S. Ahlstedt

Antibody production by human colostral cells. I. Immunoglobulin class, specificity, and quantity.

The production of antibody by human colostral cells was assayed by the hemolysis‐in‐gel technique. When sheep erythrocytes coated with O antigens from frequently encountered Escherichia coli bacteria were used as detector cells and anti‐IgA serum was added for development, numerous plaque‐forming cells (PFC) were demonstrated in all samples tested. In contrast, plaques were rarely seen in the presence of anti‐IgG developing serum. The direct (IgM) plaques occasionally noted with both antigen‐coated and uncoated sheep erythrocytes were mainly due to the production of heterophil antibodies, since they were not formed when human erythrocytes were used as O‐antigen carriers. A strikingly high number of the colostral lymphocytes formed antibodies to the E. coli antigens, up to 8% This suggests that these cells represent a rather selective population–possibly cells from the gastrointestinal tract exposed to enteric bacteria. The large number of plaques observed, the predominance of the cells forming IgA antibodies, and the marked changes in PFC number in relationship to parturition pose a number of questions relevant to the antibody‐producing colostrum cells and their relationship to the secretory immune system

A Sensitive Method for Specific Quantitation of Secretory IgA

A method to quantitate specifically secretory IgA (SIgA) has been developed using the enzyme‐linked immunosorbent assay. The IgA in the test sample was adsorbed to anti‐α antibodies attached to plastic tubes via a coat of IgA myeloma protein. The reacted SIgA was determined using anti‐secretory component antiserum conjugated with alkaline phosphatase. The technique permitted quantiations of secretory IgA in biological fluids like milk, urine, and Saliva with reproducibility of ±7%, down to 0.03 mg/l. In contrast to earlier techniques, the presence of up to 15794 of serum IgA without secretory component (SC) and free SC did not disturb the measurements of SIgA. Furthermore, variations in pH and osmolarity, within biological ranges in secretions, did not influence the estimations.

Application of the ELISA for Determination of Immunoglobulin Class-specific Escherichia coli Antibodies

Antibodies of different immunoglobulin classes possess various biological activities. Often, these are not closely related to their serological titres, determined by various techniques [l]. The reason for this discrepancy is that serological methods mostly favour antibodies of one or the other immunoglobulin class [2]. An immunological assay which reflects correctly the amounts or activities of the antibodies of the different immunoglobulin classes would be advantageous. The enzyme-linked immunosorbent assay (ELISA) is a technique with possibilities for correctly recording the capacity of antibodies to bind to a solid-phase-coupled antigen irrespective of the immunoglobulin class [l, 141. We have used the ELISA to quantitate antibodies of the different immunoglobulin classes, including antibodies carrying secretory component (SC) specific for various Escherichia coli antigens. During this work we attempted to elucidate the relation between the presence and amount of specific antibodies and their protective capacity during infections. The ELISA titres expressed in different ways were compared with the antibody titres determined with other serological techniques often used for serological diagnosis and in research. These tests included the indirect haemagglutination (IHA) method [22], the indirect haemolysis (IHL) method [22], and the ammonium sulphate precipitation (ASP) technique [7]. This communication describes some of our experiences with the ELISA for quantitation of antibodies in connection with exposure to E. coli antigens in patients and in experimental animals.

Dimeric IgA in the Rat is Transferred from Serum into Bile but not into Milk

Intravenous administration of ducts thoracicus lymph with dimeric IgA antibodies against Escherichia coli 06 to lactating rat dams did not result in transfer of IgA antibodies into the milk, although the antibodies were detectable in serum 1 min after the administration and in bile 60 min later. After intravenous injection of serum from bile‐duct‐occluded (BDO) rats Immunized in the Peyers patchs into lactating rat dams, IgA antibodies appeared in the serum and remained there up to 230 min. At this time no IgA antibodies were seen in the milk while they were present in bile. IgG and IgM 06 antibodies did not appear in bile or after intravenous administration of lymph or serum from BDO rats.

Serum antibodies against native, processed and digested cow's milk proteins in children with cow's milk protein intolerance

Children with or without cows milk protein intolerance were investigated for serum antibodies to native cows milk proteins, processed cows milk proteins and native‐ and pepsin‐digested β‐lactoglobulin. Antibodies of IgG and IgA isotypes were determined with ELISA and those of IgE isotype with RAST.

A C1q Immunosorbent Assay Compared with Thin‐Layer Gel Filtration for Measuring IgG Aggregates

A new sensitive technique measures C1q‐binding of human IgG aggregates. The method is based on the principle of the enzyme‐linked immunosorbent assay with C1g‐coated rubes. The IgG aggregates attaching to this C1g are shown by enzyme‐linked anti‐human IgG. Less than 0.01 μg of aggregates per milliliter of sample can be detected. The results with this new method showed significant correlation (P < 0.01; Spearman rank correlation test) with the estimates of IgG aggregates of 13S or more and 10S in size obtained by thin‐layer gel filtration. Both of these methods showed significant correlation with the classic hemolysis inhibition method for measuring complement fixation.

Induction of Basophilic Differentiation in the Human Basophilic Cell Line KU812

The potential for differentiation of the human basophilic leukaemia cell line KUS12 was examined by means of a panel of physiologic and non‐physiologic substances used as inducers. The phenotypic characteristics of non‐induced KU812 cells included an immature morphology with scanty cytoplasmic granulation, expression of a low amount of high affinity, but no low affinity receptors (CD 23) for IgE, and a capacity for low‐rate histamine synthesis. The differentiation process was characterized by a rapid (24 h) increase in histamine production, a slower morphological maturation with the development of Alcian blue stainable granula demonstrable after 72 h. Concomitant with the phenotypic alterations, cell growth was inhibited. Differentiation in KU812 cells was inducible by Ara‐C and to some extent by sodium butyrate, but not by dimethyl sulphoxide, retinoic acid, or gamma‐interferon. Conditioned medium (CM) from cultured peripheral blood cells from atopic individuals and 18 out of 22 analysed glioma cell lines induced differentiation of the KUS12 cells, whereas supernatant from only 1 out of 21 other cell lines, including carcinoma, melanoma, sarcoma, leukaemia, and normal fibroblasts had this activity. CM from the T‐leukaemic cell line. Mo, also induced KU812 differentiation. A primary fractionation of the active substance from this cell line by reversed phase chromatography eluted the active substance at a concentration of 42–44% acetonitrile. Our present study has shown that the KU812 may serve as an appropriate model to study differentiation of basophils. In addition, its fast and specific response to biological factors makes it suitable as a biological assay for determination of active factor produced by atopic individuals.

IgA antibodies in rat bile are not solely derived from thoracic duct lymph.

The IgA level in rat bile was significantly decreased by drainage of the thoracic duct, and passively administered IgA antibodies to Escherichia coli O antigen decreased similarity. In contrast, specific IgA antibodies against E. coli O antigen raised by immunization in the Peyers patches did not diminish significantly in the bile. Rats immunized in the Peyers patches with sheep erythrocytes had IgA‐forming cells in the thoracic lymph nodes, in the mesenteric lymph nodes, and in the spleen. Perfusion of the liver of immunized animals significantly decreased the bile levels of the IgA antibodies. It seems that IgA antibodies reach the bile not only via the thoracic duct but also via lymph ducts originating from thoracic lymph glands and the spleen.

ADCC-mediating capacity in children with cow's milk protein intolerance in relation to IgG subclass profile of serum antibodies to beta-lactoglobulin.

In a previous study sera from children with cows milk protein intolerance (CMPI) exhibiting gastrointestinal symptoms were found to efficiently induce antibody‐dependent cell‐mediated cyto‐toxicity (ADCC) to β‐lactoglobulin‐coated cells. In contrast, sera from children with coeliac disease showed a low ADCC‐mediating capacity, despite high levels of IgG anti‐β‐lactoglobulin antibodies. The study described here was undertaken to evaluate whether differences in IgG subclass profile of anti‐β‐lactoglobulin antibodies could explain the observed variations in the ADCC‐mediating capacity. Forty‐eight sera from the following groups of children were investigated: CMPI with predominantly gastrointestinal symptoms, CMPI with skin symptoms of immediate‐onset, children with untreated coeliac disease and healthy references. Absorption experiments indicated that primarily IgGl antibodies were responsible for the ADCC‐mediating capacity of the sera. Accordingly, the ADCC reactivity of individual sera correlated with their IgGl antibody levels. Sera from CMPI children with gastrointes tinal symptoms, most of which had a high ADCC reactivity, also demonstrated a distinctive subclass pattern of their anti‐β‐lactoglobulin antibodies with higher relative proportions of IgGl (ratios: IgGl/IgG, IgGl/IgG3 and IgGl/IgG4) than those from the other diagnostic groups. Using logistic regression analysis, the diagnostic potential of ADCC as well as of different IgG subclass variables for the recognition of gastrointestinal symptoms caused by CMPI was evaluated. The ADCC reactivity of sera was found to be the best predictor in this model.

Origin and kinetics of IgA, IgG and IgM milk antibodies in primary and secondary responses of rats.

IgA and IgM antibodies were detected in rat milk after immunization wish ferritin in Peyers patches (Pp) 1 day after parturition but not after intramammary gland or intravenous immunization. The antibody levels decreased from day 9 to day 17 of the nursing period and were undetectable during a second lactation period. Despite the absence of milk IgM antibodies after intramammary gland or intravenous immunization, the serum levels of the IgM antibodies were similar after all three immunization methods. IgA antibodies were not found in serum after any of the immunization methods. IgG antibodies appeared in serum and milk after Pp. intramammary gland, and intravenous immunization. Milk and serum IgG antibodies from all the Pp‐immunized animals decreased from day 9 to day 17 of the lactation period. After intramammary gland immunization, however, the IgG antibody levels increased in all the milk samples, but only in four of seven sera. The milk and serum IgG antibody levels were lower but still detectable during a second lactation period. Re‐injection of ferritin in the Pp during a third lactation period resulted in higher levels of milk IgA, IgG and IgM antibodies than after the first injection, Rats with serum IgG anlihodies against Escherichia colia 08 naturally present in their gut flora had no corresponding milk antibodies of any isotype The results suggest that milk antibodies of all three isotypes stem from local production in the mammary gland and that blood IgG and IgM antibodies originate at least partly from stimulation in Pp.