The role of IgA immunoglobulins in the passive transfer of protection to Taenia taeniaeformis in the mouse.

Normal mice were protected against infection with metacestodes of Taenia taeniaeformis when administered intestinal, colostral or serum immunoglobulins obtained from adult mice previously orally infected with the parasitie. The protective capacity of these preparations was found to be associated mainly with IgA of colostrum and intestinal secretions and IgG of serum. The removal of IgA and IgG from immune colostrum and serum, respectively, abolished the protective effect. Neonatal mice were protected against infection with T. taeniaeformis when fed purified colostral IgA and serum IgG from immune donors. The intraduodenal injection of intestinal IgA from immune donors into 4-week-old mice passively protected the recipients against infection with T. taeniaeformis, but intestinal IgG from immune donors had no protective effect when given in this manner. The protective capacity of IgA and IgG was largely eliminated by prior absorption with T. taeniaeformis antigen or hatched, activated oncospheres of T. taeniaeformis.     

IgG subclass distribution of antibodies against S. aureus teichoic acid and alpha-toxin in normal and immunodeficient donors

IgM, IgG, IgA and IgE class and IgG and IgA subclass levels were determined in 18 IgG2 deficient and six IgG3 deficient donors. IgG2 deficiency was associated with concomitant IgG4, IgA (in particular IgA2) and IgE deficiency. This pattern is compatible with a regulation defect of the downstream switch in the heavy chain locus on chromosome 14. IgG3 subclass deficiency was not associated with further deficiencies. Specific anti-teichoic acid antibodies were lacking in most IgG2 deficient donors supporting the notion that anti-teichoic acid antibodies are normally of this subclass. This was also confirmed in a subclass-specific ELISA using sera from normal donors although substantial amounts of specific IgG1 antibodies were also noted. Two IgG2 deficient donors had normal IgG titres (IgG1 in the subclass specific ELISA) and the lack of IgG1 anti-teichoic acid antibodies in most IgG2 deficient donors may suggest a lack of maturation of the appropriate idiotype. IgG antibodies to alpha-toxin, a pure protein, were within the lower normal range in a large proportion of IgG2 deficient donors but largely normal in the IgG3 deficient donors.     

Immunoglobulin subclass distribution of human anti-carbohydrate antibodies: aberrant pattern in IgA-deficient donors

The IgG and IgA subclass distribution of anti-carbohydrate antibodies in normal and immunodeficient donors was investigated. In normal donors, the specific anti-dextran antibodies were mainly of the IgG2 and IgA2 subclass, although substantial amounts of antibodies could also be of the IgG1 subclass. In children, IgG1 was the predominant subclass expressed. An aberrant IgG subclass distribution pattern of specific antibodies occurred in some IgA-deficient donors, with preferential expression of IgG1 and IgG3 anti-dextran antibodies.     

Differentiation of coeliac disease and other malabsorption diseases using specific serum antigliadin IgG subclass profiles and IgA1 levels.

The differential diagnostic potential of serum gliadin-specific IgG subclass antibodies was assessed by comparing the antigliadin IgG1, 2, 3, 4 profile at different stages of coeliac disease with that of gastro-intestinal infection and also conditions associated with increased intestinal permeability. The IgG subclass profile of untreated coeliac disease was found to be the same as in healthy controls (IgG1 approximately IgG2 > IgG3 > IgG4), with only the magnitude of the individual subclass responses being increased in coeliac patients. Coeliac adults and children on gluten-free diets had different antigliadin IgG subclass profiles with IgG2 being elevated. Increased intestinal permeability or recent gastro-intestinal infection did not alter the antigliadin subclass profile from that observed in healthy individuals. Assessment of the diagnostic potential of antigliadin IgA1 and IgG1-4 measurements in screening for coeliac disease demonstrated that measurement of subclasses of gliadin-specific IgA and IgG was less sensitive and specific compared with the combined use of total antigliadin IgA and IgG. Therefore it is suggested that IgG subclasses should not be used for routine screening for coeliac disease.     

Terminally Differentiated Human Intestinal B Cells

The relative distribution of IgA and IgG subclass-producing immunocytes was examined by two-colour immunohistochemistry in normal human distal ileum including Peyer's patches (PP), regional mesenteric lymph nodes (MLN), and peripheral lymph nodes. IgA2 cells predominated slightly over IgA1 cells in the PP dome area. There was a decreasing median proportion of IgA2 cells in the order of PP (52%), distant ileal lamina propria (40%), MLN (32%), and peripheral lymph nodes (11%). The reverse was true for IgA1 cells in independent enumerations. These results support the notion that PP-derived B cells after stimulation are seeded mainly to the lamina propria of the distal gut, but that there is a substantial retention and terminal differentiation of this migrating population in regional MLN. The median subclass proportions of IgG-producing cells in the PP dome area were in independent determinations 68% IgG1, 23% IgG2, 8% IgG3, and 9% IgG4. This distribution was fairly similar to that seen in other tissue categories, except for a trend towards increased IgG1 and reduced IgG2 proportions in peripheral lymph nodes and reduced IgG1 along with increased IgG3 in normal palatine tonsils. The data suggested an association between the expression of IgG2 (and possibly IgG4) and IgA2 in intestinal mucosal immune responses.     

Small intestine in lymphocytic and collagenous colitis: mucosal morphology, permeability, and secretory immunity to gliadin.

There is a recognised association between the "microscopic" forms of colitis and coeliac disease. There are a variety of subtle small intestinal changes in patients with "latent" gluten sensitivity, namely high intraepithelial lymphocyte (IEL) counts, abnormal mucosal permeability, and high levels of secretory IgA and IgM antibody to gliadin. These changes have hitherto not been investigated in microscopic colitis. Nine patients (four collagenous, five lymphocytic colitis) with normal villous architecture were studied. Small intestinal biopsies were obtained by Crosby capsule; small intestinal fluid was aspirated via the capsule. IEL counts were expressed per 100 epithelial cells, and intestinal IgA and IgM antigliadin antibody levels were measured by ELISA. Small intestinal permeability was measured by the lactulose:mannitol differential sugar permeability test. IEL counts were normal in all cases, median 17, range 7-30. Intestinal antigliadin antibodies were measured in six cases and were significantly elevated in two patients (both IgA and IgM). Intestinal permeability was measured in eight cases and was abnormal in two and borderline in one. These abnormalities did not overlap: four of nine patients had evidence of abnormal small intestinal function. Subclinical small intestinal disease is common in the two main forms of microscopic colitis.     

High prevalence of celiac disease in healthy adults revealed by antigliadin antibodies.

Sera from 1866 healthy blood donors and from 40 untreated adults with celiac disease were analyzed using a micro-ELISA assay. Blood donors with IgA antigliadin activity greater than 40 units corresponding to the 96.8th percentile and IgG antigliadin activity greater than 20 units corresponding to the 91.3rd percentile were selected for further investigation and jejunal biopsy. Seven of 49 blood donors with high IgA antigliadin activity showed mucosal lesions typical for celiac disease. None of the donors with high IgG antigliadin activity (35 subjects) but without high IgA activity had such mucosal lesions. A prevalence of celiac disease of at least 1/256 was observed in the donor group. There were significant age-group differences in IgA antigliadin activity. In the present study, a high IgA antigliadin activity had a positive predictive value between 18% and 25% in individuals without symptoms indicative of celiac disease depending on the way the cut-off points were chosen. In contrast, the positive predictive value of high IgG antigliadin activity alone was estimated to be 0%.     

The origin of immunoglobulins in semen

IgG and IgA, albumin and lactoferrin as well as the semen compartment parameters acid phosphatase, fructose and spermatozoa were determined in separately collected fractions of the same ejaculate of some normal donors. The distribution over the fractions per ejaculate of IgG, IgA and albumin was generally more or less similar to the distribution of acid phosphatase indicating that the bulk of these proteins enters the semen via the prostate and not via the vesicles or testis and epididymis. The distribution of lactoferrin unexpectedly was not clearly related to fructose. IgM could not be detected.

The concentrations in the (eight) total ejaculates expressed as percentages of the serum concentrations were for albumin slightly higher than for IgG, both in the order of 1% and moreover correlated with each other, indicating that IgG reached the seminal fluid in general by transudation from the circulation. The relative IgA concentrations could not be measured exactly but seemed to be slightly higher than of albumin, and not correlated to albumin concentrations, suggesting that local production of IgA may occur also.

     

IgG-, IgA-, and IgE-induced release of leukotriene C4 by monocytes isolated from patients with atopic dermatitis

Purified peripheral blood monocytes isolated from patients with atopic dermatitis (AD) and from nonallergic normal donors were compared for their abilities to release leukotriene C4 (LTC4), leukotriene B4 (LTB4) and beta-glucuronidase in response to challenge with aggregated immunoglobulins or anti-immunoglobulins. The relationship between mediator release and the number of monocytes that formed rosettes with immunoglobulin-coated indicator cells was examined. Patients with AD had twice as many IgA- and three times as many IgE-rosetting monocytes as normal donors (48 +/- 12% versus 27 +/- 10% and 40 +/- 15% versus 14 +/- 3%, respectively), and yet the amounts of IgA- and IgE-induced LTC4 released were similar for both groups. This apparent discrepancy did not result from a decreased capacity for arachidonate metabolism via the C5-lipoxygenase pathway, since stimulation of monocytes from patients and normal donors with the calcium ionophore A23187 induced similar amounts of LTC4 and LTB4 release (LTC4, 3.0 +/- 1.7 versus 3.0 +/- 1.0 ng/10(6) cells; LTB4, 5.3 +/- 0.7 versus 5.2 +/- 0.5 ng/10(6) cells, respectively). In addition, aggregated IgG-induced LTC4 release by monocytes of both groups was similar, concomitant with an equivalent number of IgG-rosetting cells. Determination of cytophilically bound IgG and IgE by flow cytometry demonstrated that monocytes from atopic patients had more IgG bound than monocytes from normal donors. Similar amounts of IgE were detected on most monocytes from both groups, despite the higher serum IgE levels of patients. However, approximately 3% to 8% of monocytes from atopic but not normal donors stained brightly for IgE, suggesting that relatively large amounts of cytophilic IgE were bound to a small percentage of the patients' monocytes. Challenge of monocytes with anti-IgE or anti-IgG induced release of similar amounts of LTC4 for both groups, despite the presence of more cytophilic IgG on monocytes from atopic donors. These data indicate that monocytes from patients with AD release LTC4 and LTB4 in response to challenge with aggregated IgE or anti-IgE, as well as aggregated IgG, IgA, and anti-IgG. However, under our in vitro conditions, stimulation of patients' monocytes with aggregated IgA or IgE was not associated with increased mediator release, despite higher percentages of IgA- and IgE-rosetting cells compared to normal donors.     

Ongoing subclinical infection of hepatitis E virus among blood donors with an elevated alanine aminotransferase level in Japan

Ongoing subclinical infection of hepatitis E virus (HEV) has not been fully studied. In the present study, serum samples were collected from 6700 voluntary blood donors with an elevated alanine aminotransferase (ALT) level of 61-476 IU/l at a Japanese Red Cross Blood Center, and were tested for the presence of IgG, IgM and IgA classes of antibodies to HEV (anti-HEV) by in-house ELISA and HEV RNA by nested RT-PCR. Overall, 479 blood donors (7.1%) were positive for anti-HEV IgG, including 8 donors with anti-HEV IgM and 7 donors with anti-HEV IgA. Among the nine donors with anti-HEV IgM and/or anti-HEV IgA, six had detectable HEV RNA. The presence of HEV RNA was further tested in 10-sample minipools of sera from the remaining 6691 donors, and three donors including one without anti-HEV IgG were found to be positive for HEV RNA. When stratified by ALT level, the prevalence of HEV RNA was significantly higher among the 109 donors with ALT > or = 201 IU/l than among the 6591 donors with ALT of 61-200 IU/l (2.8% vs. 0.1%, P < 0.0001). The HEV isolates obtained from the nine viremic donors segregated into genotype 3, shared a wide range of identities of 85.6-98.5% and were 87.3-93.9% similar to the Japan-indigenous HEV strain (JRA1), in the 412-nucleotide sequence of open reading frame 2. This study suggests that approximately 3% of Japanese individuals with ALT > or = 201 IU/l have ongoing subclinical infection with various HEV strains.     

The critical role of allergen-specific IgE,IgG4 and IgA antibodies in the tolerance of IgE-mediated food sensitisation in primary school children

Primary objective. There are about 6% of children who are either intolerant to or lose their ability to tolerate food allergens, resulting in the development of food hypersensitivity. The hypothesis that increase in food allergen-specific IgE antibody level is associated with the decrease in the levels of food allergen-specific IgG4 and IgA antibodies was used as a biomarker of food tolerance. Methods & Procedures. The Modified International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire (added gastrointestinal allergy questions) and Phadiatop infant test were used to screen one hundred 6–8-year-old allergic school children. Food allergen-specific IgE, IgG and IgA antibodies were measured by using the Phadia ImmunoCAP system radioabsorbent test (RAST). Immunoglobin E antibodies to common aeroallergens, were also detected by enzyme-linked immunosorbent assay. Main outcome. The level of analysed food specific-IgE antibody was obviously higher in the study population. Sensitivity to dust mites among the children was nearly 90%, and that to cockroach was 47%. Egg white-, cow's milk-, α-lactoalbumin-, β-lactoglobulin- and casein-specific IgG4/IgE and IgA/IgE ratios were lower in the atopic school children but not in the tropomysin-, mango- and kiwi-sensitive participants. Conclusion. The level of cow's milk- and egg white-specific IgE antibody still remained high along with a decrease in the specific IgG4/IgE and IgA/IgE ratios in our study population. Therefore, allergen-specific IgG4 and IgA antibodies are important biomarkers of tolerance establishment, and failure to establish tolerance to food allergens may be related to the regulation of the inhalant allergens encountered in late childhood stages.     

Analysis of IgG subclass production in cell cultures from IgA deficient patients and in normal controls as a function of age

IgA deficient individuals may also have low serum levels of IgG subclasses, especially IgG2. In the present study we examined the development of plasma cells producing IgM, IgA or IgG, and the IgG1 and IgG2 subclasses, following lipopolysaccharide (LPS) and pokeweed mitogen (PWM) stimulation of mononuclear cells (MNC) from normal and IgA deficient individuals as a function of age. Studies of blood MNC from 38 normal donors (age range 2-44 years) revealed an age-related distribution pattern of mu, gamma, alpha, gamma 1 and gamma 2 plasma cells produced in mitogen-stimulated and control cultures. Decreased IgA responses to both LPS and PWM were consistently observed in cultures of MNC from all of the nine children with IgA deficiency. When compared with age-matched controls the IgG response was also diminished in PWM stimulated cultures, whereas the IgM responses were normal. The IgG deficit was due to reduced responses for the gamma 1 and gamma 2 subclasses, and was most pronounced for IgG2; IgG2 plasma cell differentiation was particularly depressed in LPS cultures. In contrast to normal adult cells, blood MNC from the nine children with IgA deficiency and age-matched controls (2-17 years) yielded more IgG1 than IgG2 plasma cells in both control and LPS cultures, while the pattern of response to PWM was similar in all groups (gamma 1 greater than gamma 2). A good concordance was found between the level of secreted Ig in the culture supernatants and the relative number of IgM or, IgG and IgA plasma cells identified by immunofluorescence staining of cytoplasmic immunoglobulins.     

Functional assessment of a B cell defect in patients with selective IgA deficiency

The cellular basis of selective IgA deficiency was investigated by examining the terminal differentiation of B lymphocytes co-cultured with varying ratios of T lymphocytes in the presence of pokeweed mitogen. Eight patients were studied who had serum IgA concentrations <0·05 mg/ml, salivary IgA <0·01 mg/ml, and between 0·8 and 4% lymphocytes with surface IgA markers. Peripheral blood lymphocytes from patients and normal donors were separated into B cell (non T cell) and T cell fractions by E-rosetting. Microcultures were established at eleven B cell to T cell ratios from 100% B cells to 100% T cells. After 7 days, immunoglobulin in the supernatant fluid was measured by radioimmunoassay. Cultures containing patients' B cells and either autologous or allogeneic T cells produced very low or undetectable amounts of IgA. However, cultures from six out of eight patients contained cells with intracytoplasmic IgA. Secretion of IgM by the patients' B cells was identical to that of normal donors. Surprisingly, IgG production by patients' B cells was less than that produced by normal B cells especially in the mid-range ratios of the microcultures. Production of IgA, IgG and IgM by normal B cells from peripheral blood or tonsils was very similar in the presence of normal T cells or patients' T cells. In cultures containing optimal ratios of normal B cells, the patients' T cells not only did not suppress IgA production but also gave normal help for IgA production. It was concluded, on the basis of these studies, that a defect in patients with selective IgA deficiency was the functional inability of their B cells to produce normal amounts of IgA in vitro even when provided with normal allogeneic T cell help.     

Immunoglobulin subclass distribution of synovial plasma cells in rheumatoid arthritis determined by use of monoclonal anti-subclass antibodies

The distribution of IgG and IgA subclass plasma cells among dissociated synovial cells from 14 rheumatoid arthritis (RA) synovia was examined by immunofluorescence using mouse monoclonal anti-human subclass antibodies. Of the IgG plasma cells 81 +/- 9% were IgG1, 4 +/- 2% IgG2, 14 +/- 9% IgG3, and 0.9 +/- 0.6% IgG4. While IgG1 predominated in all 14 synovia (which is similar to what is seen in normal tissues), in 5/14 20% or greater of IgG plasma cells were IgG3, suggesting a selective increase in IgG3 production in the synovia of certain RA patients. Among IgA plasma cells 89 +/- 5% were IgA1 and 8 +/- 3% were IgA2. This distribution is similar to the distribution in normal serum.     

Terminally differentiated human intestinal B cells. J chain expression of IgA and IgG subclass-producing immunocytes in the distal ileum compared with mesenteric and peripheral lymph nodes

Two-colour immunofluorescence staining for intracellular J chain and IgA (or J chain and IgG) was performed on tissue sections of normal human ileal mucosa (eight adult kidney donors), mesenteric lymph nodes (MLN), peripheral lymph nodes, and palatine tonsils. The most prominent J chain positivity was seen for IgA (97.3%) and IgG (81.7%) immunocytes in the ileal lamina propria (LP). Moreover, the proportion of J chain-expressing extrafollicular immunocytes was significantly higher (P less than 0.05) in MLN than in peripheral lymph nodes for the IgA class (58.5% versus 25.6%); the same proportion for the IgG class was 45.9% versus 30.4%. In clinically normal palatine tonsils of adults, extrafollicular J chain expression was much lower than in peripheral lymph nodes; 14.2% for IgA cells and 5.5% for IgG cells. When related to subclass production, J chain expression was found to be higher for IgA2 than for IgA1 cells in all tissues examined (palatine tonsils excluded because of a small number of IgA2 cells), the difference being significant in MLN and ileal LP (P less than 0.05). The J chain positivity tended to be higher for all IgG subclasses in MLN than in peripheral lymph nodes; this difference was significant (P less than 0.05) for IgG2-producing immunocytes. Taking J chain expression as a marker of clonal immaturity, our results may reflect to some extent distribution of newly generated memory B cell clones from gut-associated lymphoid tissue to MLN, peripheral lymph nodes, and palatine tonsils in a strikingly decreasing order.     

Increased spontaneous secretion of rheumatoid factor by intestinal lamina propria mononuclear cells from Crohn's disease but not ulcerative colitis patients.

Increased levels of rheumatoid factors (RF) have been observed in the serum of Crohn's disease but not ulcerative colitis patients, and have been proposed to relate to an increased state of intestinal lymphocyte activation. We have therefore examined the spontaneous in vitro secretion of RF by intestinal lamina propria mononuclear cells (MNC) isolated from specimens from control and inflammatory bowel disease (Crohn's disease, ulcerative colitis) patients. Normal intestinal lamina propria MNC spontaneously secrete rheumatoid factors of different isotypes during 14 days of in vitro culture (9.7 ng/ml IgA RF, 11.6 ng/ml IgM RF and 64.6 ng/ml IgA anti-Fc (IgG)). In matched studies intestinal MNC isolated from normal large bowel exhibited significantly greater levels of RF synthesis and secretion in vitro than normal small bowel intestinal MNC. A large increase in spontaneous RF secretion was observed from Crohn's disease intestinal MNC (21.4 ng/ml IgA RF, 21.4 ng/ml IgM RF, and 108.15 ng/ml IgA anti-Fc (IgG)), when compared with normal controls. The amount of RF secreted was dependent on the amount of inflammatory activity of the bowel specimens, from which the MNC were isolated (198.3 ng/ml of IgA anti-Fc(IgG) from involved versus 50.0 ng/ml from matched non-involved tissue). Ulcerative colitis MNC released decreased amounts of RF (7.1 ng/ml IgA RF, 6.2 ng/ml IgM RF, and 42.3 ng/ml IgA anti-Fc(IgG)). These observations using isolated intestinal MNC may explain the findings of RF changes in the sera of inflammatory bowel disease patients. Our observations support the hypothesis of a heightened state of activation in normal intestinal lamina propria MNC, which is further increased in active Crohn's disease. The dissimilarities observed between Crohn's disease and ulcerative colitis may indicate fundamental differences in disease pathophysiology and will lead to further studies exploring intestinal immunoregulatory properties of RF.     

Effects of Staphylococcus aureus cell wall products (teichoic acid, peptidoglycan) and enterotoxin B on immunoglobulin (IgE, IgA, IgG) synthesis and CD23 expression in patients with atopic dermatitis.

The influence of staphylococcal cell wall products (teichoic acid, peptidoglycan) and enterotoxin B on peripheral blood lymphocytes (PBL) from patients with atopic dermatitis (AD) was investigated. The parameters studied were spontaneous and interleukin-inducible immunoglobulin (IgA, IgE, IgG) synthesis and CD23 expression. PBL from non-atopic donors served as controls. Teichoic acid and peptidoglycan induced an enhanced synthesis of IgA and IgG in normal donors. However, IgA and IgG synthesis in PBL from patients with AD was significantly suppressed by teichoic acid and enterotoxin B. The incubation of PBL from normal donors with enterotoxin B and interleukin-4 (IL-4) or IL-5 led to a significant suppression of IgA and IgG synthesis. Co-stimulation of PBL with teichoic acid or peptidoglycan and IL-4 led to a pronounced increase in IgE synthesis and CD23 expression in patients with AD. Our data indicate that cell wall products and toxins of staphylococci modulate the cytokine-dependent humoral immunity in patients with AD and may be responsible for allergic skin reactions in AD.     

Distribution of Monoclonal Antibodies in Intestinal and Urogenital Secretions of Mice Bearing Hybridoma 'Backpack' Tumours

Mice bearing IgA hybridoma 'backpack' tumours have been used to demonstrate that secretion of a single monoclonal IgA can protect against mucosal infection, but the relevance of this model to normal IgA protection is not clear. The authors analysed the distribution of specific monoclonal and total antibodies in bile, local intestinal secretions, cervical-vaginal secretions, urine and serum of mice bearing anti-cholera toxin (CT) IgA and IgG backpack tumours, with and without bile duct ligation. Backpack tumours resulted in high levels of both anti-CT and total IgA or IgG in serum, and IgA (but not IgG) in bile. Secretions recovered by absorbent filter 'wicks' from mucosal surfaces throughout the intestines of backpack tumour mice contained significant concentrations of monoclonal anti-CT IgA, but total IgA levels were as in normal mice. Neither monoclonal nor total IgA levels on mucosal surfaces were altered by bile duct ligation. Furthermore, anti-CT monoclonal IgA levels in local intestinal secretions of backpack tumour mice were comparable to specific polyclonal IgA levels previously elicited by mucosal immunization with CT. Thus, IgA-mediated protection against enteric challenge in the backpack tumour model may be a valid predictor of protection provided by natural mucosal immunization in vivo . High monoclonal IgA levels in bile, urine and the female genital tract, however, may not reflect the situation in normal immunized mice.     

A test for human cytomegalovirus-specific immunoglobulins using a modification of a commercial test kit

A technique (Ig-EIA) for the detection of CMV-specific IgG, IgM and IgA in human blood is described. Ig-EIA utilizes alkaline phosphatase-labeled goat anti-human IgG, IgM and IgA as a detection probe and CMV antigen-coated solid phase from commercial kits. Ig-EIA is compared to indirect fluorescent assay (IFA) and indirect hemagglutination (IHA) for sensitivity and specificity. On sequential samples of blood from a set of patients, Ig-EIA clearly demonstrated seroconversion in CMV-specific IgG and IgM. A test of 332 blood donors by Ig-EIA showed 177 (53%) had CMV-specific IgG and 17 (5%) had CMV IgM. Only two of the 17 donors with CMV IgM were nonreactive for CMV-IgG. The potential of CMV-IgM as an indicator of CMV infectivity is discussed.     

A method of obtaining, processing, and analyzing human intestinal secretions for antibody content

Human intestinal secretions can be readily obtained using a commercially available intestinal lavage solution. Although such secretions contained abundant protease activity, significant loss of immunoglobulins was prevented by the addition of a mixture of protease inhibitors. The total content of IgA, IgM, and IgG antibody in secretions was measured using sandwich ELISA. In the secretions of ten normal volunteers IgA was most abundant (197 micrograms/ml +/- 103 SD) followed by IgM (12.5 micrograms/ml +/- 6.8 SD) and IgG (0.24 micrograms/ml +/- 0.04 SD). The IgA in secretions was predominantly secretory IgA as shown by sucrose density centrifugation. The effect of intestinal secretions on the sensitivity of the antigen-specific ELISA was tested by adding murine myeloma IgA anti-TNP added to samples of human secretions. IgA anti-TNP activity could be detected as low as 1 ng/ml, and there was no evidence of interference with the ELISA by other constituents in the secretions. Using these methods an antigen-specific secretory IgA anti-cholera toxin B subunit response in the secretions of volunteers given an oral B subunit vaccine was readily demonstrated.