MABF2210-100UG Sigma-AldrichAnti-CD69 Antibody, clone FN61

The ability of serum factors to cross-link labeled mouse monoclonal antibody (mAb) of irrelevant specificity (mAb FN61, subclass IgG1) to different particle types coated with sheep IgG, bovine gamma-globulin, or mAb FN61 was measured simultaneously by flow cytometry. Significant interference with mAb FN61-coated particles was detected in 53 of 101 sera. Of the 30 sera showing the most pronounced interference, 23 were characterized by an even stronger cross-linking to particles coated with bovine gamma-globulin. These were designated type 1 sera. Seven sera, designated type 2, displayed a dominant interference with the mAb FN61-coated particles. The interference reaction in the two serum types was characterized by different kinetics, dependence on particle concentration, and response to blocking agents. The interference was minimized by addition of 500 micrograms of bovine gamma-globulin and 50 micrograms of mAb HH1 (IgG1) of irrelevant specificity per 10 microL of serum sample in a final assay volume of 100 microL.

The CD53 pan-leukocyte glycoprotein is a member of the recently described tetraspan family of cell membrane proteins. The structure and functional characteristics of these molecules indicate that they may play important roles in transmembrane signaling in different cells. Recently, it was reported that cross-linking of CD53 on human B cells led to an increase in cytoplasmic calcium fluxes. In the present study, we wished to further explore the possible role of CD53 in functional B cell responses. Cross-linking of CD53 with the use of the mAb MEM-53 and a polyclonal sheep anti-mouse Ig promoted activation of resting B cells into the G1 phase of the cell cycle as judged by increased expression of the early activation Ag CD69, increases in cellular volume, RNA synthesis, and c-myc protein levels, and enhanced binding of 7-aminoactinomycin D. In contrast, MEM-53 alone had no detectable effects. Cross-linking of anti-CD53 induced negligible S phase entry in the absence of other stimuli. However, cytokines, in particular IL-2 and IL-4, potentiated the DNA synthesis induced by cross-linking of CD53. Furthermore, cross-linking of the CD53 Ag induced Ig production in the presence of T cell supernatant. Taken together, the data suggest that CD53 plays an important functional role in B cell activation and differentiation.

The specialized M cells in the follicle-associated epithelium (FAE) of Peyer's patches (PP) represent an intimate interphase between luminal antigens and gut-associated lymphoid tissue (GALT). M cells form pockets that contain clusters of leucocytes probably involved in the first encounter with antigens from the gut lumen. Three-colour immunofluorescence in situ phenotyping of these leucocytes in humans revealed about equal numbers of B (CD19/20+) and T(CD3+) lymphocytes, the latter mainly CD4+ (median 73%, range 40-90%), but relatively few macrophages (CD68+). Most B cells (90%) were positive for surface IgM (sIgM) and often co-expressed sIgD (median 34%, range 6-60%). Occasional B cells (median 2%) did not express CD45RA (range 0-15%) and 13% virtually lacked HLA-DR (range 0-40%). Some B and T lymphocytes expressed the nuclear proliferation marker Ki-67 (range 1-10%). The M-cell pockets also contained occasional cells with cytoplasmic IgA or IgM. These sites thus contained a heterogeneous B-cell population with features of both follicular mantle (sIgD+ sIgM+) and marginal zone (sIgD- sIgM+) B lymphocytes. Adjacent T lymphocytes were generally of the memory phenotype (CD45RO+). Our findings suggest that the M-cell-associated B lymphocytes represent local extensions of B-cell follicles towards the gut lumen, developed topically to facilitate antigen presentation and diversification of mucosal immune responses.

We evaluated two homogeneous immunofluorometric assays (IFMAs) of alpha-fetoprotein (AFP) based on new macroporous acrylate particles combined with flow cytometry. The standard IFMA, requiring 1 h of incubation, provided a working range from 1.8 to > 900 kIU/L (CV < 10%) and a detection limit of 0.6 kIU/L. Use of overnight incubation and a lower particle concentration extended the working range by 1 decade in the lower end. Analytical recoveries for the standard IFMA varied between 97% and 108%. The slope and y-intercept of the regression line correlating measurements by the standard IFMA and a routine immunoradiometric assay were not significantly different from 1 and 0, respectively (P > 0.5), and the correlation coefficient was 0.996. High precision and warning of spuriously high measurements were obtained by including in each sample separate particle types for detecting instrument instability and measuring nonspecific binding only.

Human activation inducer molecule (AIM/CD69), a dimeric glycoprotein structure of 33 and 27 kDa, is the earliest inducible cell surface antigen expressed during lymphocyte activation and is implicated in the induction of T and B cell proliferative responses. Cross-competition monoclonal antibodies (mAb) binding assays have allowed the definition of four antigenic epitopes. Three of them (antigenic sites E1-3) are extracellular while the fourth (site I) is a sequential epitope localized intracellularly and highly conserved interspecies. Site E1 is shown to be an immunodominant antigenic determinant closely related to a functional domain of AIM important for triggering of T cell proliferation. Studies of peptide fragmentation of the two isolated AIM subunits with different proteases have demonstrated that both AIM chains are differentially glycosylated forms of a single 24-kDa core protein. Moreover, the two denatured and isolated AIM chains share common epitope(s) as demonstrated by their reactivity with an mAb by both Western blot analysis and immunoprecipitation of the separated AIM subunits. Biosynthesis studies revealed the rapid appearance of two intermediate precursor forms of 29 and 26 kDa which arise from the 24-kDa unglycosylated AIM polypeptide. This 24-kDa unglycosylated form could be also precipitated from iodinated cells pretreated with tunicamycin, indicating that glycosylation of the protein was neither required for AIM cell surface expression nor for acquisition of external epitopes E1-E3. Cell treatment with pronase resulted in the loss of the external epitopes E1-3 and the generation of a proteolytic peptide of 16 kDa that could be precipitated by the anti-AIM mAb specific for the internal site I. This proteolytic fragment retained the transmembrane and cytoplasmic regions of the molecule where both epitope I and phosphorylation sites reside. These results demonstrate that AIM is an integral membrane homodimeric glycoprotein with a large cytoplasmic domain probably involved in the activation signals transduced through this molecule to lymphocytes.