Stimulatory activity of anti-peptide antibodies against the second extracellular loop of human M_2 muscarinic receptors

Obejctive To study the activity of anti peptide antibodies against the second extracellular loop of human M 2 muscarinic receptors on cAMP production and inward calcium currents (I Ca ) in guinea pig ventricular myocytes A comparison was also made with those of a muscarinic receptor agonist Methods cAMP content was determined by radioimmunoassay and the I Ca in guinea pig single ventricular cells were recorded by the whole cell patch clamp technique Results Both the muscarinic receptor agonist, carbachol (Carb 10?μmol/L), and anti peptide antibodies (Abs 100?nmol/L) could decrease basal cAMP levels (by 46 9%±4 2% and 60 2%±4 6%, respectively) and basal I Ca Both Carb (10?μmol/L) and Abs (100?nmol/L) could also inhibit the isoprenaline induced (Iso 0 8?μmol/L) increases in cAMP production (from 108 2±7 0 to 88 4±7 2? pmol/mg·protein/min for Carb and 88 6±5 1? pmol/mg· protein/min for Abs, respectively) and the increases in I Ca The muscarinic receptor antagonist atropine (Atr) was able to prevent these effects of Carb and Abs Conclusions Anti peptide antibodies against an epitope located in the second extracellular loop of human M 2 muscarinic receptors, similar to muscarinic receptor agonist, could decrease the basal I Ca and β receptor agonist stimulated increase of I Ca by decreasing the basal and β receptor agonist stimulated increase of cAMP production, and therefore could have an effect on their target receptor These results further suggest that autoimmunity may participate in the pathogenesis of human cardiomyopathy and the second extracellular loop of human M 2 muscarinic receptor could be the main immunodominant region.

Preparation of monospecific anti-PAG antibodies for cattle pregnancy detection: use of synthetic peptides to improve specificity

Immunological methods involving pregnancy associ-ated glycoproteins (PAG) are used for cattle preg-nancy detection. The faults of these methods could be overcomed by using antibodies specific for each member of the PAG family. In order to differentiate between very similar proteins, preparation of anti-bodies specific for peptides is a method of choice. In this work, we summarize a series of considerations regarding peptide design and choose free access NCBI, Antigenicity Plot, EMBOSS Antigenic and Expasy tools to apply them. We design peptides spe-cific for different reported PAG members and obtain the corresponding polyclonal antibodies for five of them.