Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study.

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of the 2nd IUIS/WHO collaborative study.

Following the 1st IUIS/WHO Collaborative Study of monoclonal anti-IgG subclass antibodies, a panel of WHO Specificity Reference Reagents (SRR) was established [Jefferis, R., et al. (1985) Immunol. Lett., 10, 223]. At the time, the hope was expressed that further reagents particularly for IgG2, and other allotypic specificities would become available which could be applied in a wide range of assay protocols. The 2nd study reports the evaluation of nineteen anti-subclass and seven anti-allotype monoclonal antibodies. The anti-IgG1 antibody HP6187 was equivalent in performance to the SRR. Others, that were not of the mouse IgG1 isotype, may be useful for particular applications. The anti-IgG2 antibody HP6200 could be a valuable addition to the WHO SRR; it is specific for an epitope in the Fab region but does not have the light chain bias of HP6014. Antibodies of putative allotype specificity exhibited the claimed specificity when used within protocols similar to those employed by the originating laboratory. It appears to be inherent in the nature of the epitopes (allotopes) recognized that it will take several years before reagents applicable to a wide range of techniques will become available.

Analysis of antibody profiles in children with whooping cough.

Development of antibody titres in non-vaccinated children with whooping cough of different duration (all confirmed by positive culture) were investigated by ELISA using lymphocytosis promoting factor (LPF, pertussis toxin), filamentous haemagglutinin (FHA), 69 kDa protein and lipopolysaccharide (LPS) as antigens. The antibody responses occur in three different patterns: Firstly, the LPF antibody response develops very quickly starting with the first day of clinical cough with all three classes, IgG, IgM and IgA appearing simultaneously; LPF antibody appears to be a dominant feature. Secondly, FHA and 69 kDa antibodies appear, starting as IgM with the shift to IgG and IgA later. The third pattern is represented by LPS antibody, the IgA appearing early, but with IgM predominant. Higher titres of IgG reacting with LPS were observed in vaccinated children. Transplacental transfer of antibody was also studied. All antibody titres determined in maternal blood and cord blood were proportional except for anti-LPS antibody which was retarded. Most IgG antibody was IgG1 subclass; surprisingly the 69 kDa antibody consisted of a mixture of approx. 90% IgG1 and 10% IgG4.

Evaluation of immunotherapy-induced changes in specific IgE, IgG and IgG subclasses in birch pollen allergic patients by means of immunoblotting. Correlation with clinical response.

Sera from 27 birch pollen-allergic patients who had undergone hyposensitization treatment for 22-41 months were studied by immunoblotting before and after therapy, whereby the levels of IgE, IgG and IgG1-4 antibodies directed against the major allergen Bet v I and minor allergens of birch pollen were monitored. The clinical benefit of immunotherapy (IT) was evaluated using a symptom specific questionnaire. In patients with good clinical response (responders, n = 18), as defined by improvement of symptoms, anti-Bet v I IgE antibodies were found to decrease in 10/18 patients (55.5%), whereas in 6/18 (33.3%) no change and in two cases (11.2%) an increase of specific IgE was observed. In the group of patients with unsatisfactory clinical outcome (non-responders, n = 9), 3/9 patients (33.3%) showed a decrease, 3/9 (33.3%) no change and 3/9 (33.3%) an increase in levels of IgE antibodies directed against Bet v I. In the case of minor allergens, 5/18 responders (27.7%) and 8/9 non-responders (88.8%) showed specific IgE before IT. In the responder group, no increase of specific IgE could be observed after IT. In non-responders, however, an increase of IgE directed against minor allergens was seen in 3/9 patients (33.3%). In all patients, regardless of therapeutical success, IT-induced elevated levels of specific IgG, IgG1 and in particular IgG4 directed against Bet v I were found. Regarding minor allergens, a heterogeneous pattern of IgG responses without significant correlation to clinical benefit was observed. Our results indicate that changes in IgG reactivity patterns against Bet v I and minor allergens, as shown by the immunoblot technique, did not correlate with good or bad clinical outcome.

Ontogeny of the humoral response to group A streptococcal carbohydrate: class and IgG subclass composition of antibodies in children.

We determined isotypes of natural antibodies to streptococcal group A carbohydrate (A-CHO) in sera from 101 children between 1 and 16 years of age, using a calibrated enzyme-linked immunosorbent assay system. Anti-A-CHO IgM could be detected in all but one sera. Median levels increased with age and were highest between 8 and 12 years. IgG antibodies were present at low concentrations up to the age of 4 years, and consisted predominantly of the IgG1 subclass. Between 4 and 8 years, concentrations of anti-A-CHO IgG markedly increased and median levels continued to increase through age 12-16. Anti-A-CHO IgG1 levels closely followed the pattern of IgG antibody concentrations. The number of IgG2 antibody positive sera was low in young children, as expected. In the 8-12 year age group and later, anti-A-CHO IgG2 was present in more than half of the samples, and in children between 12 and 16, medians of IgG2 and IgG1 antibodies were similar. Sera containing anti-A-CHO IgG3 were rare in children up to 4 years of age, but in the group of 4-8-year-old children, this subclass was detectable in 36% and later in up to 77% of the sera. Thus, the IgG response to A-CHO showed a clear maturation during childhood, involving the subclasses IgG1, IgG2 and IgG3. There were no significant differences in A-CHO levels between boys and girls.

[IgG subclass distribution in the serum of patients with homozygous alpha1-antitrypsin deficiency]. / IgG-Subklassenverteilung im Serum von Patienten mit homozygotem alpha 1-Antitrypsinmangel.

In 12 patients with homozygotic ZZ-phenotype alpha 1 antitrypsin deficiency, the serum levels of the IgG subclasses IgG1-4 were studied. In four of these patients, isolated or combined disorders or defects in the distribution of the IgG subclasses were detected; in cases with low IgG2 and absent IgG3, the clinical course of the disease was complicated by bronchiectasis.

Serum immunoglobulin levels and natural antibodies to Haemophilus influenzae in hemodialysis patients: evidence for IgG subclass imbalances.

Humoral immune parameters were studied in 13 patients with end-stage renal failure on maintenance hemodialysis. Serum IgA, IgM and IgG concentrations were comparable to control values from 14 healthy blood donors. IgG subclass analysis revealed significantly increased IgG1 levels in the patients when compared to controls (p less than 0.01). In 3 patients, IgG2 deficiency was found, in one case associated with low IgG3 level. Concentrations and subclass composition of naturally occurring antibodies to Haemophilus influenzae type b (Hib) polysaccharide (PS) were measured using an indirect ELISA. In patients IgM and IgG, including IgG1 and IgG2 antibodies to Hib, presented no difference from controls. Subclass analysis of Hib specific IgG antibodies revealed that IgG2 accounted for a substantial amount of the anti-Hib PS antibody response in controls as well as in patients. We conclude that patients on maintenance hemodialysis present imbalances of immunoglobulin levels. However, the antibody response to certain PS antigens could remain unaffected by renal failure.

Ig allotype-linked regulation of class and subclass composition of natural antibodies to group A streptococcal carbohydrate.

To determine the importance of genes located in or near the Ig constant regions in regulating the human antibody response, we correlated Ig allotypic markers with total Ig concentrations and natural antibody concentrations to the streptococcal group A carbohydrate (A-CHO) in 193 healthy adult blood donors. The major correlations between Ig allotypes and total Ig and specific antibody concentrations were observed with the Gm(f;n;b) haplotype. When compared with Gm(f;n;b) negative individuals, Gm(f;n;b) positives had significantly higher concentrations of total IgG2 (p less than 0.001) and IgG2 anti A-CHO (p less than 0.05), lower concentrations of total IgG1 (p less than 0.001) and IgG1 anti A-CHO (p less than 0.001), and lower concentrations of total IgM (p less than 0.001) and IgM anti A-CHO (p less than 0.05). We conclude that individuals with the Gm(f;n;b) haplotype respond preferentially with IgG2 rather than IgG1 subclass antibodies. This increased capacity to respond with IgG2 antibodies may be reflected in the magnitude of the total antibody response when the IgG2 subclass comprises a major proportion of the response, as occurs in the adult response to many polysaccharide Ag.

Immunoglobulin G subclass distribution in three human intravenous immunoglobulin preparations.

In immunodeficiency patients the lack of immunoglobulins (Ig) can be total or partial with a specific IgG subclass imbalance masked by normal values for total IgG. In the latter case therapy with intravenous IgG preparations (IVIG) is generally beneficial, provided the IVIG preparations used originate from large pools of normal blood donors and exhibit a normal IgG subclass distribution. We have analyzed the subclass distribution of three IVIG products: Sandoglobulin (SAGL), GamimuneN (GI), Gammagard (GG), 6-10 lots each, in four different laboratories. The competitive enzyme immunoassays and radial immunodiffusion methods used different monoclonal and polyclonal antibodies specific for IgG1, IgG2, IgG3, and IgG4, respectively. Despite minor interlaboratory differences, the results show that the slightly lower IgG1 content of SAGL versus GI and GG was quantitatively compensated by a higher proportion of IgG2, that no differences existed in IgG3 levels, but that one preparation (SAGL) contained 2-3% of IgG4 compared to 0.5-1.5% in GI and below 0.5% in GG. This difference was significant, the two latter preparations being at or below the lower limit of what are considered to be normal values found in human adults. Such differences may have important clinical consequences.

Jacalin: a new laboratory tool in immunochemistry and cellular immunology.

In recent years, a new lectin--jacalin--has raised the interest of immunologists because of its original properties with respect to human immunoglobulins and lymphocytes. Its structure and carbohydrate binding specificity are now well documented, and it can be purified easily from jackfruit seeds by ion exchange or affinity chromatography. The binding and precipitating specificities of jacalin with heavy chains of human immunoglobulins allow its use as a diagnostic (IgA subclass typing) and preparative tool (purification of IgA and IgD, removal of IgA from biologic samples and preparations). Other possible applications of jacalin's binding properties also can be envisaged. In addition, the lectin displays a mitogenic activity specific for human CD4 T-lymphocytes; consequently, the proliferative response induced by jacalin appears to represent a new and interesting assay for a functional study of CD4 cells, with obvious applications in primary and acquired, especially AIDS, immune deficiency states.

Electrophoretic properties of human IgG and its subclasses on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and immunoblots.

Unreduced human immunoglobulin G (IgG) which was not aggregated showed anomalous apparent molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It migrated mainly as three distinct bands with apparent molecular masses from 190 to 240 kDa on gels containing 8% polyacrylamide, when denatured at 37 degrees C. Generation of this banding pattern has two reasons: (a) the pattern is a superposition of bands originating from the four IgG subclasses that differ in molecular masses and structures; and (b) the complexity of the band pattern is further increased, because IgG myeloma proteins of the IgG1 and IgG2 subclass migrated as doublets, while IgG3 and IgG4 formed primarily one band with slightly different apparent molecular masses. These properties were independent of the type of light chain in all myeloma proteins studied. Generation of doublets suggests heterogeneities of monoclonal proteins. The two separable protein populations from IgG1 differ in their susceptibility to reduction. Reduction at 37 degrees C cleaved the larger into heavy and light chain, while it generated heavy chain dimer and light chain from the smaller species. Hence, it is possible that monoclonal IgG1 are comprised of at least two subpopulations of molecules with different S-S bonds. Doublet formation of IgG2 remains unexplained, since both species were equally sensitive to reduction. Knowledge on the anomalous properties of IgG on SDS-PAGE is a prerequisite to run immunoblots from unreduced cellular antigens without confounding cell-associated IgG with cellular antigens.

Constant isotype pattern of anti-dsDNA antibodies in patients with systemic lupus erythematosus.

The isotype profile, particularly emphasizing IgG subclass distribution, of dsDNA antibodies in patients with systemic lupus erythematosus was evaluated using an especially adapted ELISA technique. Anti-dsDNA antibodies were quantified with class-specific antisera and subclass-specific monoclonal antibodies. IgG subclass specificity was proven with 20 myeloma proteins differing in light chains and allotypes. The standardization with myeloma proteins proved to be useful and reliable. Results from more than 100 anti-dsDNA positive sera from SLE patients showed specific antibodies within the three subclasses (IgG1: 52-100%, IgG2: 0-39%, IgG3: 0-48%). IgG4 was not detected in significant amounts. No correlation to the subclass distribution of total IgG was found. Each patients' serum displayed an individual isotype pattern that remained constant in longitudinal studies, independent of anti-dsDNA titre fluctuations. These results suggest a stable population of autoreactive clones in the progress of the disease.

Differences among available immunoglobulin preparations for intravenous use.

Today almost all IgG preparations for intravenous use (IVIG) fulfill the basic requirements for a preparation given intravenously (sterility, pyrogenicity, antibody content but also anticomplementary activity, etc.). However, there are still marked differences among such preparations caused by the method of preparation: (1) Enzymatically treated IVIGs (by pepsin and plasmin) have a shorter biologic half-time and a disturbed IgG subclass composition; (2) in chemically treated IVIGs (beta-propiolactone, reduced or sulfonated IgGs) the IgG3 subclass is lacking and some of the Fc-related functions are altered; and (3) the IVIGs purified by anion exchangers are poor in the IgG4 subclass. The three main preparations sold in the United States (Gamimune N, Gammagard and Sandoglobulin) belong to the nonmodified preparations and, with the exception of the IgG subclass representation, show similar Fab- and Fc-related properties (antibody content, interaction with Fc receptors on monocytes, phagocytosis-promoting activity, etc.) In none of these preparations, an elevated level of undesired contaminants (prekallikrein activator, irregular anti-erythrocyte antibodies) are found.

Survival of antigen-specific antibody following administration of intravenous immunoglobulin in patients with primary immunodeficiency diseases.

To measure the survival of IgG, IgG subclasses and antigen-specific antibody in immune-deficient patients, we infused 4 patients with X-linked agammaglobulinemia (XLA) and 6 patients with common variable immune deficiency (CVID) with modified immunoglobulin at a dose of 400 mg/kg per month until steady state was reached. Following the 8th monthly infusion, serial samples were obtained and analyzed for serum concentration of IgG, IgG subclasses and for specific antibody activities against a battery of antigens. Half-lives for IgG and IgG subclasses were between 30 and 40 days except for IgG3 which appeared to consist of two populations of molecules, one showing a rapid decay, the other disappearing at a rate suggesting a half life of 22-24 days. Antigen-specific antibodies, including antibodies to HBsAg, cytomegalovirus, pneumococcal polysaccharides and streptococcal group A carbohydrate were similar to that for total IgG. These studies demonstrate that protective antibody titers to infective agents can be maintained for several weeks following high-dose intravenous immunoglobulin infusion.

Changes of house dust mite-specific IgE, IgG and IgG subclass antibodies during immunotherapy in patients with perennial rhinitis.

House dust mite-specific IgE, IgG and IgG subclass antibody responses were evaluated during immunotherapy in perennial allergic rhinitis patients. It was found that IgG4 antibodies steadily increased during treatment, while IgG and IgG1 antibodies reached a plateau 6 months after the initiation of immunotherapy. IgE antibodies also increased during treatment. It was also found that the good clinical outcome was associated with the rate of increase of IgG4 antibodies, but not with IgG, IgG1 or IgE antibodies.

IgG subclasses and antibodies to group B streptococci, pneumococci, and tetanus toxoid in preterm neonates after intravenous infusion of immunoglobulin to the mothers.

High doses of intravenous immunoglobulin were given to seven pregnant women between the 27th and 36th wk of gestation who were at risk for preterm delivery. Determinations of IgG subclasses and of antibodies against group B streptococcal serotypes, pneumococcal polysaccharides, and tetanus toxoid were done in maternal serum before and after intravenous IgG infusion and after delivery in cord serum. Substantial transplacental passage of the infused material could be observed in five cases where delivery occurred at the 34th wk or later. After the 36th wk of gestation, IgG subclass and antibody concentrations in cord serum were increased up to the levels in the maternal serum.

Susceptibility to infections in children with selective IgA- and IgA-IgG subclass deficiency.

This study included 36 children with IgA-deficiency, increased susceptibility to infections and/or other disorders. Recurrent, usually bacterial infections were noticed in 23 out of 26 patients (88%) with complete and in 7 out of 10 patients (70%) with partial IgA-deficiency. All patients with severe infections had complete IgA-deficiency. Complete IgA-deficiency was also present in the six children who had autoimmune disorders associated with recurrent infections. In 22 out of the 36 patients studied the serum could be analysed for concomitant IgG subclass deficiencies: one patient had marked decrease of IgG2. In a second patient IgG4 was not detectable. Two patients had combined IgG2-IgG4-deficiency. In a girl with severe acute and chronic infections and relapsing idiopathic thrombocytopenic purpura, IgA-IgG2-IgG4-deficiency was found to be the prodromal stage of common variable immunodeficiency with panhypogammaglobulinaemia.