Monoclonal antibodies to the molluscan small cardioactive peptide SCP_B: Immunolabeling of neurons in diverse invertebrates

Abstract

We reported a development of murine monoclonal antibodies to a molluscan small cardioactive peptide (SCPB) and their application to immunolabeling of neurons in several molluscan and arthropod species. In vitro stimulations of mouse lymphocytes with SCPB conjugated to a carrier protein yielded exclusively IgM class antibodies; in vivo stimulation resulted in generation of both IgM and IgG classes of antibodies. Monoclonal antibodies of the IgM class labeled identified SCP-containing neuron B11 in the frozen sections of the buccal ganglia of Tritonia diomedia. These antibodies failed to stain any neurons in whole mount preparations. A monoclonal antibody of IgG1 subclass selectively labeled neurons in both frozen sections and whole mount preparations of diverse invertebrate species. Thus, neurons B11, B12, and GE1 and several other neurons of the buccal and gastroesophageal ganglia of T. diomedia bound the antibody, and a similar pattern of immunolabeling was found in the closely related gastropod Tritonia festiva. We also observed SCPB-like immunoreactivity in the central neurons of other nudibranch and pulmonate molluscs and in examples of insect (Acheta domesticus and Tehrmobia domestica) and crustacean (Semibalanus cariosus) classes of the Arthropoda. Our results suggest a specific pattern of distribution of SCPB-like immunoreactivity in the gastropod nervous system and a broad occurrence of SCPB-like antigenicity in the diverse invertebrates.

Allatostatin‐like‐immunoreactive neurons of the tobacco hornworm, Manduca sexta , and isolation and identification of a new neuropeptide related to cockroach allatostatins

“…The sequence of the last five amino acids of the C-terminus (AFPRVamide) of CAP 2b is identical to that of SCP B , except that the terminal amino acid of SCP B is methionine rather than valine. Masinovsky et al (1988) concluded that the epitope of the SCP antibody includes all or part of a six amino acid sequence located at the C-terminus of SCPs. We have shown that the SCP antibody can be preadsorbed with synthetic CAP 2b ; these results probably are due to the similarity of the C-terminus of CAP 2b to that of the SCP B .…”

“…The anti-FMRFamide serum has been characterized by O'Donahue et al (1984). The SCP B antibody was characterized by Masinovsky et al (1988) and Arbiser and Beltz (1991). Cells immunostained by the anti-FMRFamide serum will be referred to as FMRFamide-ir, and those immunostained by the SCP B antibody are referred to as SCP-ir.…”

Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling inDrosophila

“…Tissue was blocked with 5% normal donkey serum for 15 minutes followed by transfer to a solution of primary antibodies and incubation overnight. Primary antibodies used were monoclonal mouse anti-SCP (Masinovsky et al, 1988), rat anti-mCD8 (1:200, CALTAG) and mouse anti-EcR (IID9.6, common) (Talbot et al, 1993). After several rinses with PBS-Tx, the tissues were incubated overnight in secondary antibody (1:200 in PBS-Tx of Texas red conjugated donkey anti-mouse IgG, Texas red conjugated donkey anti-rabbit IgG, or FITC conjugated donkey anti-rat IgG; Jackson ImmunoResearch).…”

Development of serotonin-like immunoreactivity in the embryos and larvae of nudibranch mollusks with emphasis on the structure and possible function of the apical sensory organ

“…Positive controls for our immunocytochemical experiments were incubated as described above with a murine monoclonal antibody specific for small cardioactive peptides (anti-SCP; Masinovsky et al, 1988) diluted 1:20, 1:60, or 1:80 in GS-PBS ϩ followed by secondary labeling with goat, antimouse IgG conjugated to RITC. Negative controls were incubated as described above either 1) in a 1:1,000 dilution of polyclonal rabbit anti-DBH (dopamine-b-hydroxylase) antibody (IncStar, Stillwater, MN), 2) in a 1:1,500 dilution of anti-5-HT primary antibody that had been preincubated for ϳ30 hours with a bovine serum albumin (BSA)-5-hydroxytryptamine conjugate (IncStar; cat.…”