R. van Nieuwmegen

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Detection of specific antibody-producing cells in the spleen and simultaneous determination whether or not they produce immunoglobulin G antibodies.

Rabbits were primed intravenously with human serum albumin (HSA) and boosted with the same antigen 2 months later. Cells producing specific antibodies against HSA could be detected in vivo and it could be determined whether or not they belonged to the immunoglobulin (Ig) G class using a combined peroxidase (HRP) and alkaline phosphatase (AP) immunocytochemical technique. HRP-HSA conjugate was used for detection of anti-HSA-producing cells and AP-sheep anti-rabbit IgG (SRIgG) was used to determine the IgG class of the antibodies produced by these cells in the same spleen section. After performing both HRP and AP cytochemistry, cells with a red-stained cytoplasm represent anti-HSA-producing cells not stained for their antibody class and cells with a blue-stained cytoplasm represent cells producing IgG antibodies not directed against HSA. Cells with a double-stained cytoplasm represent cells producing anti-HSA antibodies belonging to the IgG class. We also attempted to determine whether or not part of the anti-HSA-producing cells belonged to the IgM class using AP-sheep anti-rabbit IgM (SRIgM). In this case no double-stained cells were detected, indicating that the affinity of intracellular IgM-anti-HSA antibodies is too low to allow detection using the present technique.

Association of an albumin antigen with phosphatidylcholine liposomes alters the nature of immunoglobulins produced during the immune response against the antigen

Abstract Human serum albumin has been injected intravenously in rabbits either free in solution or associated with liposomes. Blood samples were obtained from the rabbits at various time intervals after injection, and two different antibody determinations were performed in each sample. Whereas a haemagglutination technique was applied for determination of predominantly IgM anti-human serum albumin antibodies, a second technique, using antigen-coated Sepharose beads and horseradish peroxidase-conjugated anti-rabbit IgG, was used to detect the IgG anti-human serum albumin antibodies. Liposomes appeared to enhance strongly the IgM response against human serum albumin. No such marked differences were found, however, between the IgG response against liposome-associated or free human serum albumin. The conclusion is drawn that the immunoadjuvant effect of liposomes during the primary immune response against an albumin antigen is mainly due to an enhanced IgM antibody production.

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Simultaneous detection of cells producing monospecific antibodies and cells producing cross-reacting antibodies in the spleen of rabbits after injection of two related antigens.

Rabbits were injected simultaneously with both human gamma globulin (HGG) and bovine gamma globulin (BGG). Sections of spleen tissue were prepared from spleen biopsies taken during the primary or secondary immune response, and incubated simultaneously with horseradish peroxidase (HRP)-HGG conjugate and alkaline phosphatase (AP)-BGG conjugate in order to detect cells containing specific antibodies against one or both of the antigens. After both HRP and AP cytochemistry, cells with a red-stained cytoplasm, cells with a blue-stained cytoplasm, and cells with a violet-stained cytoplasm were detected in the spleen. The red-stained cells had bound the HRP-HGG conjugate, indicating that these cells contained anti-HGG antibodies. The blue-stained cells had bound the AP-BGG conjugate, indicating that these cells contained anti-BGG antibodies. The violet-stained cells had obviously bound both the HRP-HGG conjugate and the AP-BGG conjugate, indicating that these cells contained antibodies cross-reacting with both antigens. Results are compared with earlier studies on the antigenic similarities and differences between HGG and BGG when used as antigens in rabbits.

Peroxidase Immunocytochemistry for the Detection of Specific Anti-Hapten (Penicilloyl) Antibody Producing Cells in the Spleen, After Injection of a Hapten-Carrier Conjugate

Cells producing specific antibodies against the hapten penicilloyl (Pen) are demonstrated in tissue sections of the spleen, using peroxidase immunocytochemistry. Horseradish peroxidase-human serum albumin-penicilloyl (HRP-HSA-Pen) conjugate, prepared by coupling Pen to HRP-HSA conjugate, was used for detection of anti-Pen producing cells in the spleen after intravenous injection of bovine gamma globulin-penicilloyl (BGG-Pen) conjugate. HRP-HSA conjugate was used as a control to exclude the possibility that positive cells contained antibody against a common determinant in both HSA and BGG. The method may be applied for the detection of cells producing antibodies against other haptens as well.

The Development of Specific Antibody Producing Cells in the Spleen of Rabbits During the Primary and Secondary Immune Response

Until recently, no suitable method was available to detect specific antibody producing cells in the lymphoid tissues, except for cells producing antibodies against the antigen horseradish peroxidase (HRP). The major disadvantage of HRP is that it is a weak antigen and has to be administered together with a strong adjuvant such as aluminum phosphate1 or Freund’s complete adjuvant2. An intravenous injection e.g., in order to study the immune response in the spleen could not be given. Moreover, the antigen HRP when adsorbed to aluminum phosphate or emulsified in Freund’s complete adjuvant may be released from the injection site only slowly. In that case it is difficult to determine, at what time interval after injection the primary immune response finishes and the secondary immune response starts. Recently we introduced a new approach for the detection of specific antibody producing cells in lymphoid tissues. An antigen-HRP conjugate or an antigen-alkanline phosphatase (AP) conjugate was used for detection of cells containing specific antibodies in their cytoplasm3–11. The aim of the present contribution was to review our recent findings on the development of specific antibody producing cells during the primary and secondary immune response in the spleen. Specific antibody producing cells were found in various compartments of the white pulp of the spleen.

Evidence for a clonal development of specific antibody forming cells in the spleen.

The clonal development of antigen triggered lymphoid cells has been studied extensively in vitro. No technique was available until now to distinguish between two different antibody producing cells in vivo. It could not be investigated for that reason whether or not after simultaneous administration of two different antigens, specific antibody producing cells develop in the lymphoid organs of the responding animal randomly. When, after contact with the antigens, the antigen reactive cells and/or the specific antibody producing cells that develop during the immune response against the antigen, migrate not at all or only slowly, specific antibody producing cells arising from one precursor will remain close together forming a group of cells, all producing the same antibodies. When, in the contrary, they show a relatively strong migration, specific antibody producing cells arising from different precursors will be distributed randomly. It is shown in a series of recent experiments, reviewed in the present contribution, that after intravenous injection of two different antigens, clones of similar specific antibody producing cells develop in the spleen of rabbits. Double immunocytochemical staining, using both horseradish peroxidase (HRP) and alkaline phosphatase (AP) was performed for the simultaneous detection of two or three different specific antibody producing cells, after injection of two different protein antigens1–3.