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.     

An enzyme-linked immunosorbent assay for the quantitation of human IgG subclasses using monoclonal antibodies

An enzyme-linked immunosorbent assay was established for the quantitation of human IgG1, IgG2, IgG3 and IgG4 using IgG subclass-specific monoclonal antibodies. The method could detect 1-10 ng/ml of the Ig subclasses. The technique is suitable for measuring IgG subclass concentration in sera of healthy adults and in supernatants from human lymphocytes cultured in the presence of pokeweed mitogen.     

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.     

The subclasses of human IgG antibodies against tetanus toxoid.

The subclass of IgG antibodies against tetanus present in the serum of thirty-five human individuals, who received an injection with tetanus toxoid, was determined. Six successive serum samples were obtained from twenty-five normal individuals (laboratory personnel) 0, 3, 7, 14, 28 days and 2-3 months after the injection with tetanus toxoid had been given. Another ten serum samples were obtained from ten persons with a positive IgE-RAST, taken 2 weeks after the injection. Antibodies were determined with a quantitative immunofluorescence method known as the defined antigen substrate spheres (DASS) system. The normal individuals in whose serum a clearly positive IgG binding was found (nineteen) showed activity in all four subclasses. The binding activity in all individuals reached a maximum between 2 and 4 weeks after the injection. The antibody activity in the serum of four individuals whose serum gave weak IgG binding was confined to IgG1. Two individuals did not show any IgG binding activity at all. In the ten persons with a positive IgE-RAST and three of the normal individuals, who also had a positive IgE-RAST, the distribution of the antibodies over the subclasses was the same as in the others.     

Use of monoclonal antibodies to quantify subclasses of human IgG. II. Enzyme immunoassay to define antigen specific (anti-filarial) IgG subclass antibodies

Because of their single epitope specificity, monoclonal antibodies (Mcabs) may perform with different levels of efficiency in immunoassays depending on the accessibility of the particular epitope recognized. In order to develop assays capable of detecting specific antibodies of each of the four human IgG subclasses, we have evaluated by ELISA the performance characteristics of a panel of Mcabs raised to the subclass proteins. At least one Mcab to each of the four subclasses was identified that was specific in its ability to capture its own relevant IgG subclass without any associated light chain, allotype or isoallotype activity and that was able to function effectively as a probe in an optimized, quantitative ELISA. When IgG subclass antibodies were measured in sera from patients with filariasis using specific filarial antigen, the sensitivities of each subclass antibody assay varied; for IgG1 and IgG4 antibodies the sensitivity of detection was 50 ng/ml and for IgG2 and IgG3, 10 ng/ml. The potency of the Mcab, determined by its titration for use as a probe, did not correlate with the sensitivity of the assay. These Mcabs were also capable of defining IgG subclass antibody responses qualitatively in immunoblot analyses with little or no non-specific binding. The availability of such highly characterized Mcabs for use in quantitative and qualitative definition of specific IgG subclass antibody responses should greatly improve our detection and subsequent understanding of the role of these IgG subclasses in various disease states.     

IUIS/WHO notice. Appropriate uses of human immunoglobulin in clinical practice.

There is a consensus amongst clinical immunologists that an effort should be made to define, for non-specialists, criteria and indications for administering immunoglobulin (gammaglobulin, immune serum globulin [ISG]) to patients. In addition to the well established immunoglobulin preparations for intramuscular injection, preparations rendered suitable for intravenous administration have recently become available, opening new prospects for the use of this material. The present report has been prepared by members of the Clinical Immunology Committee of the International Union of Immunological Societies in collaboration with the World Health Organization. It aims to delineate current recommendations for the use and dosage of immunoglobulin in the prophylaxis of viral and bacterial infections and in replacement of antibodies in immunodeficient patients. It is a further aim of this report to point out clearly those situations where immunoglobulin is not deemed to be useful or is contraindicated. Inappropriate use of this valuable material results in excessive and unnecessary costs for health services, carries some risk and is wasteful. This report also summarizes current knowledge on adverse reactions to immunoglobulin injections and those qualities which would be most desirable, if not essential, for safe and useful immunoglobulin preparations.     

Hinge region of human IgG2 protein: conformational studies with monoclonal antibodies.

We previously produced three anti-human IgG2 mAbs with high specificity and found that they recognize distinct epitopes in the hinge region and neighboring residues in human IgG2: HG2-6A was reactive with the hinge region (Glu216-Pro230); HG2-56F with the Pro234 residue and HG2-30F with the Val235 residue. In this study, we evaluated the reactivities of those three mAbs with human IgG2 protein under various conditions. The results obtained using HG2-6A mAb indicated that the hinge region was concealed in the native form, but exposed after heat treatment at 63 degrees C, or chemical treatment with 3 M KSCN, 3 M guanidine, 30% CH3CN, 8 M urea or acid at pH 2.0 as well as by adsorption onto polystyrene beads. The IgG2 hinge region was also exposed after binding to specific antigens. The Pro234 residue recognized by HG2-56F mAb was exposed under all conditions studied. The neighboring Val235 residue recognized by HG2-30F, however, was completely concealed in the native and antigen-bound states. Only treatment with 3 M guanidine and acid at pH 2.0, or physical adsorption induced conformational changes to partially expose the Val235 residue.     

Monoclonal antibodies specific for rat IgG1, IgG2a, and IgG2b subclasses, and kappa chain monotypic and allotypic determinants: reagents for use with rat monoclonal antibodies

Mouse monoclonal antibodies to rat IgG were obtained by fusion of immune SJL mouse spleen cells to NSI myeloma cells. Seven monoclonal antibodies have been labeled with 125I and studied as to specificity and avidity by using a panel of rat monoclonal antibodies both as inhibitors and target antigens in soft well plate and indirect cell binding assays. All MAb were selected for high avidity of 4 X 10(7) to greater than or equal to 2 X 10(9) M-1. Four MAb were subclass-specific. RG11/39, RG7/1, and RG7/11 were absolutely specific for the Fc' region of IgG1, IgG2a, and IgG2b, respectively. RG9/6 showed specificity for the Fab' region of IgG2a but crossreacted with lower avidity with IgG2c. Three MAb reacted with rat kappa chains. RG7/9 defined a monotypic (common) kappa chain determinant. RG11/15 and RG7/7 were specific for allelic kappa 1a and kappa 1b determinants, respectively. The monotypic and kappa 1a allotypic determinants are topographically separated. The antibodies can be used as screening reagents in indirect cell binding assays. They have sensitivity similar to affinity-purified rabbit anti-rat IgG and more defined specificity. They do not crossreact with mouse or human IgG, making them particularly suitable companion reagents for rat anti-mouse or anti-human MAb. One Mab, RG7/7, strongly crossreacts with Syrian hamster IgG.     

Subclasses of IgG on the surface of human lymphocytes: a study with monoclonal antibodies

Cells from sIgG+ B lymphoblastoid cell lines, sIgG+ B cell neoplasms and from tonsils, adult and cord blood and fetal spleen were tested for IgG subclass expression using a panel of monoclonal antibodies. IgG1 was in all cases the commonest subclass except on blood lymphocytes, when approximately equal numbers of cells expressing IgG1 and IgG2 were found. No evidence for multiple subclass isotype expression was found.     

Production of human monoclonal IgG and IgM antibodies with anti-D (rhesus) specificity using heterohybridomas

Heterohybridomas secreting human IgM and IgG anti-D antibodies of the rhesus blood group system have been established by fusion of EBV-transformed anti-D secreting cells with the mouse myeloma cells X63-Ag8.653. Both classes of antibody reacted with all Rh-positive cells, some Du cells but not with Rh-negative or DB cells. Concentrations of both antibodies reached between 25 micrograms/ml and 50 micrograms/ml in the culture supernatants. The cell lines have been maintained in culture for 14 months and have been shown to be suitable for large-scale production of antibody.     

Similar specificity of IgG1 and IgG2a antibodies in individual mice.

In this study the previous finding of similar variable regions in individual IgG1 and IgG2a antibody populations is extended by the demonstration of similar fine specificity of IgG1 and IgG2a combining sites. Antibody populations from individual mice directed against oligo-D-alanine determinants were analyzed in their cross-reactivity towards 5 heterologous dipeptides. This was done by mixing antibody and hapten followed by determination of free antibodies in a kinetic red cells sensitization assay. The comparison of hyperimmune sera from 10 mice showed that genetically identical mice can differ significantly in their cross-reaction pattern. Within each serum the cross-reaction pattern was determined for IgG1 and IgG2a. With a few exceptions the same individual pattern was found in both IgG1 and IgG2a antibody populations. This was taken as evidence that the combining sites of IgG1 and IgG2a anti-oligo-D-alanine antibody populations in an individual mouse are similar.     

Modulation of the murine immune response to human IgG by complexing with monoclonal antibodies. II. Antibody responses to idiotopes of the human IgG paraprotein and of the mouse monoclonal antibodies.

Immune sera have previously been shown to play a positive role in immune protection against murine experimental brucellosis. The protective properties of a panel of five anti-Brucella monoclonal antibodies (Mabs) were therefore assessed by estimation of the acceleration of the blood clearance of intravenously inoculated Brucella and of the reduction of splenic infection on Day 7 after infection. Three 'strongly protective', one 'weakly protective' and one 'non-protective' Mabs were identified. As a first step towards the study of the mechanism of this humoral protection, these Mabs were further compared for structural and functional properties such as immunoglobulin isotype, anti-Brucella specificity, anti-Brucella in vitro bacteriostasis, Brucella agglutination and complement fixation when complexed with tyndallized Brucella. No correlation was found between protection and either agglutination or direct bacteriostasis. On the other hand, the results observed suggest that isotypes (and especially the IgG2a isotype) could play an important role in in vivo immuno protection and that complement may be involved. However, the fact that one of the protective Mabs belongs to the IgA isotype, does not cross-react with the others in anti-Brucella epitopic specificity and does not fix complement underlines the probable diversity of the mechanisms involved.     

The development of difference turbidimetric analysis for monoclonal antibodies to human IgG

Individual monoclonal antibodies to human IgG have been shown to form immune complexes of defined stoichimetry. These complexes are non-precipitating and do not exhibit turbidity. Combinations of two monoclonal antibodies directed against spatially distinct antigenic determinants produce complexes exhibiting marked turbidity. Such combined monoclonal antibody ‘cocktails’ may be applied to quantitative techniques.     

Production and characterization of monoclonal antibodies against human IgG in Balb/c mouse

Immunoglobulin G (IgG) is the main immunoglobulin in natural human serum. It constitutes 70 to 75 percent of all immunoglobulins. Monoclonal antibodies have many applications in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgG are used in most diagnostic kits. For production of monoclonal antibodies against human IgG, spleen cells of the most immune mouse were fused with SP2/0 (myeloma cells) using Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. The suitable clones were selected for limiting dilution (L.D). Then, the supernatant of suitable monoclones were assessed for cross reactivity with IgM & IgA by ELISA and confirmed by immunobloting. The subclasses of the selected monoclonal antibodies were determined and the clones were frozen and kept in liquid nitrogen. Finally, suitable monoclone was injected into the mouse, intraperitoneally, that has been primed with Pristane. In this study, 127 clones were obtained of which 15 clones had absorbance more than 1 which two of them with absorbance about 1.5 were selected for limiting dilution. The yield of limiting dilution was 6 clones with absorbance about 1.8 which did not show cross reactivity with IgM & IgA. Among these clones, G2 monoclone with IgG1 subclass was selected as suitable one and it was reproduced in FCS free RPMI 1640. For large scale production in invivo, the suitable clone was implanted in the peritoneum of the Balb/c mouse and its titer was measured, which showed 1/100,000 dilution for ascitic fluid, having no cross reactivity with IgM & IgA. Monoclonal antibody was purified by chromatography, confirmed by SDS- PAGE, conjugated with enzyme and applied for diagnostic kits.     

In vitro reactions of anti-Rh antibodies depending on the pattern of IgG subclasses.

In 47 serum samples obtained from 31 women immunized with D antigen of the Rh system the level of anti-Rh D antibodies was measured by the papain and antiglobulin tests and the IgG subclasses of anti-Rh antibodies were determined. If the antibodies were of the IgG1 subclass or if this subclass prevailed in the IgG subclass pattern, the antibody level determined by the papain test of L?w was higher or equal to their level found in the antiglobulin test. In all cases, in which the antibodies titer was higher in the antiglobulin test, the antibodies were either of the IgG3 subclass exclusively, or this subclass prevailed quantitatively. This was due to partial inactivation of antibodies belonging to the IgG3 subclass by papain added to the serum in the L?w's test. In the test with papainized erythrocytes (called two-stage test) the IgG subclasses could not be differentiated, and the results obtained then were always higher than in the antiglobulin test. The differences in the reactions of antibodies performed routinely for diagnostic purposes can be explained by differences in immunoglobulin subclasses produced during immunization.     

Evidence for an absence of cross-reactivity between the human beta2-microblobulin and human IgG allotypes and subclasses.

Six beta2-microglobulins were isolated from the urine of six patients with renal tubular dysfunction. Cross-reactivity between these beta2-microglobulins and antigenic determinants of different IgG allotypes and subclasses was investigated. No evidence of cross-reactivity was found. Some hypotheses are discussed concerning these results.     

Implications for the assay and biological activity of interleukin-8: results of a WHO international collaborative study

Eight ampouled preparations of interleukin-8 (IL-8) have been evaluated for their suitability to serve as an international standard for IL-8 by 30 laboratories in 12 countries in an international collaborative study. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the study that different recombinant preparations of IL-8 can have different biological specific activities, even though all were produced using E. coli. It is of interest that the intra-laboratory variability of estimates provided by several neutrophil degranulation bioassays was less than that of the immunoassays, suggesting that these bioassays can be as precise, if not more so, than immunoassays. In addition, immunoassay estimates of IL-8 preparations differed from those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine. The large reduction in the inter-laboratory variability of estimates in terms of a common reference preparation clearly illustrates the need for a standard for IL-8. On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-8 (89/520) was established as the International Standard for IL-8 with an assigned unitage of 1000 IU/ampoule.     

Elisa test system with monoclonal antibodies to human IgG4 for the detection of allergen-specific antibodies in pollinosis

G. N. Gabrichevskii Moscow Research Institute of Epidemiology and Microbiology. Institute. of Experimental Cardiology, All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. I. Vorob'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 11, pp. 574–577, November, 1989.     

Human Leucocyte Differentiation Antigen nomenclature: update on CD nomenclature. Report of IUIS/WHO Subcommittee

The 7th International Workshop on Human Leucocyte Differentiation Antigens (HLDA7) studied a number of newly characterised molecules relevant to human leucocyte differentiation and function. The HLDA organisation, which devised and continues to maintain the CD nomenclature, is responsible, under the auspices of IUIS and WHO, for the nomenclature of all leucocyte differentiation markers. The 7th Workshop redefined a number of (principally carbohydrate) molecules, and assigned CD names to approximately 80 new molecules. This update lists, in tabular form, the redefined and newly assigned names, together with antibodies, which have been confirmed under Workshop conditions as specific for the new and redefined molecules. The major features of the cellular expression patterns are summarised, and a LocusLink accession number provided to enable the reader to access more detailed information through http://www.ncbi.nlm.nih.gov/LocusLink.