Charles B. Reimer

Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative study.

Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG sub-classes (59) or Gm allotypes (4) have been evaluated for reactivity and specificity in eight laboratories employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities McAb have been produced that can be satisfactorily applied in most methodologies employed and have potential as reference reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated demonstrating apparent assay restriction and whilst performing well in some assays proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable variability rather than capricious behaviour by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the physical and chemical procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.

Two-site immunoenzymometric assays for serum IgG subclass infant/maternal ratios at full-term.

Improvements were made in the range, precision, convenience, automation, and reliability of our previously published two-site immunoenzymometric assays using mouse monoclonal antibodies specific for human IgG and its subclasses. The serum concentration ratios for these immunoglobulin isotypes were measured in neonatal/maternal paired sera from 119 normal full-term deliveries. These ratios are significantly different than 1.0 (P = 0.001) for total IgG, IgG1, and IgG2 (show non-equality of paired neonatal/maternal sera concentrations) but are not significantly different than 1.0 for IgG3 and IgG4. On average, IgG1 is elevated 61%, and IgG2 is depressed 11% in the full-term neonate with respect to its own mother. In some pregnancies, active transport of IgG1 may be selectively enhanced by low material IgG1 concentration and selectively inhibited by high levels.

Human antibody responses to bacteriophage φX 174: Sequential induction of IgM and IgG subclass antibody☆

We studied the appearance of antigen-specific immunoglobulin classes and IgG subclasses in normal adult human subjects in response to primary, secondary, and tertiary immunization with the T-cell-dependent neo-antigen bacteriophage phi X 174. To complete the study we developed a sensitive, specific, and reproducible ELISA assay which was closely comparable to the widely used neutralization assay for total antibody (r = +0.97) and for IgG antibody (r = +0.93), and reasonably comparable for IgM antibody (r = +0.76). We confirmed that the initial response to primary immunization was predominantly, but not exclusively, IgM antibody. The secondary and tertiary responses demonstrated memory, amplification, and switch from IgM to IgG antibody. There was an orderly appearance of phage-specific IgG subclasses. IgG3 and IgG1 antibodies appeared 2 to 6 weeks after primary immunization. In all subjects there was a marked increase in IgG1 and IgG3 antibody after secondary immunization, and IgG2 antibody followed closely; IgG4 antibody appeared in some subjects. IgM antibody persisted in significant amounts (approx 50%) throughout the secondary response period. Following tertiary immunization, IgG1, IgG2, and IgG3 antibody consistently increased, and IgG4 antibody appeared in all subjects; IgG1 antibody predominated. Low levels of IgM antibody (approx 1% of total) persisted during the tertiary response. The persisting antibody on long-term follow-up (median 4 years after immunization) was virtually all (greater than 90%) IgG1.

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.

Use of monoclonal antibodies to quantify subclasses of human IgG: I. Development of two-site immunoenzymometric assays for total IgG subclass determinations

Dissection of the IgG antibody response into its subclass components has been difficult largely because of the lack of adequate supplies of specific reagents. The development of monoclonal antibodies (Mcab) promises to overcome this problem, but the use of such antibodies has certain inherent problems. It has been shown recently that Mcabs which were avid, potent and specific for well defined epitopes may partially or completely lose their activity depending on the assay system in which they were used. In order to identify Mcabs that would be specific and useful as capture antibodies in a simple two-site enzymometric assay, a panel of 18 Mcabs was screened and one Mcab to each of the four IgG subclasses was identified for quantitation of subclass levels in human serum.

Serum immunoglobulin G subclasses in children infected with human immunodeficiency virus type 1

We studied serum concentrations of IgG subclasses in 47 human immunodeficiency virus 1-infected (17 asymptomatic and 30 symptomatic) children. Thirty-nine of 47 (83%) had an abnormality of at least 1 subclass. Sixteen had only elevated IgG1, 6 had only elevated IgG3 and 12 had elevated IgG1 and IgG3 concentrations. IgG2, IgG4 and combined IgG2-IgG4 deficiency was found in 3, 4 and 4 patients, respectively. IgG2 concentrations did not differ between patients with (n = 23) or without (n = 24) bacterial infections. Additionally the number of bacterial infections was similar between the patients with normal or low IgG2 and/or low IgG4. These data indicate that IgG subclass abnormalities are found in most children with human immunodeficiency virus 1 infection, but quantitative deficiencies of specific subclasses do not appear to explain the high frequency of bacterial infections occurring in these patients.

Phase I study of intravenous gamma globulin in multiple myeloma

Seventeen patients with multiple myeloma were given intravenous immunoglobulin at doses ranging from 150 mg/kg to 500 mg/kg in a phase I study. The intravenous immunoglobulin was well tolerated with only three transient episodes of mild clinical toxicity during 27 infusions. In no instance was hepatic or renal toxicity seen. Marked biologic variability over the one month study period in total IgG levels in patients with non-IgG myeloma and IgG subclasses in many of the patients was observed, making intravenous immunoglobulin half-life determinations based on IgG or IgG subclass levels problematical. The decay of functional antibody to hepatitis B surface antigen was determined. Analysis of the hepatitis antibody data suggested that intravenous immunoglobulin half-life was in the range of seven to 20 days for the entire study group and was not related to the isotype of the myeloma paraprotein or to the baseline levels of IgG. No infections were observed in the study group during the study period, but the potential for infection prophylaxis by intravenous immunoglobulin in myeloma patients must be evaluated in a randomized, prospective, controlled phase III study.

An indirect solid-phase microradioimmunoassay for human IgM-anti-IgG (rheumatoid factor).

In order to satisfy the general need for a more precise measurement of the serum concentration of rheumatoid factor (RF) than is presently obtainable with the latex test, we developed a rapid indirect solid phase micro radioimmunoassay for RF determination. The assay involves the binding of IgM-anti IgG (RF), to the polyclonal, native or denatured human IgG dried on the bottom surface of microtiter plates; the amount of antiglobulin bound is then determined by adding 125I-labelled goat anti-human IgM (125I-AHIgM). Variations of reagents and their concentrations, temperatures and incubation times were studied to find the optimal conditions for test sensitivity, specificity and reproducibility. The test has a within day and between day average coefficient of variation of 9 and 15% respectively, showing its advantages over the latex test. The results obtained by studying 100 human sera from patients with positive or negative latex test show that the test should prove valuable in diagnosis and research.

A monoclonal antibody may show cross-reactivities in Ouchterlony assays but not in other assays

A monoclonal antibody to human IgG was tested with myeloma proteins of the four IgG subclasses. When tested by immunofluorometric assay, enzyme-linked immunosorbent assay, hemagglutination and hemagglutination inhibition assays, the antibody reacted with IgG3 but not with the other three IgG subclasses. When tested by Ouchterlony assays in the presence of polyethylene glycol, the antibody formed lines with all four IgG proteins. The line with IgG3 was sharp and stable, but the lines with the other three IgG subclasses tended to blur with time and with the lower PEG concentrations. These findings show that Ouchterlony assays can reveal cross-reactions of a monoclonal antibody that can be missed by more sensitive assays.

Immunoglobulin class and subclass-specific monoclonal antibody sandwich ELISA for the detection of antibodies against coxsackieviruses B, types 1–5

Immunoglobulin subclass-specific ELISAs were developed for human IgG1, IgG2, IgG3, IgG4, IgAtotal, and IgM directed against Coxsackie B (CB) virus types 1, 2, 3, 4, and 5. In all the assays the solid phase was coated with immunoglobulin class/subclass-specific monoclonal antibodies, followed by an incubation with the serum specimens. Incubation with one of the CB viruses, as well as an incubation with biotinylated serotype-specific monoclonal antibodies to the same virus type provided the virus specificity. Finally, there were incubations with peroxidase labeled Extravidin and the substrate-chromogen system. This ELISA method eliminated the competition between the immunoglobulin classes and subclasses. IgG3 and/or IgG1 were seen most frequently of the IgG subclasses, but IgG2 and IgG4 were also present infrequently. The viral specificity of the antibody subclass assays seems to be predominantly at the enterovirus group level, but this remains to be evaluated in a larger study. IgA and IgM were seen almost exclusively in specimens from patients with acute enteroviral infections, except in the assays with the crude CB5 antigen. This indicates the possible suitability of the IgA and IgM assays as diagnostic tests for enteroviral infections. A larger study is necessary to confirm this finding.