Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin

The aim of this study was to investigate the recognition pattern of bovine serum albumin (BSA), a major dietary protein by serum IgG and IgA antibodies. Anti-BSA IgG and IgA antibodies were measured by ELISA technique in 3 different cohorts: 578 unselected persons, 84 new-onset insulin-dependent diabetes mellitus (IDDM) patients and 103 atopic persons. In order to characterize the recognition pattern of the different BSA domains, recombinant BSA and recombinant fragments covering the 3 BSA domains were produced. BSA digestion was monitored in simulated gastric fluid experiments by means of domain specific monoclonal antibodies. IgG and IgA antibody titres to native BSA were highest in IDDM patients. The three major BSA domains were equally well recognized by IgG antibodies of the three cohorts. Interestingly all three study groups showed a dissociation of their IgG and IgA antibody response to the first BSA domain. The ratio of IgG to IgA antibodies recognizing this domain was 93%/42% in controls, 92%/37% in IDDM patients and 80%/47% in atopic persons. In simulated gastric fluid experiments, the first BSA domain was the first to become undetectable to specific monoclonal antibodies during digestion. In conclusion humoral IgG and IgA antibodies recognize the major BSA domains with different frequencies. The N-terminal domain of BSA, the first to be degraded during simulated gastric digestion is less well recognized by IgA antibodies. This suggests that early digestion is negatively correlated to the IgA antibody response and that the IgA response associated to the gut associated lymphoid tissue (GALT) and the systemic IgG antibody responses are independent.     

Prevalence of norovirus GII-4 antibodies in Finnish children

Noroviruses (NoVs) are the second most common cause of viral gastroenteritis after rotavirus in children. NoV genotype GII-4 has emerged as the major type not only in outbreaks of NoV gastroenteritis but also endemic gastroenteritis among infants and young children worldwide. Using baculovirus-insect cell system virus-like particles (VLPs) of NoV genotype GII-4 and an uncommon genotype GII-12 were produced. These VLPs were used in enzyme-linked immunosorbent assays (ELISA) for detection of NoV-specific immunoglobulin G (IgG) and IgA antibodies in 492 serum specimens from Finnish children 0-14 years of age collected between 2006 and 2008. NoV IgG antibody prevalence was 47.3% in the age group 7-23 months and increased up to 91.2% after the age of 5 years. Avidity of NoV IgG antibodies was low in the primary infections while high avidity antibodies were detected in the recurrent infections of the older children. In GII-4 infections, the homologous antibody response to GII-4 VLPs was stronger than to GII-12 VLPs but cross-reactivity between GII-4 and GII-12 was observed. Binding of GII-4 VLPs to a putative carbohydrate antigen receptor H-type 3 could be blocked by sera from children not infected with NoV during a waterborne outbreak of acute gastroenteritis. Therefore, protection against NoV infection correlated with strong blocking activity.     

Induction and persistence of local rotavirus antibodies in relation to serum antibodies

The induction and persistence of local rotavirus antibodies, including stool IgA, jejunal IgA, and jejunal neutralizing antibody, were evaluated in 14 adult volunteers infected with the CJN strain of human rotavirus. In addition, the relationships between local rotavirus IgA and serum rotavirus IgA, IgG, and neutralizing antibody were determined. Both stool and serum rotavirus IgA appeared to have similar kinetics. Both antibodies peaked by days 14-17 after inoculation in all subjects, then decreased rapidly. By days 26-28, titers had fallen to 13% and 30% of their respective peaks. Serum rotavirus IgG peaked somewhat later, occurring in five subjects on days 26-28. Serum neutralizing antibody peaked on days 26-28 in all but three subjects. Both serum IgG and neutralizing antibodies also declined more slowly than rotavirus IgA. Although all antibody concentrations had decreased to only a fraction of their peak responses by days 270-365 after rotavirus inoculation they remained higher than baseline levels. For example, stool rotavirus IgA concentrations were 13.5-fold higher than baseline, while jejunal rotavirus IgA and neutralizing antibody were 8.9- and 4.3-fold above baseline, respectively. Similarly, serum antibodies remained 3.7- to 11.2-fold higher than baseline at 270-365 days after rotavirus inoculation. These studies imply that serum rotavirus IgA is a good indicator of local antibody responses. Furthermore, although both serum and local antibody titers peaked within 2-4 weeks after infection, these antibodies persisted at above baseline concentrations for at least 9-12 months after infection.     

Bacteroides gingivalis-specific serum IgG and IgA subclass antibodies in periodontal diseases

The level of serum IgM, IgG and IgA antibodies including IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 subclass-specific antibodies to Bacteroides (Porphyromonas) gingivalis fimbriae and to lipopolysaccharide (LPS) were analysed in patients with different forms of periodontal disease (PD) and control subjects by ELISA. Among PD subjects, sera obtained from adult periodontitis (AP), rapidly progressive periodontitis (RPP) and gingivitis contained high titres of fimbriae-specific IgG antibodies (7500-15,000 ELISA units) followed by IgA (90-700 units) and IgM (30-90 units). In contrast, sera from localized juvenile periodontitis (LJP) subjects exhibited much lower titres of fimbriae-specific IgG (89 +/- 11 units), IgA (31 +/- 5 units) and IgM (17 +/- 3 units) antibodies. A similar response pattern was also seen in sera from normal subjects aged 35-41 years who practice normal oral hygiene, while sera of younger adults (aged 18-24) with superior hygiene did not have any antigen-specific antibodies. Analysis of IgG subclass anti-fimbriae responses revealed that the major response was IgG3 followed by IgG1, IgG2 and IgG4 in AP, RPP and gingivitis. Although lower, a similar pattern of IgG subclass titre was seen in LJP and normal subjects aged 35-41 years. When IgA subclass responses were measured in AP and RPP, higher titres of the fimbriae-specific response were noted with IgA1 when compared with IgA2. However, lower but approximately equal levels of fimbriae-specific IgA1 and IgA2 titres were seen in other PD groups. When anti-B. gingivalis LPS-specific responses were measured, the sera of AP patients contained high levels of IgG antibodies (2265 +/- 224 units) followed by IgA (411 +/- 90 units) and IgM (214 +/- 56 units). Further, IgG anti-LPS responses were mainly IgG2 followed by IgG4, IgG3 and IgG1. For IgA subclass responses, higher titres of anti-LPS-specific antibodies were noted in IgA2 subclass over IgA1. These results showed that higher anti-B. gingivalis antibody responses occur in PD when compared with healthy individuals and protein and lipid-carbohydrate antigens of B. gingivalis induce distinct patterns of antigen-specific IgG and IgA subclass responses.     

Cytomegalovirus IgG and IgA serum antibodies in a study of HIV infection and HIV related diseases in homosexual men.

The significance of serum IgG and IgA antibodies to cytomegalovirus (CMV) at various stages of human immune deficiency virus (HIV) infection was studied in 175 homosexual men. Sera were obtained from 123 HIV seropositives [41 asymptomatic, 29 with lymphadenopathy associated syndrome (LAS), 22 with AIDS related complex (ARC), and 31 AIDS patients], 17 HIV seroconverters, and 35 HIV asymptomatic seronegatives. The sera were tested blindly for CMV IgA and IgG antibodies using the immunoperoxidase assay (IPA) and CMV infected human embryo cells. Cross-sectional analysis of CMV IgG antibodies at a titer of greater than or equal to 20 showed 87% and 100% prevalence in the HIV seronegative groups and in the HIV seropositive groups, respectively (P less than 0.05). CMV IgG antibodies at a titer of greater than or equal to 80 were present in significantly higher proportions among the HIV seropositive subjects of the various groups as compared with the HIV seronegative homosexual men. However, in the HIV seronegatives who later seroconverted to HIV, a significantly higher prevalence of CMV antibodies (35%) was detected before HIV seroconversion, as compared with the persistently HIV seronegative subjects (14.3%) (P less than 0.05). The HIV seronegatives pre-HIV seroconversion also exhibited a significantly higher geometric mean titer (GMT) of CMV IgG antibodies (62.17 +/- 0.64) as compared with the persistently HIV seronegatives (34.0 +/- 0.6) (P = 0.03). Significantly higher GMTs of CMV IgG antibodies were detected in all the HIV seropositive groups as compared with the persistently HIV seronegative group. CMV IgG antibodies were not detected in the HIV seronegative subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and characterization of antibodies specific to food antigens (gliadin,ovalbumin and β-lactoglobulin) in human serum,saliva, colostrum and milk

Antibodies against food antigens are usually produced in healthy people. This humoral response can be detected both in serum and secretions. The characterization of this response can be useful for a better understanding of food-related immunological alterations. In this study, IgA and IgG antibodies specific to ovalbumin, β-lactoglobulin or gliadin were measured in serum, saliva, colostrum and milk from 40 healthy breast-feeding women. Specific IgA and IgG to the three antigens were measured by indirect ELISA. Specific IgG levels were highest in serum and very low in the other biological fluids. No correlation between the IgG specific to the different antigens was found. Specific IgA reactivity was found in all the samples analysed. Levels observed were higher in colostrum and milk than in serum and saliva. In spite of being three different unrelated food antigens, a correlation between the levels of specific IgA was found in saliva, colostrum and milk samples of all subjects studied. The specificity of IgA anti-gliadin antibodies from serum, saliva and colostrum was analysed by immunoblotting of SDS–PAGE-separated wheat proteins. Each sample presented a unique pattern of recognition. No common pattern of recognition was found either among the same biological fluids of the different subjects tested, or among the different samples—either serum, colostrum or saliva—of the same individual. Different degrees of specificity to wheat proteins among IgA from colostrum, saliva or serum were observed, suggesting that the local IgA-producing populations are functionally different in the different tissues of the organism.     

Effects of bovine serum albumin on antibody determination by the enzyme-linked immunosorbent assay

The effect of bovine serum albumin (BSA) post-coating and addition of BSA to serum diluent was studied in an IgG and IgA Candida antibody enzyme-linked immunosorbent assay (ELISA). BSA post-coating decreased the background readings and increased the specific Candida antibody activity in most sera. However, in a few sera post-coating alone increased background readings and this effect was probably due to the occurrence of BSA antibodies. It could be abolished by the addition of BSA to serum diluent. It is therefore suggested that BSA post-coating should only be used in combination with BSA addition to serum dilution buffer in ELISA.     

Class and subclass distribution of hantavirus-specific serum antibodies at different times after the onset of nephropathia epidemica

Sera from Dutch and Belgium individuals who suffered from nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), were tested for the distribution of classes and subclasses of Hantavirus (HV)-specific antibodies at different times after the onset of the disease, with class- and subclass-specific Ig capture enzyme-linked immunosorbent assays (ELISAs). In the acute, early convalescent, and convalescent phases, predominantly specific IgA, IgM, and IgGS antibodies were detected. Specific IgG2 antibodies were only detected at low levels in the early convalescent and convalescent phases. In the late convalescent phase specific IgG1 and IgGS antibodies were found, whereas in the late post convalescent phase only specific IgG1 antibodies proved to be present. Specific IgG4 antibodies were not detected in any of the respective phases. These data show that the simultaneous determination of classes and subclasses of HV specific serum antibodies allows the estimation of the time elapsed after the onset of NE. © 1994 Wiley-Liss, Inc.     

An enzyme-linked immunosorbent assay (ELISA) for IgG and IgA antibodies to respiratory syncytial virus in low dilutions of secretions of human serum and secretions

An enzyme-linked immunosorbent assay (ELISA) has been developed for titration of IgG and IgA antibodies to respiratory syncytial (RS) virus in low dilutions of human serum, colostrum, and nasopharyngeal secretions. Previously the sensitivity of RS virus ELISA on such specimens has been limited by nonspecific absorption of antibody, particularly IgA, to crude antigen preparations. For IgG antibody estimation in infant sera, this unwanted binding was reduced to workable levels by increasing the serum, salt, and detergent concentration of the diluent. Residual nonspecific binding of IgA in colostra appeared mainly due to antigen lipids or to lipoproteins. This was markedly reduced by partitioning Triton X-100-treated infected cell lysate antigens in Arklone. Using the modified ELISA technique for anti-RS virus IgA, good correlations were found with unfixed cell membrane immunofluorescence (MIF) for colostra (r = 0.81, P less than 0.001) and nasal secretions from adult volunteers. In several samples nonspecific absorption of antibody precluded MIF assay, but did not affect the ELISA. Although there was an overall correlation between ELISA for anti-RS IgG antibody in sera, the complement fixation test (r = 0.75, P less than 0.001), and MIF test (r = 0.82, P less than 0.001), the sensitivity of ELISA for antibody responses in convalescent sera of infants from 3 months to 2 years was poor. Conversely, the sensitivity of ELISA for antibody in the sera of older children and for transplacentally acquired antibody in very young infants was higher than that for the other two tests. ELISA was thus less reliable than either CF or MIF for detecting antibody rises in paired infant sera, particularly where maternally acquired antibody remained in the acute serum. The reasons for this apparent disparity are discussed.     

Specific IgG and IgA antibodies to herpes simplex virus (HSV)-induced surface antigen in patients with HSV infections and in healthy adults

Herpes simplex virus (HSV)-specific IgG and IgA antibody response in patients with HSV infection and in healthy adults was studied by the immunoperoxidase antibody-membrane antigen (IPAMA) technique. In all HSV infections in which specific IgM antibodies were detected by enzyme-linked immunosorbent assay (ELISA), a significant rise in the titer of HSV IgG and IgA antibodies was found. In contrast, in patients with recurrent herpes labialis in which no specific HSV IgM antibodies were detected by ELISA, HSV IgG and IgA antibodies were not found to fluctuate significantly during the course of infection. A higher geometric mean titer (GMT) for HSV IgG and for IgA antibodies was found in seropositive individuals with a previous history of recurrent HSV than in seropositive individuals without a previous history of recurrent HSV infection. Nineteen of 26 HSV IgG seropositive healthy medical students without a previous history of recurrent HSV infection had HSV IgA antibodies to membrane antigen. The significance of this finding in understanding the mechanism of latency in healthy seropositive individuals without previous history of HSV recurrent infections is discussed.     

Severity of infections in IgA deficiency: correlation with decreased serum antibodies to pneumococcal polysaccharides and decreased serum IgG2 and/or IgG4

In order to define abnormalities of humoral immunity which determine susceptibility to respiratory tract infections in IgA-deficient adults, serum IgG subclass concentrations, and serum concentrations of pneumococcal antibodies and Haemophilus influenzae type B (Hib) antibodies sera from IgA-deficient adults with and without susceptibility to respiratory tract infections were compared. Infection susceptibility was not related to the degree of IgA deficiency, but was related to deficiency of IgG4 and, to a lesser extent, IgG2, as well as to low basal serum concentrations of pneumococcal polysaccharide antibodies. The combination of IgG2 and/or IgG4 deficiency and a non-protective basal serum concentration of antibody against two or more pneumococcal polysaccharides was present in the serum of six of 12 (50%) patients with severe infections, but only one of 44 (2%) patients without infections. Furthermore, the preservation of antibody responses against the most immunogenic pneumococcal polysaccharide type 3, but not against the less immunogenic types 7F, 9N and 14, in patients with severe infections suggested that abnormalities of pneumococcal polysaccharide antibody responses might include defects of affinity maturation.     

Porphyromonas gingivalis-specific serum IgG and IgA antibodies originate from immunoglobulin-secreting cells in inflamed gingiva.

Patients with adult periodontitis (AP) exhibit elevated serum antibody levels to Porphyromonas (Bacteroides) gingivalis; however, it is not known whether these antibodies originate from plasma cells in the local disease site or from peripheral lymphoid tissues. We studied the isotype and subclass levels and origin of antibodies to P. gingivalis fimbriae, since elevated serum anti-fimbriae responses were seen when compared with sera of healthy controls. IgG anti-fibriae titres were dominant and the subclass response was IgG3 much greater than IgG1 greater than IgG2 much greater than IgG4; however, some IgA anti-fimbriae antibodies were also seen. The IgA subclass fimbriae-specific response was mainly IgA1; however, significant IgA2 anti-fimbrae antibodies were seen. We also assessed numbers of anti-fimbriae antibody producing cells from peripheral blood mononuclear cells (PMBC) and from either healthy or inflamed gingiva of AP subjects. Gingival mononuclear cells (GMC) of AP patients exhibited high numbers of immunoglobulin-producing (spot-forming) cells (SFC) including fimbriae-specific antibody secreting cells in a pattern of IgG greater than IgA greater than greater than greater than IgM. However, low numbers of SFC were seen in GMC from healthy gingiva; further, no anti-fimbriae SFC responses were noted in healthy GMC. Although no fimbriae-specific immunoglobulin-producing cells were seen in PBMC, low numbers of antigen-specific SFC were found in pokeweed mitogen-triggered PBMC from AP subjects. Treatment of AP patients for plaque and surgical removal of inflamed gingiva resulted in significant reductions in serum anti-fimbriae responses. These studies show that AP patients exhibit brisk serum IgG and IgA subclass anti-fimbriae antibodies, whose origin appear to be the plasma cells present in the localized inflamed tissues.     

Isotypic analysis of grass-pollen-specific antibodies in human plasma. III. Relationship to autoantibodies to IgE

Since it has been shown that autoanti-IgE may be mistaken for antiallergen antibodies, thus appearing as pseudo-allergen-specific antibodies, it is crucial to separate true- from pseudo-allergen-specific antibodies and to determine to what extent autoanti-IgE appeared as pseudo-allergen-specific antibodies. For this purpose, human Ig pools were affinity-purified successively on a grass-pollen column and then on an antihuman-IgE column. IgG1–4, IgA, and IgM antibodies that were eluted from the grass-pollen column separated into pseudo- (∼30–40%) and true-allergen-specific antibodies that were coretained and not coretained, respectively, with the IgE on the anti-IgE column. Levels of autoanti-IgE were determined in individual plasma samples by surface plasmon resonance and statistically compared to the concentrations of allergen-specific antibodies obtained previously in the same plasma samples. A positive correlation between IgM autoanti-IgE levels and grass-pollen-"specific" IgM concentrations ( P < 0.0002), and negative correlations between IgA autoanti-IgE and both IgE anti-grass pollen and IgG2 autoanti-IgE levels ( P < 0.03, in both cases) were observed for the first time. This supports the contentions that: (1) autoanti-IgE antibodies appeared as pseudo-grass-pollen-specific antibodies, (2) they hid IgE antibodies when the latter were measured, and (3) they compete with one another in binding IgE. Lastly, a model of large Ig complexes is discussed.     

Detection and persistence of specific IgA antibodies in serum of patients with hepatitis A by capture radioimmunoassay

The serum immunoglobulin A (IgA) response to hepatitis A virus (HAV) was investigated with a sensitive capture radioimmunoassay. In serial serum samples drawn from 15 patients with viral hepatitis A, IgA anti-HAV antibodies reached their highest titer between 1-2 weeks after onset and peak titers ranged from 10,000-20,000. Serum samples were available from six patients 30-32 months after onset of illness. These samples were all positive for IgA anti-HAV and some had titers similar to peak titers during illness. However, the height of the titration curves, expressed as the binding ratio (BR) at a dilution of 1/1000, was in all cases significantly lower at 30-32 months than during acute illness and early convalescence. The significance of the persistence of the IgA anti-HAV and possible reasons for the change in the BR are discussed.     

Specific IgG and IgA antibodies to herpes simplex virus and varicella zoster virus in acute peripheral facial palsy patients

The role of herpes simplex virus (HSV) and varicella zoster virus (VZV) in acute peripheral facial palsy (APFP) was evaluated in 153 patients. The sera of patients were examined for IgG and IgA antibodies to HSV and VZV by the immunoperoxidase antibody-membrane antigen (IPAMA) technique. The prevalence of IgG and IgA seropositivity to HSV was significantly higher in the APFP patients than in the matched control group. No significant difference was found in the geometric mean titer (GMT) to HSV antibodies in the APFP patients compared to the matched control groups. The prevalence of IgA antibodies to VZV was significantly higher in the APFP patients group than in the matched control group. The GMT for VZV IgG antibodies was significantly higher in the APFP patients than in the matched control group. No significant difference was found in the GMT of VZV IgA antibodies. Eleven of 83 patients for whom paired sera were available had significantly increased or decreased IgG and IgA antibody titers to VZV on subsequent examination. Four of these patients did not show any evidence of zosterian eruption. These studies support the concept that VZV and HSV might have a role in the etiopathogenesis of APFP in some of the patients.     

Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus.

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.     

Characterization of the homo- and heterotypic immune responses after natural norovirus infection

Noroviruses (NoV) are a genetically and antigenically diverse group of viruses that are common causes of outbreaks of gastroenteritis in humans of all ages. Limited information has been obtained on type specificity of the NoV immune response. In this study, we characterized the homologous and heterologous antibody responses in adults from 13 outbreaks, representing 4 different NoV genotypes. NoV specific IgG and IgA antibodies were determined as well as the increase of antibody avidity. In addition, antibody-mediated blocking of NoV binding to its putative receptor was evaluated. Both homologous and heterologous serological responses were detected after NoV infection. The avidity of antibodies could not be used to distinguish between homologous and heterologous antibody responses. However, a homologous blocking response but not a heterologous response was detected after infection with NoV belonging to genogroup II.4 by a NoV ligand binding inhibition assay. Infection with NoV induces antibodies that can block virus ligand interactions. In contrast with all currently known antibody detection assays for NoV, this can be used as a type specific assay and may be an alternative for studying neutralizing antibodies.     

Detection by ELISA of IgA and IgM antibodies in secretion and IgM antibodies in serum in primary lower respiratory syncytial virus infection

Twenty-six infants and children with primary lower RS virus infection, diagnosed by the detection of RS virus in nasopharyngeal secretion (NPS) by use of immunofluorescent antibody (FA) technique, were studied with respect to the presence of IgA and IgM antibodies. Samples of NPS and serum obtained during the first 3-4 months following the beginning of illness, were investigated. Employing a reverse ELISA technique, we found IgM antibodies in the acute, but not during the convalescent, phase of illness in NPS from 20 of the patients and in serum from 21 of the patients. The majority of the IgM antibody conversions observed occurred in NPS as well as in serum on days 5-8 following the illness. RS virus IgA antibodies, also detected by a reverse ELISA technique, were demonstrated in NPS in 22 of the patients, with antibody conversions being found in 19 of the patients on days 5-8 following the beginning of the illness. Two patients still had IgA antibodies in NPS approximately 3 months FSOI. By comparison, RS virus was detected in acute-phase NPS by double-antibody sandwich ELISA in 25 of the 26 patients investigated.     

The protective capacity of immune sera in experimental mouse salmonellosis is mainly due to IgM antibodies

Passive immunization was used to protect mice against a general infection caused by Salmonella typhimurium and our purpose was to compare the protective capacity of different immunoglobulin isotypes (classes and subclasses). Three antisera were studied, one pool of mouse serum against the envelope of rough bacteria, and two rabbit sera against smooth bacteria. Three different methods were used to separate isotypes. The consistent finding was that only IgM antibodies protected efficiently. A unit of IgG antibodies had an effect that was 1/50th of the IgM effect or less. This effect could have been due to a contamination by IgM. IgA appears to be non-protective like IgG. In two of the antisera a considerable proportion of protective antibodies were against a defined antigenic determinant (anti-0–4,5 or anti-0–9). IgG antibodies of these sera measured by the solid phase assay were also predominantly anti-0–4,5 or anti-0–9, respectively. This argues that the failure of IgG antibodies to protect cannot be explained by assuming that unlike IgM antibodies they are directed against “non-protective” determinants. We conclude that the observed difference between the protective capacities of IgM and IgG antibodies is due to C-region differences between the μ- and γ-chains.