O. Mäkelä

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.

Inherited Immunoglobulin Idiotypes of the Mouse

A-CHO, group A streptococcal polysaccharide; cap; epsilon-aminocaproyl; Cg gene and Cj^ gene, the structural germ-line gene for the H chain or the L chain constant polypeptides; HPI, Haptenated Phage Inactivation method for detecting anti-hapten antibodies; IEF, isoelectric fociising; NBrP, (4-hydroxy-5-bromo-3-nitrophenyl) acetyl; NIP, (4-hydroxy-5-iodo-3-nitrophenyl) acetyl; NNP, (4-hydroxy-3, 5-dinitrophenyl) acetyl; NP, (4-hydroxy-3-nitrophenyl) acetyl; PC, phosphorylcholine; TMV, tobacco mosaic virus; V region, in this text, the part of an immunoglobulin molecule consisting of a Vg and a V^ domains or polypeptides (= Fy piece); Vg gene and V^ gene, structural germ line gene for the H chain or the L chain variable polypeptides.

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 hulk of IgG2a bearing allotypes a or j eluted already at pH 5, in contrast to IgG2a bearing allotype b. Another newfinding 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 al 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.

Establishment of anti-TNP antibody-producing human lymphoid lines by preselection for hapten binding followed by EBV transformation.

Human lymphoblastoid cell lines that produce specific antibody against the haplen trinitrophenyl (TNP) have been established by selecting TNP‐binding human B lymphocytes by TNP‐rosetting and Ficoll‐lsopaque separation, followed by Epstein‐Barr virus (EBV) immortalization. Derived lines secreted polyclonal anti‐TNP antibodies and contained relatively small numbers of specific rosette‐ and plaque‐forming cells against TNP‐RBC. Following rerosetting with TNP‐RBC, the frequency of rosette‐forming cells increased from 2% to 75%. In parallel, the frequency of plaque‐forming cells increased from 0.4% to 30%. The antibody titres in the supernatants increased from 64 to 512 and from 48 to 192, as measured by TNP agglutination and haeniolytic assays, respectively. The antibodies were I9S, IgM. The specificity of the anti‐TNP antibody was confirmed by the hapten inhibition test, in comparison and cross‐reactivity tests with the supernatant of the previously established. EBV‐transformed anti‐4‐hydroxy‐3,5‐dinitrophenacetic acid (NNP) antibody‐producing cell line. Both antibodies were specific: the homologous hapten inhibited them but the heterologous hapten did not.

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 vaccines 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 und 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).

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 geneticallyG1mf-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.

Release of Sialic Acid and Carbohydrates from Human Red Cells by Trypsin Treatment

It was found that trypsin treatment of red cells, as commonly used in blood group laboratories for demonstrating incomplete antibodies, releases about one‐half of the sialic acid as well as about 15% of the hexosamine and structural sugars of the cells.

Proportions of Ig Classes and Subclasses in Mumps Antibodies

Mumps antibodies of 34 human beings were studied, 12 patients with natural mumps infection, 15 subjects vaccinated with a live mumps vaccine, and seven subjects vaccinated with an inactivated vaccine. Small amounts of antibodies reacting with mumps antigen were found in the prevaccination sera. An immunization with either the live or the killed vaccine caused an increase in the mumps antibodies (range, from 1.1‐fold to more than 50‐fold; geometric mean, approximately sevenfold). IgG1 was the major isotype in all post‐vaccination sera; the averag share was 61%. Next came IgM (28%), followed by IgA (9%), and IgG3 (2% of total). The patient samples had 10 (acute phase) or 20 times (convalescent phase) more mumps antibodies than the prevaccination samples. IgGl was the predominant isotype in the acute phase sera (average 42% of all antibodies). Next came IgM (41%) followed by IgA (13%), and IgG3 (4%). In convalescent sera IgGl was also the predominant isotype (average 67%), followed by IgM (19%). The minor isotypes in the second samples were IgA (12%) and IgG3 (3%). Small amounts of IgG2 antibodies were found in 1 patient and 1 vaccinee. IgG4 antibodies were not detected.

Relative Immunogenicity in Mice of Different Regions of the Human IgG

Monoclonal mouse antibodies to human IgG myeloma proteins were produced and characterized by determining their binding to a series of different purified myeloma proteins. Two types of immunization schedules were used. When the same myeloma protein was used for priming and boosting the mouse, all determinants of the molecule were effectively immunogenic. Of the 353 clones originating from these experiments 42% secreted anti‐Fv antibodies, 57% anti‐CH antibodies, and 0.85% anti‐CL, antibodies. In another schedule only the CL and CH regions were the same in priming and boosting; 270 anti‐CH (94%), 18 anti‐CL (16%), and no anti‐Fv hybridomas were found. Our results indicate that a unit mass of the Fv region was 1.3 times more antigenic than a unit mass of the CH region. A unit mass of the CH region was nearly five times more antigenic than a unit mass of the CL domain when the antigenicity of the Fv region had been excluded. When the antigenicity of the Fv region had not been excluded, a unit mass of the CH region was about twenty times more antigenic than a unit mass of the CL domain. The data suggest that an antigenic competition was taking place between different parts of the molecule and that the strongly antigenic region (Fv) was more efficient in competing out the nearest neighbour (CL) than in competing out other parts of the molecule.

Immunogenic Properties of α(1 6) Dextran, Its Protein Conjugates, and Conjugates of Its Breakdown Products in Mice

Mice were immunized with α(1–6) dextran, either as such or coupled to protein carriers, and their anti‐dextran response was measured by a solid‐phase radioimmunoassay and the Farr assay. Like earlier investigators we found that protein‐conjugated dextran was more antigenic than plain dextran. Our novel findings were that (1) a standard dose (30 μg of dextran per injection) coupled to strongly antigenic protein (chicken serum albumin (CSA)) was three times more antigenic than dextran coupled to weakly antigenic bovine serum albumin (BSA); (2) dextrans of low molecular weight (1000–10,000 daltons) coupled to CSA induced at least ten times stronger secondary responses than did a similarly coupled macromoleculur dextran (5–40 million daltons); (3) variation of the CHO/protein ratio from 0.3 to 1 had little effect on the antigenicity of the dextran. Increase of the ratio from one appeared 10 decrease immunogenicity when BSA was the carrier but not when CSA was ihe carrier.