PubMed · 3489880
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F Shakib. 1986. The IgG4 subclass.. https://pubmed.ncbi.nlm.nih.gov/3489880/
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The existence of two IgA subclasses in humans has been reliably shown by biochemical, immunochemical and genetic means. IgA is unique among immunoglobulins in the regular occurrence of both monomeric and polymeric forms. In order to be able to study the relationship between monomeric and polymeric IgA1 and IgA2 concentrations in the circulation and mucosal compartment i.e. secretions, it is essential that the methods used are not biased by the molecular size of the IgA under investigation. We validated IgA and IgA subclass measurements in serum and saliva by sandwich enzyme-linked immunosorbent assay (ELISA). Coating reagents were specific mAbs against IgA (clone 4E8), IgA1 (clone 69-11.4) or IgA2 (clone 16-512-H5 and clone IF8.58). Pooled normal human serum and purified dimeric IgA1 (d-IgA1) or IgA2 (d-IgA2) myeloma proteins were used to standardize the assays. Polymeric and monomeric forms of IgA in sera from volunteers and patients with myelomatosis were assayed in fractions separated by high performance liquid chromatography (HPLC). Dithioerythritol (DTE) was used to determine the influence of the quarternary structure of IgA on its detection by mAbs. We found that mAbs 4E8, 69-11.4 and 16-512-H5 reliably measured d-IgA, d-IgA1 and d-IgA2 respectively, independent of the standard employed. Clone IF8.58 underestimated the concentration of d-IgA2 (correction factor +/- 2) with increased sensitivity in the presence of DTE. This difference is probably explained by the composition of the immunogen against which the mAb was raised. We conclude that no reliable conclusions can be made concerning the subclass ratio in biological fluids unless the monoclonal antibodies used have been appropriately validated.
Although stem cell factor (SCF) has been identified as a critical cytokine for the development of human mast cells from their progenitors, the effects of other cytokines on human mast cells are less well understood. We examined the effects of several cytokines on the survival of human mast cells of 100% purity generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of 100 ng/mL recombinant human (rh) SCF and interleukin-6 (IL-6). Mast cells suspended in conventional serum-containing medium died over a period of 2 to 6 days after the withdrawal of SCF and IL-6. The cells became pyknotic and underwent DNA fragmentation characteristic of apoptosis. The addition of SCF, IL-3, IL-4, IL-5, or IL-6 to the cultures in both serum-containing and serum-free medium prolonged their survival in a dose-dependent manner. Some other cytokines, such as IL-2, IL-9, IL-10, IL-11, tumor necrosis factor-alpha, transforming growth factor-beta 1, and nerve growth factor, had no survival-promoting effect at 100 ng/mL. Preincubation of mast cells with SCF, IL-4, IL-5, or IL-6 for 24 hours during sensitization with IgE enhanced IgE/anti-IgE antibody-induced histamine release from mast cells, whereas IL-3 showed a negligible effect. Polymerase chain reaction amplification of alpha-chains of IL-3 receptor (R), IL-4 R, IL-5 R, and IL-6 R yielded products of the correct size predicted from the sequence of each receptor. The binding assay using 125I-labeled IL-3 indicated that these mast cells bear receptors for IL-3. These findings suggest that IL-3, Il-4, IL-5, and IL-6, which are mainly produced by T-helper 2 lymphocytes, might regulate the functions of human mast cells in vivo via specific receptors in allergic reactions.
The efficiency of radioimmunotherapy with iodine-labelled antibodies is often limited by intracellular internalisation and catabolism after initial binding to the cellular targets. We have developed a technique called affinity enhancement system (AES) which uses bi-specific antibodies to target radiolabelled bivalent haptens to cells. This targeting method has been applied successfully to tumour imaging in colorectal cancer patients and is now considered for therapy. We have investigated the potential of this technique to target iodine radioisotopes by comparing it to targeting with covalently iodine-labelled antibodies in a rapidly internalising antigenic system, the surface IgM of a B-lymphoma cell line. A 5-fold increase in the intracellular retention time of activity as compared to 125I-labelled F(ab')2 or IgG was observed. The radiolabelled hapten did not undergo any catabolism after internalisation. Resistance to cellular proteases and failure of recognition of the hapten by amino acid transporter systems may be potential explanations for these observations. This should make non-covalent targeting, particularly the AES, a method of choice to target modulating antigens for the therapy of malignant hemopathies.