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.     

The primary immune response in patients with selective IgA deficiency

The primary immune response in vivo of 20 patients with selective IgA deficiency was studied and compared to controls. The primary cellular immune response tested by dinitrochlorobenzene (DNCB) was decreased in many patients. The primary humoral immune response was elicited by immunization with the test immunogen Helix pomatia haemocyanin (HPH). Using a direct ELISA technique antibodies against HPH of the IgA, IgG and IgM class were measured. Two weeks after immunization no response of IgA anti-HPH was seen except in three patients who showed a low but detectable antibody level. In spite of normal or even elevated serum IgG and IgM levels there was a significantly lower response of the IgG and IgM anti-HPH antibodies at 2 weeks after immunization as compared to the controls followed by a further decline at 6 weeks. We conclude that selective IgA deficiency is often accompanied by more general disturbances in humoral and cellular immunity to newly encountered antigens.     

Serum immunoglobulin A antibody to varicella-zoster virus in subjects with primary varicella and herpes zoster infections and in immune subjects.

Immunoglobulin A (IgA) antibodies to varicella-zoster virus (VZV) were measured in sera from subjects with acute varicella and herpes zoster, VZV-immune subjects remote from infection, and recipients of a live attenuated varicella vaccine, using a solid-phase radioimmunoassay. Primary infection with VZV was associated with early production of IgA antibodies. Among 36 subjects with varicella tested 1 to 5 days after onset, 22 had detectable IgA, and all of the negative sera were obtained before day 3 of the varicella exanthem. VZV IgA was detected in one of three sera obtained more than 60 days after onset of the illness. Four of five sera obtained from subjects within 1 week of the onset of herpes zoster had measurable levels of IgA. Between 1 and 4 weeks after onset of zoster, all 10 subjects tested had detectable IgA to VZV. VZV IgA was detected as late as 63 days after the onset of herpes zoster. Of 10 vaccine recipients, 5 developed VZV IgA which was detected as early as 4 weeks and persisted for as long as 16 weeks after vaccination. VZV IgA was not detected in sera from 42 children who had no detectable IgG antibody to VZV. VZV IgA was found on only 3 of 23 sera from adults who had varicella more than 20 years before.     

Experimental immunization with Pseudomonas aeruginosa alginate induces IgA and IgG antibody responses.

We tried experimentally to induce a specific antibody response against Pseudomonas aeruginosa locally in the airways and systemically in rats by three different routes of immunization; intragastric feeding, intratracheal inoculation or subcutaneous vaccination. Three groups of rats were immunized with live mucoid P. aeruginosa PAO 579 by intragastric feeding or with killed PAO 579 intratracheally or subcutaneously. Three other groups were immunized with purified P. aeruginosa alginate either by intragastric feeding, intratracheally or subcutaneously. At weekly intervals for four weeks animals were sacrificed and serum and bronchial fluid were obtained. The specific IgA and IgG antibody response in lavage fluid and serum was measured. Only traces of antibodies could be detected in the bronchial lavage fluids. Anti-alginate IgA and IgG antibodies developed in all rats immunized with alginate but no antibodies against other P. aeruginosa antigens were detected. The highest IgA and IgG titer against alginate was induced by the subcutaneous immunization. IgA and IgG antibodies against other P. aeruginosa antigens developed in rats immunized with liver and sonicated bacteria. The highest IgA and IgG titers were obtained after intratracheal and subcutaneous immunization with sonicated bacteria. The present work has shown that IgA and IgG antibodies develop with high specificity after immunization. The different titers obtained do not necessarily reflect different degrees of protection.     

Detection of virus-specific IgA antibodies in serum of kidney transplant patients with recurrent cytomegalovirus infection by enzymeimmuno and radioimmunoassay techniques.

The feasibility of using human cytomegalovirus (CMV)-specific IgA antibody determinations as a signal for early detection of recurrent CMV infections in eight renal transplant recipients was analyzed. Solid phase radioimmunoassay (RIA), enzyme-liked immunosorbent assay (ELISA) and immunoperoxidase assay (IPA) techniques were used for IgA antibody determinations. In parallel, IgG antibodies to CMV were studied by immunoperoxidase assay. A significant rise of CMV-specific IgG antibody titre was observed in all of these patients between 5 and 53 weeks post-transplantation. CMV-specific IgA antibody production was detected close to the time a rise in CMV IgG antibody was observed in seven out of eight patients studied by RIA and ELISA, and in six out of eight patients studied by IPA. In two patients specific CMV IgA antibodies were detected by all three methods before a significant rise of CMV IgG antibody titre was demonstrated. In these patients CMV IgA was detected by RIA earlier than by ELISA and IPA. The potential application of CMV-specific IgA antibody determination for early detection of recurrent CMV infection in renal transplant patients is discussed.     

Comparison of rabies humoral antibody titers in rabbits and humans by indirect radioimmunoassay, rapid-fluorescent-focus-inhibition technique, and indirect fluorescent-antibody assay.

Rabies humoral antibodies were induced in eight New Zealand rabbits by a single intramuscular injection of inactivated suckling mouse brain rabies vaccine. The primary response to immunization was measured in blood samples taken at selected intervals for 6 months. The anamnestic response was measured in blood samples obtained 2 weeks after the rabbits received a booster immunization. The humoral antibody concentrations were measured by the rapid-fluorescent-focus-inhibition technique (RFFIT), indirect fluorescent-antibody assay (IFA), and indirect radioimmunoassay (RIA). The maximal neutralizing antibody titers as measured by RFFIT were attained by the 4th week and persisted into the 24th week. After booster immunization the antibody response was almost 10-fold higher than the highest level attained in the primary response. The antibody levels as measured by IFA and RIA were similar, but the titers as measured by either procedure were almost 10-fold lower than those determined by RFFIT. After booster immunizations the antibody levels, as measured by IFA and RIA, were three- and sixfold higher, respectively, than the maximal levels attained in the primary response. Twenty-two human serum specimens were tested by the same serological procedures, with disparate results. Both RIA and RFFIT effectively differentiated antirabies-positive sera from antirabies-negative sera.     

Virus-specific IgA in serum, saliva, and tears of children with measles.

Measles-specific IgA antibody titres were determined by radioimmunoassay (RIA) for serial serum, saliva and tear samples obtained from 21 children with measles infection, from onset of rash until up to 14 months later. Serum IgA titres rose rapidly after onset of illness and remained detectable throughout the follow-up period. Virus-specific salivary IgA titres peaked at 4 to 7 days after onset of rash and decreased thereafter. Measles-specific lacrimal fluid IgA antibodies remained elevated for long periods of time; however, secretory component-bearing measles-specific antibodies in tears became for the most part undetectable by 1 month after onset of rash. These data raise anew the question of whether some form of viral latency is associated with the presence of virus-specific IgA antibody, or whether such antibody is simply a reflection of immune memory.     

Immunoblot analysis of humoral immune responses following infection with Bordetella pertussis or immunization with diphtheria-tetanus-pertussis vaccine.

To help develop better diagnostic tests for pertussis, we examined the serologic response to whole-cell proteins of Bordetella pertussis after natural infection or vaccination with diphtheria-tetanus-pertussis vaccine. Serum specimens collected during a pertussis outbreak investigation and from uninfected persons were used in Western blot (immunoblot) analyses to determine the presence of immunoglobulin G (IgG) and IgA antibodies to specific B. pertussis proteins. IgG antibodies to proteins of molecular masses 220 and 210 kilodaltons (kDa) were detected in 14 of 18 serum samples obtained from patients with culture-confirmed pertussis greater than or equal to 40 days after the onset of coughing. IgA antibodies were detected in 15 of the 18 samples. Of 19 serum samples obtained from patients who had not been ill with pertussis, 6 contained IgG antibodies to these proteins and 1 contained IgA antibodies. The two proteins bound antiserum specific for filamentous hemagglutinin and comigrated with purified filamentous hemagglutinin. IgG antibodies to two additional protein bands of molecular masses 84 and 75 kDa were associated with previous vaccination. Antibody to the 84-kDa protein was detected in 15 of 17 vaccinated, never-infected persons, and antibody to the 75-kDa protein was detected in 16 of the 17. None of 11 nonvaccinated, never-infected persons tested had antibodies to either protein. All seven fully vaccinated persons with culture-documented infection had antibodies to both proteins. Antibodies to the 84-kDa protein were detected in 6 of 22 nonvaccinated and infected persons, and antibodies to the 75-kDa protein were detected in 8 of the 22. Use of Western blot analysis in this study allowed us to distinguish antibody responses to infection and immunization.     

The immunostimulating complex (ISCOM) is an efficient mucosal delivery system for respiratory syncytial virus (RSV) envelope antigens inducing high local and systemic antibody responses

ISCOM is an efficient mucosal delivery system for RSV envelope proteins as measured by antibody responses in respiratory tract secretions and in sera of mice following two intranasal (i.n.) administrations. Intranasally administered RSV ISCOMs induced high levels of IgA antibodies both in the upper respiratory tract and in the lungs. In the lungs, a prominent and long-lasting IgA response was recorded, which still persisted 22 weeks after the second i.n. immunization when the experiment ended. Subcutaneous (s.c.) immunization only induced low IgA titres in the upper respiratory tract and no measurable response to RSV was found in the lungs. Differences were also noticed in serum between the i.n. and s.c. modes of immunization. ISCOMs given intranasally induced earlier, higher and longer lasting IgM and IgG1 serum anti-RSV antibody responses than those induced by the s.c. mode of administration. A low serum IgE response was only detectable at 2 weeks after i.n. immunization with ISCOMs and after s.c. immunization with an inactivated virus, but no IgE response was detectable after s.c. injection of ISCOMs. The serum IgA response was more pronounced following s.c. injection of inactivated virus than after i.n. application of ISCOMs, and a clear-cut booster effect was obtained with a second immunization. Virtually no serum IgA response was detected after the s.c. administration of ISCOMs. In conclusion, the high immune responses induced by RSV ISCOMs in the respiratory tract and serum after i.n. administration indicate prominent mucosal delivery and adjuvant properties of the ISCOMs, warranting further studies.     

Specific IgM antibodies in the saliva of mumps patients

Antibodies to mumps virus were detected in 63.5% of saliva samples from mumps patients. The secretory antibody response was of primary type. Specific IgM antibodies were found in some samples collected early after the onset of disease. Specific IgA were detected in later obtained samples. Persons over 15 years of age reacted more often and more promptly than the children. The authors discuss the possible significance of prior antigenic stimulation by related paramyxoviruses (namely parainfluenzaviruses) for the intensity of local antibody response to mumps virus infection.     

甲型肝炎减毒活疫苗及灭活疫苗灌胃免疫小鼠后的抗体应答

目的 :测定甲肝减毒活疫苗及灭活疫苗灌胃免疫小鼠后的抗体应答效应。方法 :将活或灭活的甲肝疫苗加或不加明胶 ,经胃免疫小鼠 ,于末次免疫后 2w取血清及肠液 ,用间接ELISA法分别检测其中的特异性IgG和IgA抗体水平 ,并与空白及肌注组比较。结果 :实验组特异抗体水平明显高于肌注组 (P <0 0 1) ;加明胶组的免疫效果较不加者好 (IgG :P <0 0 0 1,IgA :P <0 0 5 )。结论 :甲肝活疫苗及灭活疫苗经消化道免疫小鼠后 ,均可诱导全身及局部的抗体应答 ;明胶有增强抗体产生的作用 ,可作为一种安全、廉价的粘膜佐剂被进一步开发与利用。     

Platelia-Toxo IgA, a new kit for early diagnosis of congenital toxoplasmosis by detection of anti-P30 immunoglobulin A antibodies.

With the aim of achieving earlier diagnosis of congenital toxoplasmosis, anti-P30 immunoglobulin A (IgA) antibodies were assayed by using a Platelia-Toxo IgA kit with samples from 72 children born to mothers who seroconverted during pregnancy. A total of 148 serum samples and 1 cerebrospinal fluid samples were from 23 congenitally infected children (2 serum samples were collected from fetuses), and 74 serum samples were from 49 uninfected children. Among the 23 infected children, anti-P30 IgA antibodies were present in all infants either at birth or in the following weeks, whereas anti-P30 IgM antibodies were present in 13 from the 23 infected children either at birth or in the following weeks. Serum samples collected in utero from two infected children were also tested. One of these samples was positive for both anti-P30 IgA and anti-P30 IgM antibodies, whereas both children were negative at birth for these antibodies. Neither anti-P30 IgA nor anti-P30 IgM antibodies were detected in 47 of 49 uninfected children. These results suggest that detection of anti-P30 IgA antibodies by the Platelia-Toxo IgA kit is a very effective method for early diagnosis of congenital toxoplasma infection.     

Influence of maternal murine immunization with Dermatophagoides pteronyssinus extract on the type I hypersensitivity response in offspring

BACKGROUND: Maternal exposure to environmental ubiquitous allergens could exert an influence on the newborn's immune repertoire and the later development of allergy. The aim of this study was to investigate the effects of maternal immunization with Dermatophagoides pteronyssinus (Dp) on the hypersensitivity response and IgG subclass production in offspring using a murine model. METHODS: A/Sn mice were immunized with Dp before mating with normal A/Sn males. Diaplacental serum samples were collected from newborn mice delivered by cesarean section, and maternal milk samples were extracted from the stomachs of newborn mice. Groups of offspring 25 or 45 days old were Dp immunized and boosted on the 10th day after sensitization. The animals were bled 7 days after the booster. RESULTS: High levels of anti-Dp IgG subclasses - mainly IgG1, but also IgG2a and IgG2b - were transmitted by immunized mice via the placenta to the offspring. In the milk from immunized mothers, significant levels of anti-Dp IgG subclasses and anti-Dp IgM and IgA antibodies were detected. Moreover, the increase in total IgA antibodies in the milk of the immunized females correlated with a significantly increased level of TGF-beta1. TGF-beta2 levels were markedly higher than the beta1 isoform in the milk, although no difference was observed between the groups. When offspring from immunized mothers were sensitized at 25 days, a significant decrease in total and anti-Dp IgE antibodies as well as total and anti-Dp IgG1, IgG2a and IgG2b subclasses was observed compared to normal female offspring, whereas when offspring were sensitized at 45 days, both offspring groups showed similar levels of IgE and IgG subclasses. CONCLUSIONS: Our study showed that maternal immunization with Dp promotes the transference of specific antibodies and/or TGF-beta, which can negatively modulate the allergic response in offspring, and suggests that maternal preexposure to allergen before mating can protect mice during the early phase.     

Antibody responses in arthritic and uncomplicatedYersinia enterocolitica infections

Concentrations of antiyersinia antibody isotypes IgG1, IgG2, IgG3, IgG4, IgA and IgM were measured in 33 patients with yersiniosis using a solid-phase radioimmunoassay. Sixteen patients had a complicating reactive arthritis. Throughout the observation period IgG1 and IgM antibodies both constituted approximately one-third of the total antibodies, while IgA accounted for 10%, IgG3 accounted for 1%, and IgG4 antibodies could not be detected. IgG1, IgM, and IgA antibodies (and the total titer) had reached their peak at the beginning of the observation period (ca. day 20 after the onset of symptoms). The levels then gradually decreased; the total titers averaged 40 times the background at the beginning of the observation period and 4 times the background on day 350. IgM antibodies could be detected as late as a year after the infection. The concentration of IgG2 antibodies varied greatly from patient to patient. In most patients it increased until a plateau was reached approximately 2 months after the onset of symptoms. A decline was observed later. Five arthritic but no nonarthritic patients had a pronounced IgG2 response (more than half of the IgG antibodies were IgG2 in one or several samples).     

Humoral immunity against Francisella tularensis after natural infection

Forty-two subjects with acute tularemia were studied for the occurrence of C-reactive protein (CRP), and 73 subjects with acute tularemia or experience of the disease within the last 11 years were studied for immunoglobulin M (IgM), IgA, and IgG class-specific antibodies, agglutinating antibodies, and complement-fixing antibodies to Francisella tularensis by using an enzyme-linked immunosorbent assay (ELISA), the tube agglutination test, and a complement-fixing ELISA. The incubation time between infection and the outbreak of symptoms varied from 1 to 10 days, averaging 6.5 days. Elevated CRP concentrations were found in all samples taken in the first 6 days of illness, when the antibodies generally were absent. The highest CRP values, up to 165 mg/liter, occurred in the earliest samples and then decreased rapidly, being undetectable (less than 1 mg/liter) from 1 month after the onset of symptoms. Simultaneous though individually varying formation of IgM, IgA, and IgG class-specific antibodies to F. tularensis was demonstrable by ELISA in all the tularemia patients during the acute stage. In most cases, these antibodies appeared 6 to 10 days after the onset of symptoms, i.e., about 2 weeks after infection, reached their highest values at 4 to 7 weeks, and, despite a decreasing trend in their level, were still present 0.5 to 11 years after onset of tularemia, as demonstrable by the agglutination test and by the complement-fixing ELISA. Of the three methods used, ELISA for IgM, IgA, and IgG proved to be the most efficient for the early serodiagnosis of tularemia.     

Use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection

An indirect immunofluorescent assay (Euroimmun AG, Luebeck, Germany) was used to investigate the avidity of immunoglobulin G (IgG), IgM, IgA, and total Ig (IgGAM) antibody responses to severe acute respiratory syndrome coronavirus (SARS CoV) infections. Serial serum samples from eight patients collected during the first, third, and ninth months after the onset of infection were evaluated. It was found that low-avidity IgG antibodies were detected in 15/15 (100%), 1/5 (20%), and 0/8 (0%) serum samples collected during the first, third, and ninth months after the onset of symptoms, respectively. Low-avidity antibodies of IgA and IgM subclasses were detected in 14/14 (100%) and 3/14 (21%) serum samples, respectively, collected in the first month after the onset of infection. However, IgA antibodies remained low in avidity in a proportion of patients even during late convalescence. As a consequence, IgG antibody avidity assays gave better discrimination between acute-phase and late-convalescent-phase serum samples than IgM, IgA, or IgGAM assays. In two of these patients, sequential serum samples were also tested for IgG avidity against human CoV strains OC43 and 229E in parallel. While SARS CoV infections induced an anamnestic IgG antibody response to the 229E and OC43 viruses, these cross-reactive antibodies remained of high avidity from early (the first month) postinfection. The results showed that assays to detect low-avidity antibody may be useful for discriminating early from late antibody responses and also for distinguishing anamnestic cross-reactive antibody responses from primary specific responses. This may be useful in some clinical situations.     

Cholera antibody production in vitro by peripheral blood lymphocytes following oral immunization of humans and mice.

We have studied specific antibody production from peripheral blood lymphocytes (PBL) after oral cholera immunization of humans and mice. Two oral immunizations with cholera toxin (CT) in mice or a single dose of the combined cholera B-subunit/whole cell vaccine in humans gave rise to PBL which spontaneously secreted cholera-specific antibodies when cultured in vitro. A high proportion of IgA antibodies was seen in contrast to antibodies produced by PBL after parenteral immunization which were predominantly IgG. Cultured PBL produced antitoxin as well as anti-lipopolysaccharide antibodies after oral immunization, whereas serum only revealed titre rises for anti-CT. Antibody-secreting PBL appeared in the blood 2-4 days after immunization and persisted for about two weeks with a peak after 6-8 days. Mitogen stimulation in vitro of PBL from multiply-orally vaccinated humans activated a population of specific IgM antibody-secreting cells which persisted for several months following immunization, suggesting the presence of long-lived memory cells. The analysis of IgA antibody production from in-vitro cultured PBL seems to be a promising technique to assess the local immunogenicity of oral vaccines.     

The humoral immune response of mice to intra-mammary immunization with ovalbumin.

Groups of lactating mice were immunized intra-mammarily on the second day of lactation with 20 micrograms, 150 micrograms or 400 micrograms of ovalbumin (OVA). This resulted in the appearance of IgG in serum, and IgA and IgG in milk. In serum, no IgA antibodies were detected 16 days after immunization in any of the groups. The serum response of IgG was variable and not related directly to the immunizing dose. Both IgA and IgG antibodies were absent in milk 5 days after immunization and IgG antibody level in milk increased significantly throughout lactation as measured 10 and 15 days after inoculation. No IgA antibodies appeared in the milk of the 20 micrograms and 150 micrograms group; however, responses appeared in milk with the highest dose (400 micrograms), but the number of responders for IgG increased in milk but not in blood. The results suggest that intra-mammary immunization can provoke a local IgA response in milk, and that serum is not a major source of IgG in that fluid. Moreover, the kinetics of the IgA and IgG responses differ.     

Antibody avidity following varicella-zoster virus infections.

The avidity of IgG antibodies following varicella-zoster virus (VZV) infections was investigated using urea treatment of antigen-bound serum antibody by indirect radioimmunoassay (RIA) and immunoblotting techniques. Sequential sera from 16 patients with varicella and 17 patients with zoster were tested, as well as sera from 80 seropositive individuals without a recent history of VZV disease. Both types of assay showed that low-avidity antibodies predominate early after primary infection, but that antibody avidity increases markedly during convalescence. Using RIA, all sera taken up to 12 weeks after the onset of varicella showed greater than 50% reduction in antibody titre after treatment with 8 M urea but thereafter the proportion of urea resistant antibody increased with time. In contrast, after recurrent infection, high avidity antibodies were found to predominate at all times. Only 6 of 47 sera tested from zoster cases showed greater than 30% reduction after urea treatment and all these were taken within 2 weeks after onset of rash. Immunoblotting also showed that the highly immunogenic p32/p36 nucleoproteins appear to induce predominantly low avidity antibodies, even after recurrent VZV infection. The results of this study indicate that treatment with 8 M urea in RIA for IgG antibodies may be a simple and reliable method for distinguishing primary and anamnestic antibody responses against VZV.     

Secretory immune responses to Mycoplasma pulmonis.

Formalinized Mycoplasma pulmonis, along with aluminum hydroxide as an adjuvant, was used to subcutaneously immunize rats in the vicinity of the salivary gland to examine the characteristics of the secretory immune response to this pathogen. The induction of specific antibody to this microorganism was detected in serum and the exocrine fluids, namely, saliva and lung lavage fluid. Both immunoglobulin G (IgG) and IgA isotype antibodies were detected in each of these fluids after primary and secondary local immunizations. Serum responses from immunized animals were significantly greater than in the control group, but a dose response was not observed in either IgG or IgA antibody at the dosages selected for immunization. Salivary IgG antibody responses peaked early after both the primary and secondary immunizations, exhibiting a clear dose response. Salivary IgA in immunized groups was significantly greater than that in the control group but displayed little dose-dependent kinetics, and, at the termination of the experiment, this response had not yet peaked. Lung lavage IgG and IgA were minimal after the primary immunization when the antibody was normalized to total protein but displayed dose-dependent kinetics after a secondary challenge. IgG peaked immediately after a secondary challenge, while IgA peak responses were observed only after 20 days. A positive correlation was noted between the serum, saliva, and lung lavage fluid IgGs after both primary and secondary immunizations and only after a secondary challenge for IgA. In this study we were able to elicit a secretory immune response, consisting of both IgG and IgA, which exhibited a dose-dependent characteristic in lung lavage fluid to this immunogen. Additionally, a positive correlation of antibody levels between saliva and lung lavage fluid suggests that saliva could be used as an indicator for monitoring specific antibody to M. pulmonis in lung lavage secretions without requiring invasive, deleterious procedures.