study we demonstrated that CneF contained components which were serologically and electrophoretically similar to the three previously described cryptococcal culture filtrate fractions. The MP fraction elicited significantly stronger delayed-type hypersensitivity responses than did the GXM or GaIXM fraction when used in mice immunized either with the CneF in complete Freund adjuvant or whole heat-killed Cryptococcus neoformans yeast cells. These findings were confirmed when the footpads of immunized mice were challenged with GaIXM and MP preparations from a culture filtrate of a C. neoformans acapsular mutant that does not produce GXM. Thus, we concluded that the MP was the primary component recognized by the anticryptococcal cell-mediated immune response in mice.     

Quantitation and biological properties of released and cell-bound lipooligosaccharides from nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media in children. NTHi releases lipooligosaccharide (LOS) as outer membrane fragments during its growth. The release of LOS may play an important role in the pathogenicity of otitis media caused by this organism. The amounts of LOS in bacterial cells and growth media for five NTHi strains were determined by quantitative silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These strains were estimated to have 1.6 x 10(6) to 4.8 x 10(6) LOS molecules per bacterium. During a 3-day growth period, these NTHi strains released variable but significant amounts of LOS into the growth medium. Cells started to release detectable amounts of LOS into the medium at 2 to 5 h and continued to do so for up to 48 or 72 h. The concentrations of LOS in the culture supernatants released by these five strains were 10 to 55 micrograms/ml at 24 h and 40 to 100 micrograms/ml at 72 h, which was 34 to 189% of the cell-bound LOS concentration. The biological properties of released and cell-bound LOSs from two representative strains were compared. Released LOS showed an approximately 10-fold increase in inducing human monocytes to produce tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6, a 13- to 28-fold increase in mouse lethal toxicity, and a 16- to 37-fold increase in the clotting of Limulus amebocyte lysate. These results suggested that released LOS or its inflammatory mediators play a more important role than the LOS in bacteria in the pathogenicity of otitis media caused by this organism.     

Biologic activities of antibody to a peptidoglycan-associated lipoprotein of Haemophilus influenzae against multiple clinical isolates of H. influenzae type b.

A peptidoglycan-associated lipoprotein of about 15 kilodaltons was purified from the outer membranes of Haemophilus influenzae by using nondenaturing detergents. To assess its vaccine potential, rabbit antiserum to the purified protein was obtained. The antiserum was specific for the peptidoglycan-associated lipoprotein in whole cell lysates of H. influenzae and was bactericidal for H. influenzae types a, b, d, e, and f and for 181 of 182 H. influenzae type b clinical strains isolated in widely dispersed geographic areas. The antibody protected infant rats from challenge with each of five clinical H. influenzae type b isolates and was additive to and did not interfere with bactericidal and protective activities of antibody against the type b capsule. These data indicate that the purified peptidoglycan-associated lipoprotein is a potentially valuable vaccine candidate for H. influenzae type b disease and may enhance the effectiveness of preexisting anticapsular antibody.     

Serological epidemiology and the function of serum banks

Summary Techniques and principles which are used in serological epidemiologic investigations are described, and it has been pointed out that the World Health Organization has recently established Serum Reference Banks to expand the potentialities in this field.Serologic and hematologic surveys of diverse populations living in different environments have been studied recently and have yielded information regarding the fields of the epidemiology of infectious and non-infectious diseases, anthropology, and genetics.Examples of the application of serologic epidemiology to surveys of populations in several parts of the world are given.Dedicated to the Honor of the 60th birthday of ProfessorSven Gard.The work of this Serum Bank has been supported jointly by grants from the World Health Organization and by several grants from U.S. Federal agencies and private Foundations.     

Opsonizing and bactericidal effects of normal human serum on nontypable Haemophilus influenzae.

The observation that nontypable (NT) Haemophilus influenzae causes serious infection in adults has stimulated interest in mechanisms that may protect the human host against NT H. influenzae infection. Incubating NT H. influenzae with normal human serum (NHS) caused dose- and time-dependent killing that varied with the individual NHS and NT H. influenzae. Adsorption of NHS with NT H. influenzae removed bactericidal activity against the adsorbing isolate but not necessarily that against others, suggesting antigenic diversity and supporting recent studies that show different outer membrane protein profiles among NT H. influenzae. Heating NHS to 56 degrees C for 30 min abolished bactericidal activity; this activity was not restored by complement-rich guinea pig serum or NT H. influenzae-adsorbed NHS. This is analogous to the "third factor" needed for intraleukocytic killing of pneumococci. Optimal opsonization of NT H. influenzae for phagocytosis by human polymorphonuclear leukocytes required antibody and complement, but other serum factors also played a role. Bactericidal activity generally, but not uniformly, correlated with opsonizing activity of individual NHS. Humoral factors may be important in host defenses against NT H. influenzae infection; their emergence during convalescence warrants further study.     

Serological association of lupus autoantibodies to a limited functional domain of 28S ribosomal RNA and to the ribosomal proteins bound to the domain.

Site-specific anti-RNA antibodies were sought in 120 sera of patients with autoimmune diseases by ribonuclease-protection assay using six fragments covering 28S ribosomal RNA (rRNA) as antigens. Fifteen of 90 sera from patients with systemic lupus erythematosus (SLE), but none of 30 sera of the other autoimmune diseases, provided a 60 nucleotide fragment within a region termed the 'GTPase domain' of 28S rRNA. These sera had potency to precipitate 0.42-69.3 nmol of the RNA domain per ml serum, which was higher than 15 control sera of healthy donors. No other specific antigenic site was detected in 28S rRNA under conditions used. All of the 15 sera having this anti-RNA antibody showed reactivity to ribosomal P proteins (anti-P), and two of them contained an additional antibody to ribosomal protein L12. These results suggested a strong association of the production of these three antibodies. Since P and L12 proteins form a stable complex with the GTPase domain, this serological association may result from an immune response to epitopes clustered on a single RNA-protein complex domain in ribosomes.     

Multiple biological activities of homogeneous human alpha interferon.

To identify the active molecule in human alpha interferon preparations, we performed several studies, using both partially purified interferon and homogeneous interferon. Our results indicated that there is little difference between the partially purified and the homogeneous interferon preparations in terms of antiviral activity, inhibition of deoxyribonucleic acid synthesis in human neoplastic cells, and enhancement of human natural killer cell activity.     

Naturally occurring antibodies in human sera that react with Haemophilus influenzae type b ribosomal vaccine.

Sera from populations of normal adults and children as well as sera from children with systemic Haemophilus influenzae type b disease were tested for antibodies reacting with ribosomes from H. influenzae type b. Adults generally had high titers of antibody, with 90% having titers greater than 1:64. The distribution of titers approximated a normal curve. Among normal children, there was more variability between individual titers, with the median titers ranging between 1:64 and 1:128. In contrast, acute-phase sera from children with systemic H. influenzae type b disease all had titers of 1:16 or less. Two convalescent-phase sera had high titers. Absorption experiments ruled out cross-reaction between ribosomes and type b capsular material. Ribosomes from two unrelated type b strains were completely cross-reactive, whereas absorption with ribosomes from a type c strain led to significantly decreased titers in three of four sera. Absorption of sera with ribosomes from Pseudomonas aeruginosa and Streptococcus pneumoniae also decreased titers, indicating that these antibodies may have been induced by ribosomes of other bacteria.     

Serological and biological characteristics of "Streptococcus milleri" isolates from systemic purulent infections.

Ninety-one Streptococcus milleri strains isolated from various systemic purulent lesions of 68 patients were examined by physiological and serological tests. Most strains formed a smooth colony (66 strains), did not form spontaneous aggregation of cells in BHI broth culture (79), were non-beta-haemolytic (alpha-35 or non-41), and belonged to biotype Ia (49) or Ib (34) and to API taxa S. milleri I (41) or II (38). Almost all of the beta-haemolytic strains as well as two-fifths of the non-beta-haemolytic belonged to API taxon I; strains of API taxa II and III were non-beta-haemolytic and non-haemolytic, respectively. Two-fifths (38) of the isolates belonged to one of eight serotypes, a-g and k, and more than half (47) to Lancefield groups A, C, F or G, the most frequent being type b (19) and group F (33). Fifteen strains carried simultaneously type a/group A, b/C, c/C, e/G, f/F or k/G antigens. Nineteen were neither typable nor groupable. All the 38 serotypable isolates were non-beta-haemolytic and not members of API taxon III, and were serologically and physiologically similar to oral S. milleri. The isolates from various infected sites--sputum, thorax, abdomen, urogenitalia, skin, eye and dental--exhibited distinct combinations of biological and serological properties. These results suggest that serotyping, haemolytic properties and API taxon, and their combinations, would be useful methods to trace oral S. milleri in systemic infections.