Mouse sera are harvested at different time points before and after immunization. using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry,in vitroculture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting. Keywords:Immunology and Infection, Issue 139, Thymus-dependent (TD) antigen, thymus-independent (TI) antigen, immunoglobulin (Ig), Ig isotype, memory response, immunization, adjuvant, enzyme-linked immunosorbent assay (ELISA) Download video stream. == Introduction == B lymphocytes are the principal player in humoral immunity and the only cell type in mammals that are capable of producing antibodies, also termed as immunoglobulins (Ig)1,2. Antibodies secreted by B cells provide a formidable defense against invading pathogens via diverse mechanisms including neutralization, opsonization and complement activation, leading to protective immunity3. Secretion of antibodies by B cells is only achieved after full activation of specific B cells, which normally requires two distinct signals3. Signal 1 is relayed by direct binding of the antigen (Ag) to the B cell receptor (BCR) expressed on the surface of specific nave B cells3. Depending on the source of Signal 2, B cell activation can be GDC-0980 (Apitolisib, RG7422) divided into thymus-dependent (TD) or thymus-independent (TI)3,4. In a TD antigen response, Signal 2 is provided by activated cognate CD4 T helper (TH) cells, which express CD154, the ligand for the co-stimulatory receptor CD40 expressed on B cells1,2,3. In a TI antigen response, Signal 2 comes from either engagement of Toll-like receptors (TLRs in the case of type 1 TI Ag) or extensive cross-linking of the BCRs (in the case of type 2 TI Ag) on the B cells3,4. Type 1 TI (TI-1) antigens are microbial ligands of TLRs, including bacterial lipopolysaccharides (LPS), viral RNAs, and microbial CpG DNA4,5. Type 2 TI (TI-2) antigens have highly repetitive structure, and are able to deliver prolonged and persistent signaling to the B cell by GDC-0980 (Apitolisib, RG7422) multiple cross-linking of the BCRs4,6. Typical examples of TI-2 antigens include pneumococcal polysaccharides and hapten-conjugated polysaccharide6,7. Both TD and TI antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) with the help provided by antigen presenting cells (APCs) such as dendritic cells (DCs)1,2,3. Furthermore, both TD and TI antigens are able to induce memory responses with the help of APCs, but TD antigens are more efficient at inducing memory B GDC-0980 (Apitolisib, RG7422) cell generation3,8. In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) and TNP-polysaccharide (neutral, highly branched and high-mass), respectively9,10,11. TD antigens are usually used with an adjuvant to enhance the production of antibodies12. Here in our protocol, TNP-KLH is injected with alum, a commonly used adjuvant in immunization studies12. Other examples of adjuvants that can be used include complete or incomplete Freund’s adjuvant (CFA or IFA), monophosphoryl-lipid A/trehalose dicorynomycolate (“Ribi” adjuvant), and CpG oligodeoxynucleotides,etc.13,14. After immunization, mouse sera are harvested at different time points and TNP-specific antibodies in sera are quantified using Ig isotype-specific enzyme-linked immunosorbent assay (ELISA)9,10,11. ELISA is a plate-based assay that is widely used as a diagnostic tool in medicine and also as an analytical tool in biomedical research15,16. Rabbit Polyclonal to HP1gamma (phospho-Ser93) It is used to detect and quantify analytes including antibodies, hormones, cytokines, chemokines, and various antigens,etc. ELISA can be performed in several different formats, including direct, indirect, sandwich and competitive ELISA15,16. In general, it involves the immobilization of the antigen to a solid surface, usually a 96-well microtiter plate, which is incubated with a primary antibody. After incubation, the unbound antibody is washed away. In a direct ELISA, the primary antibody is directly conjugated to an enzyme (typically horseradish peroxidase or alkaline phosphatase), which can cleave a chromogenic substrate to yield a visible color change detected by a signal-detection instrument such as a spectrophotometer15,16. In contrast, if an enzyme-linked secondary antibody is used to bind the primary antibody, then this is considered as an indirect ELISA15,16. Direct ELISA is faster whereas indirect.