本试剂盒应用双抗体夹心酶标免疫分析法测定标本中mAChRs水平。用纯化的抗体包被微孔板，制成固相抗体，往包被抗体的微孔中依次加入mAChRs、生物素化的抗大鼠mAChsR抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的mAChRs呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。
Intended use
This immunoassay kit allows for the specific measurement of rat muscarinic acetylcholine receptor,M-AChR concentrations in cell culture supernates, serum, and plasma.
Introduction
Muscarinic receptors are those membrane-bound acetylcholine receptors that are more sensitive to muscarine than to nicotine. Those for which the contrary is true are known as nicotinic acetylcholine receptors. Muscarine and nicotine are both alkaloids. Many drugs and other substances (for example pilocarpine and scopolamine) act as agonists or antagonists of only muscarinic or only nicotinic receptors, making this distinction useful.
Muscarinic acetylcholine receptors are also present and distributed throughout the central nervous system, in post-synaptic and pre-synaptic positions. There is also some evidence for postsynaptic receptors on sympathetic neurons allowing the parasympathetic nervous system to inhibit sympathetic effects.
Muscarinic acetylcholine receptors belong to a class of metabotropic receptors which use G proteins as their signalling mechanism. There are known to be a large number of these G-protein-coupled receptors for neuroreceptors, hormones, and other substances. G proteins are also present in taste, and odour detecting cells, in the retina, and in many other systems.
In such receptors, the signalling molecule (the ligand) binds to a receptor which has seven transmembrane regions, in this case the ligand is ACh. This receptor is bound to intracellular proteins, known as G proteins, which begin the information cascade within the cell.
By contrast nicotinic receptors use an ion-gated mechanism for signalling. Sufficient ligands cause an ion channel to open, filling (or evacuating) a cell of a particular ion.By the use of selective radioactively-labelled agonist and antagonist substances, four subtypes of muscarinic receptors have been determined, named M1-M4 (using an upper case M and subscript number). For example, the drug pirenzepine is a muscarinic antagonist (decreases the effect of ACh) which is much more potent at M1 receptors than it is at other subtypes. The acceptance of the various subtypes has proceeded in numerical order: therefore, sources exist which only recognise the M1/M2 distinction, more recent studies tend to recognise M3, and the most recent M4.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for mAChRs has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any mAChRs present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for mAChRs is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of mAChRs bound in the initial step. The color development is stopped and the intensity of the color is measured.
