Cell based assays and kits for assessing serum cholinergic receptor activity

A non-radioactive method for determining the level of muscarinic acetylcholine receptor subtype-1 (M1 receptor) cholinergic or anticholinergic activity in a blood serum sample, the method comprising:

• removing protein from the blood serum sample by treatment with perchloric acid (PCA) to produce a PCA-treated serum sample;
• incubating the PCA-treated serum sample with a membrane preparation from cultured cells expressing the M1 receptor, the M1 receptor loaded with a calcium sensitive fluorophore or dye;
• collecting fluorescence measurements from the cells for a first period of time;
• adding an aliquot of carbachol solution or another acetylcholine receptor agonist to produce maximal or near maximal fluorescence of the cells from release of calcium;
• collecting fluorescence measurements from the cells for a second period of time;
• comparing pre-carbachol, or another acetylcholine receptor agonist, activity of the blood serum sample to pre-carbachol activity of a control blood serum sample, wherein the control blood serum sample is known to contain no cholinergic agonists or other drugs with cholinergic activity;
• comparing post-carbachol, or other acetyl choline receptor agonist, activity of the blood serum sample to post-carbachol activity, or other acetylcholine receptor agonist, of the control blood serum sample,
• wherein comparing pre-carbachol activity provides a measure of pure agonist properties of a subject's serum and comparing post-carbachol activity provides a measure of the subject serum's net agonist and antagonistic anticholinergic properties.

A non-radioactive method for determining the level of muscarinic acetylcholine receptor subtype-1 (M1 receptor) cholinergic or anticholinergic activity in a blood serum sample, the method comprising either A) or B):

• A) removing protein from the blood serum sample by treatment with perchloric acid (PCA) to produce a PCA-treated serum sample;
• incubating the PCA-treated serum sample with a membrane preparation from cultured cells expressing the M1 receptor, the M1 receptor loaded with a calcium sensitive fluorophore or dye;
• collecting fluorescence measurements from the cells for a first period of time;
• comparing acetylcholine receptor agonist activity of the blood serum sample to a control blood serum sample, wherein the control blood serum sample is known to contain no cholinergic agonists or other drugs with cholinergic activity;
• wherein comparing the activity of the blood serum sample to a control blood sample provides a measure of pure agonist cholinergic properties of a subject's serum;
• or;
• B) removing protein from the blood serum sample by treatment with perchloric acid (PCA) to produce a PCA-treated serum sample;
• incubating the PCA-treated serum sample with a membrane preparation from cultured cells expressing the M1 receptor, the M1 receptor loaded with a calcium sensitive fluorophore or dye;
• adding an aliquot of carbachol solution or another acetylcholine receptor agonist to produce maximal or near maximal fluorescence of the cells from release of calcium;
• collecting fluorescence measurements from the cells for a period of time;
• comparing post-carbachol, or other acetyl choline receptor agonist, activity of the blood serum sample to post-carbachol activity, or other acetylcholine receptor agonist, to a control blood serum sample, wherein the control blood serum sample is known to contain no cholinergic agonists or other drugs with cholinergic activity;
• wherein comparing post-carbachol activity provides a measure of the subject serum's net agonist and antagonistic cholinergic properties.