Concentrations of serum immunoglobulins and antibodies to pneumococcal capsular polysaccharides in patients with recurrent or chronic sinusitis.

A study was carried out to search for underlying immunoglobulin deficiencies in 25 patients with recurrent or chronic sinusitis. The mean duration of the patient histories of recurrent or chronic sinusitis was 7.2 years. Concentrations of serum immunoglobulins and specific pneumococcal antibodies were measured in the patients and in 25 age- and sex-matched control individuals. The mean serum IgA concentration (1.6 g/L) was lower in the patients than in the control individuals (2.1 g/L, p = .024). On the other hand, the mean serum concentration of IgG antibodies to pneumococcal type 14 polysaccharide was higher in the patients (2.54 microg/mL) than in the control individuals (0.92 microg/mL, p = .008). However, elevated concentrations of IgG antibodies to pneumococcal type 14 polysaccharide were detected mainly in patients with the highest serum IgA concentrations. The results suggest that in a subpopulation of patients with a long-lasting history of sinusitis, a low serum IgA concentration may be associated with a susceptibility to sinusitis.

Allotype-associated differences in concentrations of human IgA2.

Concentrations of immunoglobulin (Ig)A2 were determined in 176 Finnish blood donor sera. Their IgA, IgM, IgE, IgG1, IgG2, IgG3 and IgG4 concentrations and Gm allotypes had been determined earlier. The mean concentration of IgA2 was higher in individuals carrying the Gm(ax) allele (0.15 g/l) than in those negative for Gm(x) (0.103 g/l). The difference was statistically significant (P = 0.006). As > or = 70% of IgA was usually IgA1, its concentration could be calculated fairly reliably by subtracting the IgA2 value from the IgA value. The mean IgA1 concentration (2.03 g/l) seemed to be independent of the Gm allotypes.

Proportion of protein A bindable molecules in human IgM and IgA antibodies to seven antigens.

Human IgM or IgA antibodies to seven antigens (tetanus and diphtheria toxoids, and five bacterial polysaccharides) were studied by determining what proportion of these antibodies were bound by staphylococcal protein A. This alternative binding is a marker of VH genes of family 3. Each response was studied in an average of nine individuals. The binding proportion of antibodies to the two toxoids resembled that of total serum immunoglobulins; 13-14% of IgA and 40% of IgM antibodies were bound by protein A. All anti-polysaccharide antibodies had higher proportions of protein A bindable molecules than serum IgM or IgA indicating a bias for VH genes. This excess was high in antibodies to Haemophilus influenzae type b (Hib) and pneumococcal type 14 polysaccharides (> two-fold). It was moderate but statistically significant in antibodies to pneumococcal types 18C and 3 capsular polysaccharides and to C polysaccharide. All vaccinated Finns exhibited the VH3-preference in antibodies to Hib and type 14 polysaccharide.

Low concentrations of Gm allotypic subsets G3 mg and G1 mf in homozygotes and heterozygotes.

Serum concentrations of IgG3, IgG1, and of the Gm allotypic subsets of these two isotypes were measured in adult homozygotes and heterozygotes. Alleles G3 mb and G3 mst of the IgG3 locus, and alleles G1 ma and G1 max of the IgG1 locus were found to associate with a high concentration of the allotypic product. Alleles G3 mg (IgG3) and G1 mf (IgG1) were associated with a low concentration of the product. This was true regardless of the haplotype; for example, allele G3 mb was associated with a high concentration of the product in all haplotypes f;n+;b f;n-;b and fa;n+;b. One dose of allele G3 mg was associated with a characteristic mean concentration of the product (g-type IgG3). This rule was valid regardless of the other allele of the subject, thus, heterozygotes and G3 mg/g homozygotes had mean concentrations of 0.10 and 0.20 g/liter, respectively, of g-type IgG3. Products of the IgG1 alleles were also simply additive: one dose of allele G1 ma(x) or G1 mf was associated with mean concentrations of 3.63 and 2.84 g/liter, respectively, and two doses with twice these amounts. Only allele G3 mb did not completely follow this rule. We also studied the serum concentrations and the allotype distribution of 41 IgG1 and 31 IgG3 myeloma proteins. The results suggested that the allotype-associated differences in serum concentrations are caused by different numbers of B cells producing allotypic subsets of IgG1 or IgG3, not by different rates of synthesis per B cell.

Half-life of the maternal IgG1 allotype in infants.

The residence time of maternal IgG1 in the circulation of infants was measured by monitoring f-allotypic IgG1 or f-positive tetanus toxoid antibody in genetically G1mf-negative infants. G1ma-positive maternal tetanus toxoid antibody was similarly monitored in genetically a-negative infants. Blood samples were taken from infants at the age of 1-3 days, ca. 4 months, and ca. 6 months. An exponential decay at the same rate took place from age 1-3 days to 4 months and for the 2 subsequent months. The average concentration of the maternal IgG1 had dropped to ca. 10% of the 1- to 3-day value in 4 months and to ca. 3% in 6 months. The drop was due mainly to clearance but partly also to the weight increase of the child (doubling in 6 months). By correcting for the weight increase, we calculated that ca. 17 and 7% of the original maternal IgG1 was still present at ages 4 and 6 months, respectively. The average half-life of the maternal IgG1 was thus 48.4 days. The concentration of endogenous IgG1 in the cord blood was determined by studying a separate series of mother-newborn pairs. Assuming that cross-reactions of antiallotype reagents had no effect, the highest measured concentration of f-positive IgG1 in infants of f-negative mothers was 10 mg/L, half a percent of adult heterozygote values. Crossreaction may have played a role, however, and the value must be considered the upper limit of the true concentration.

Gm allotypes influence the production of IgG3 but the effect is age-dependent.

Serum concentrations of IgG3 were found to be higher in Gm-f-positive (= b-positive) than in f-negative individuals except in young children. Young children aged 3-4 months had a mean concentration of 0.24 g/l of IgG3 regardless of allotype. The concentration gradually rose with age in f-positive individuals to a geometric mean of 0.56 g/l in adults but it remained essentially unchanged in f-negative people. A corresponding allotype effect was seen in influenza-specific antibody responses. While the total IgG response (mainly IgG1) was equally strong in f-positive and in f-negative patients, f-positive (= b-positive) patients produced more IgG3 antibodies than f-negative patients. The difference between geometric mean values of opposite homozygotes (f/f versus f-negative) was 2.3-fold (p = 0.0113). This finding indicates that the b-positive gamma-3 allele is more productive than the g-positive allele.

Susceptibility to invasive Haemophilus influenzae type b disease and the immunoglobulin G2m(n) allotype.

There has been considerable controversy about the role of the immunoglobulin G2m(n) allotype and risk of invasive Haemophilus influenzae type b (Hib) disease. This allotype was studied in a large cohort of Finnish children (178) with invasive Hib disease. The G2m(n) allotype distribution was similar to that in the normal white Finnish population. No increased risk of Hib disease could be associated with the n-/n- genotype [i.e., lack of G2m(n) allotype]. Thus, the G2m(n) allotype does not seem to be a major determinant of susceptibility to Hib infection among white populations in industrialized countries.

Allotype-associated differences in concentrations of human IgG subclasses.

The concentrations of seven immunoglobulin isotypes (IgA, IgE, IgM, IgG1, IgG2, IgG3, and IgG4) were measured in the sera of 207 Finnish blood donors, and they were allotyped with anti-Gm antibodies: anti-f, anti-a, anti-x, and anti-n. The above population could be divided into 12 phenotypes, and significant differences in isotype concentrations between different phenotypes were observed. They are best explained by postulating that the following alleles of different loci are associated with a high concentration of the product of the locus: a(x)-IgG1, n-IgG2, b-IgG3, and perhaps 4b-IgG4. The following concentration differences between the low and the high homozygotes were found: IgG1, 1.2-fold; IgG2, 1.5-fold; and IgG3, 2.6-fold. No significant allotype-associated differences in the concentrations of IgA, IgM, or IgE could be detected.

Effect of Gm allotypes on IgG2 antibody responses and IgG2 concentrations in children and adults.

Earlier studies have suggested that in adults the n-positive allele of the human IgG2 gene is more productive than the n-negative allele. This superiority was seen to be manifested in IgG2 antibody responses to polysaccharides, in the higher serum concentration of total IgG2 in the n/n than in -/- individuals, and in the higher concentration of n-positive than n-negative IgG2 in heterozygotes. The present study shows that in 1- or 2-year-old children, the concentration of IgG2 was independent of allotype G2m(n), and both alleles of a heterozygote contributed an average of one-half of the total IgG2. On the other hand, the superiority of the n-positive allele was also seen in young children in IgG2 antibody responses induced by the Haemophilus influenzae type b polysaccharide (Hib). The effect of allotype n on antibody responses was evident only when the immunogen was the Hib polysaccharide. When the immunogen was a conjugate of Hib and diphtheria toxoid, the IgG2 antibody responses of n-positive and n-negative vaccinated individuals were almost equal, both in adults and in children.

One allele [G2m(n)] of the human IgG2 locus is more productive than the other.

Earlier investigators have shown that a high serum concentration of IgG2 and a high IgG2 antibody response to several polysaccharide antigens is associated with the allotype G2m(n) of the IgG2 subclass. We now show that the total IgG2 concentrations are significantly higher in G2m(n) than in G2m(-n) homozygotes (difference of means was 1.26-fold, p = 0.002). Furthermore, in G2m(n) heterozygotes, the G2m(n) allele contributed more than the G2m(-n) allele to specific IgG2 antibody responses and to the total IgG2.

IgG subclasses of pneumococcal antibodies--effect of allotype G2m(n).

Antibody responses to three pneumococcal polysaccharides (types 3, 14, and 18C) were analysed after vaccination with a 23-valent polysaccharide vaccine. Antibodies to all three polysaccharides could be detected before immunization. Clear cut IgG, IgA, and/or IgM antibody responses to the polysaccharides were seen in three-quarters of the vaccinees. IgG2 was the predominant and IgG1 the second most abundant subclass of anti-pneumococcal IgG antibodies both before and after the vaccination. The relative proportions of IgG2 and IgG1 antibodies exhibited a continuous variation from 1:0 to approximately 0:2. After vaccination, G2m(n)-positive homozygotes had about four times more IgG2 antibodies (anti-14 and anti-18C) than G2m(n)-negative vaccinees. Heterozygotes occupied an intermediate position. The same pattern was seen less clearly in type 3 antibodies after vaccination, and in all three antibodies before vaccination. The G2m(n) allotypes had no detectable effect on the levels of IgG1, IgG4, or IgM antibodies, and possibly a weak effect on IgG3 and IgA antibodies (G2m(n)-positive homozygotes responded strongly).

Human antibody responses to two conjugate vaccines of Haemophilus influenzae type B saccharides and diphtheria toxin.

Antigenicity of two Haemophilus influenzae type b (Hib) conjugate vaccines was studied by immunizing adults and 2-year-old children. Both vaccines induced strong anti-Hib responses and strong antibody responses to diphtheria toxin (DT), the protein part of the conjugate. The adults' responses were stronger than the children's. A conjugate of Hib oligosaccharide and mutant diphtheria toxin (HbOC) emerged as slightly superior to a conjugate of Hib polysaccharide and diphtheria toxoid (PRP-D). HbOC induced somewhat higher total anti-Hib responses and significantly higher IgG1 anti-Hib responses than PRP-D. IgG1 and IgG2 were the main IgG subclasses of the anti-Hib antibodies, whereas IgG1 and IgG4 were the main subclasses of the anti-DT antibodies. Within this main rule, the ratio IgG1/IgG2 of anti-Hib antibodies varied between individuals. The average ratio was higher than five in children but approximately one in adults. It was lower in adult recipients of the polysaccharide conjugate (0.69) than in adult recipients of the oligosaccharide conjugate (1.55). A large interindividual variation was observed in concentrations of IgG2 of Hib specificity, perhaps reflecting a small number of IgG2-committed B-cell clones participating in the response.

Anti-DNA antibodies: the choice of assays for routine diagnostic work.

Sixty-six sera from 23 patients with systemic lupus erythematosus (SLE), 26 sera from patients with rheumatoid arthritis (RA), and 22 sera from normal healthy subjects were tested for the presence of antibodies against native (ds) DNA by the Crithidia luciliae immunofluorescence test and by the Farr assay, and for the presence of antibodies against denaturated (ss) DNA by the enzyme-linked immunosorbent assay (ELISA). Anti-dsDNA antibodies were detected in 57% of the SLE patients by the Crithidia test and in 65% by the Farr assay. Two of the RA sera were positive in the Crithidia test, whereas all were Farr negative. Anti-ssDNA antibodies of IgG class could be detected in 74% of the SLE patients and in none of the RA sera, while anti-ssDNA antibodies of IgM class were found in 26% of the SLE patients and in one RA serum. There was a good correlation between the results of the Farr assay and the IgG-anti-ssDNA ELISA but no agreement was found between the results of the Farr assay and the Crithidia test. We also measured the amount of C-reactive protein (CRP) in the sera but no correlation was seen between the levels of CRP and anti-DNA antibodies. We conclude that the demonstration of anti-ssDNA antibodies of IgG class is a good screening method in the diagnosis of SLE, and that antibodies against native DNA should be determined, preferably both by the Crithidia test and the Farr assay to confirm the diagnosis and in the follow-up of the patients.

IgG subclass distributions in anti-hapten and anti-polysaccharide antibodies induced by haptenated polysaccharides.

Mice were immunized with hapten [NIP, (4-hydroxy-5-iodo-3-nitrophenyl)acetyl or TNP (2,4,6-trinitrophenyl)] conjugates of Ficoll or pneumococcal polysaccharide type 14 (S14), and they were bled on days 10 or 14. Anti-hapten and anti-polysaccharide antibodies were determined from the sera or from fractions (IgM + IgA). IgG1, IgG2a, IgG3 or IgG2b separated by a gradual acid elution from protein A. Approximately one-half of both anti-hapten and anti-polysaccharide antibodies was found in the IgM + IgA fraction. The subclass distribution of the IgG antibodies was dependent on the antigenic determinants. Polysaccharide antibodies were mostly in the IgG3 fraction (36-62%) and in the IgG1 fraction (18-36%). Hapten IgG antibodies were mostly in the IgG1 fraction (38-74%): each of the other three subclasses contributed the average of 13%. These results provide the first evidence that antibodies to different determinants of one antigen have grossly different isotype distributions.

Mouse IgG antibodies have subclass associated affinity differences.

Subclasses of IgG were separated from pools of mouse sera by letting immunoglobulins absorb on protein A-Sepharose and by eluting with buffers of decreasing pH. Most donor mice were immunized with a conjugate of a hapten (NIP) and chicken gamma globulin 20 days previously. The results indicate that concentrations of IgG varied from 5.1 to 8.6 mg/ml in the pools of immune sera and was 3.0 mg/ml in one normal serum tested. One half of this was IgG1, ca. 20% of IgG2a and IgG2b each, and 10% IgG3 in the pools of BALB/c sera. IgG2a and IgG3 could not be separated from C57BL sera (due to allotype b), but their combined share of IgG appears to be higher than in BALB/c. Immune sera contained 0.5-1.6 mg/ml of anti-NIP antibodies. Of this 90-98% was IgG1 and the remainder was split between the other subclasses. Up to one half of the protein in the IgG1 fraction was anti-NIP antibody. This surprising finding was confirmed by demonstrating that nearly 50% of the u.v.-light absorption was specifically removed by a NIP-immunosorbent. Subclass-associated affinity-differences were observed. IgG1 anti-NIP had a greater average affinity than IgG2a anti-NIP antibodies. The difference was ca. 1.5-fold when the equilibrium dialysis was focusing on the high-affinity bracket of the total population (concentration of free hapten 16-200 nM). At higher hapten concentrations the trend was the same but the data are fewer. Antibodies in subclasses IgG2b and IgG3 appear to share the lower affinity of IgG2a.

The range and fine specificity of the anti-hapten immune response: phylogenetic studies.

Heterodontus francisci (horned shark) and Pseudopleuronectes americanus (winter flounder) were immunized with furyl-oxazolone (furyl-Ox) and phenyl-oxazolone (phenyl-Ox) coupled either to bacteria or protein carriers. The antibodies produced were measured by inactivation of furyl- or phenyl-Ox conjugated bacteriophage, and their affinity and fine specificity were estimated by inhibition of phage inactivation with a series of structurally related hapten analogues. In both species, post-immunization peak titres were 100 to 2000 times higher than preimmunization titres. A number of unique features distinguished Heterodontus antibodies from Pseudopleuronectes or mammalian antibodies. Heterondontus antibodies exhibited a lower affinity for the immunizing hapten (furyl-Ox or phenyl-Ox) and a reduced ability to distinguish the homologous immunogenic hapten from its structural analogues. In addition, Heterodontus antibodies exhibited a lower level of inter-individual variation in affinity and fine specificity than did Pseudopleuronectes or mammalian IgM antibodies; this was especially prominent in anti-furyl-Ox responses. Typically the affinity and fine specificity of Heterodontus antibodies did not change over the 146-day period of immunization and were not influenced by the nature of the carrier. The implications of these findings in terms of the phylogenetic origins of antibody diversity are discussed.

The four subclasses of IgG can be isolated from mouse serum by using Protein A-Sepharose.

We confirmed the findings of Ey and colleagues that mouse IgG is absorbed by protein A-Sepharose at pH 8.0. Also confirmed was their finding that IgG1 mainly elutes from such a column by means of a buffer with pH 6.0 and that the corresponding pH values for IgG2a and IgG2b were 4.5 and 3.5. We made the new finding that the bulk of IgG2a bearing allotypes a or j eluted already at pH 5, in contrast to IgG2a bearing allotype b. Another new finding was that IgG3 was mainly eluted at pH 4.5 regardless of the allotype. All four subclasses of IgG could thus be physically separated if the allotype was a or j (the only known exception is allotype b). Separation of IgG2a and IgG3 was achieved even when the allotype was b by using a pH gradient for elution. IgG2a came out at a slightly higher pH than IgG3. Mouse IgG antibodies against group A streptococcal polysaccharide belonged mostly to IgG3 and, to a lesser extent, to IgG2a and IgG2b.

An immunological characterization of the basic proteins of rodent sciatic nerve myelin.

Radioimmunoassays (RIA) for the myelin basic protein (MBP) and P2 protein together with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to establish the identities of and relationships between the basic proteins (BP) of rodent peripheral nervous system (PNS) myelin. The PNS myelin proteins studied, in order of increasing mobility of SDS-PAGE, are P1, PR (R = rodent) and PB (B = breakdown). The majority of the acid extractable proteins of rodent PNS myelin are MBP related as shown by MBP-RIA. When tested individually, rodents P1, PR and PB were each found to cross-react in the RIA for MBP but not that for P2. The acid extracts of rodent PNS myelin were found to contain P2, although in minute quantities. P2 accounts for approximately 0.05-1.0% of the acid extractable protein in rodent PNS myelin.