The complement regulators, decay accelerating factor, membrane cofactor protein, and CD59 are present in human glomeruli. Crry is the rodent analogue to the former two proteins. In this study, we examined complement regulation in cultured rat glomerular endothelial cells (GEnC) and mesangial cells (MES). Immunoprecipitation of 125I-labeled membrane proteins and Western blotting studies were performed with anti-Crry and anti-CD59. In both GEnC and MES, Crry was present as 53, 65, and 78 kD proteins. The 20 kD CD59 was apparent in GEnC. CD59 was also present in MES, but in relatively smaller quantities. By Northern analyses, 1.8 kb CD59 mRNA was present in GEnC as well as in RNA from isolated rat glomeruli. mRNA for Crry was present in both GEnC and MES as 2.2 kb species. The functional significance of these proteins was evaluated next. Anti-Thy 1.1 IgG was used to activate the complement classical pathway in MES. To inhibit the function of the complement regulators, anti-CD59 and/or anti-Crry F(ab')2 antibodies were added with anti-Thy 1.1. Inhibition of Crry function led to enhanced cytotoxicity, while there was no effect when CD59 function was inhibited. The complement alternative pathway was studied by adding complement in Mg-EGTA buffer. Inhibition of Crry led to productive alternative pathway activation, which was accentuated by anti-CD59 when Crry was incompletely inhibited. Alternative pathway regulation was also evaluated in GEnC. Inhibition of CD59 function alone had no effect in GEnC, while inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. Therefore, Crry is present in both GEnC and MES and restricts the complement alternative pathway in both cell types. Crry also regulates the classical pathway in MES. CD59 is present and functionally active in GEnC, while it appears to have a minor role in MES.

The consumption of cetacean meat is geographically common and often of undetermined sustainability. Besides, it can expose humans to contaminants and zoonotic pathogens. The illegality of possessing cetacean meat was likely under-reported in some countries due to lack of attention paid by the officials although DNA analysis of market products helped to show such practices. We developed two monoclonal antibodies against synthetic peptides of myoglobin (Mb) for constructing a rapid immune colloidal gold strip. Only cetacean Mb is capable of binding to both antibodies and presents positive signal while the Mb from other animals can bind only 1 of the antibodies and presents negative result. The strip for cetacean meat would be an applicable and cost-effective test for field inspectors and even the general public. It contributes to increase the reporting capacity and coverage of illegal cetacean meat possession, which has implications for global cetacean conservation and public health.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

In the mouse, MCR1 and MCR2 on B lymphocytes are encoded by alternatively spliced Cr2 gene transcripts. Immune adherence receptors that bind C3 are present on mouse platelets and unstimulated neutrophils, yet they are not MCR1 or MCR2. To examine C3b- and C3d-binding proteins on mouse platelets and neutrophils, we performed C3b and C3d affinity chromatography as well as immunoprecipitation studies using previously described Ab to MCR1/MCR2 (mAb clones 8C12, 7G6, and 7E9 and polyclonal Ab BRN-1). Mouse neutrophils contained a 190-kDa membrane protein that specifically bound to C3b-Sepharose. Preabsorption of neutrophil proteins with anti-MCR1/MCR2 Ab did not affect the recovery of the 190-kDa C3b-binding protein by subsequent C3b affinity chromatography. Thus, this protein is immunologically distinct from the previously described MCR1 and MCR2 proteins. By virtue of its size and C3b-binding capacity, the 190-kDa protein was named C3bR-190. C3bR-190 was also apparent on platelets, but in reduced amounts. BRN-1 anti-MCR1/MCR2 Ab immunoprecipitated proteins of 125 and 150 kDa from surface-radiolabeled mouse platelets, which specifically bound to C3d-Sepharose. However, these proteins were not identified by mAb to MCR2, thus distinguishing them from previously described MCR2. These proteins were named C3dR-125 and C3dR-150. Therefore, we have identified a 190-kDa C3b-binding protein on mouse neutrophils and 125- and 150-kDa C3d-binding proteins on mouse platelets. These appear to be distinct from the heretofore identified mouse B lymphocyte MCR1 and MCR2. The identity of these C3b- and C3d-binding proteins on mouse neutrophils and platelets awaits further study.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG 1 ). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.