Enumeration of human peripheral blood lymphocytes secreting immunoglobulins of major classes and subclasses in healthy children and adults

Abstract

The reverse enzyme-linked immunospot assay was modified to enumerate peripheral blood mononuclear cells (PBMC) secreting IgG1-4, IgA1-2, and IgM. Anti-human IgG F(ab')2 and mouse monoclonal antibodies specific to each of the isotypes were used as solid-phase capture antibodies and developing antibodies, respectively. Although attempts were also made to detect IgD- and IgE-secreting cells (SC), their numbers in the peripheral blood were too few to be reliably estimated. The assay was applied to study healthy subjects including 21 neonates within 3 days of birth, 44 1- to 48-month-old children, and 32 adults. Sixty percent of these neonates had IgM SC in small numbers (less than 20 per 10(6) PBMC), but only three neonates had IgSC of other isotypes. In contrast, by 1-2 months of age children had IgSC of all isotypes, including IgA2 and IgG4, often in higher numbers than adults. The relative frequencies of IgSC were IgA1 greater than IgG1 greater than IgM greater than IgG2 greater than IgG3 greater than IgG4 greater than IgA2 in the children and IgA1 greater than IgG1 greater than IgA2 greater than IgM greater than IgG4 greater than IgG2 greater than IgG3 in the adults. The order of the serum concentrations in the adults was IgG1 greater than IgG2 greater than IgA greater than IgM greater than IgG4 greater than IgG3. No correlation was found between the serum level and the IgSC number of individual isotypes (except for serum IgA and IgA1-SC). This new methodology should facilitate investigating the current status of immunoglobulin synthesis and the Ig repertoire in adults and children, in health and in disease.