Immunoassay technique employing sacs having a lysable membrane and at least one determinant or epitopic site recognizable by an antibody. Enclosed in the sacs are water soluble stable free radical compounds, which are capable of escape from the sac upon lysis. The immunoassay can be employed for the determination of an epitope containing compound, antibodies or complement. Two basic modes are employed. The first mode depends upon the difference in the EPR spectrum of the stable free radical when enclosed in the sac, at relatively high localized concentration, and when free in solution, at relatively low concentration. The amount of the free radical compound free in solution is dependent upon available antibody bound to the sacs and available complement. The second mode can be used for the determination of an epitope containing compound or antibody. In this mode, the antibody agglutinates the sacs, the agglutinated sacs are separated from the non-agglutinated sacs and either or both of the differentiated sacs lysed and the amount of free radical determined. The immunoassay is extremely sensitive and has a high multiplication factor, because a single event results in the freeing of a large number of free radical molecules.
CROSS REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of application Ser. No. 339,755, filed Mar. 12, 1973, whose disclosure to the extent that it includes matter not included in this application, is incorporated herein by reference.
A method of forming an analyte-functionalized liposome for use in immunoassay techniques employs a phosphotriester intermediate of a phospholipid derivatized with the desired analyte in a process of forming a liposome which is functionalized on its outer surface with the analyte and which carries an enzyme marker. The method is also used to functionalize the liposome with a ligand which acts as a leash between the analyte and the liposome. Liposomes produced by the method include penicillin-G-functionalized liposome.
Compounds are provided for use in assays of organic compounds, where organic compounds of biological interest are determined at extremely low concentrations by combining in a medium, the composition to be determined, hereinafter referred to as ligand, a high molecular weight material of at least 10,000 molecular weight, which has a site spatially characterisitc of the ligand, hereinafter referred to as receptor, and an analog of the ligand having a free radical functionality hereinafter referred to as "ligand analog". The ligand analog and ligand in the medium compete for the receptor site, the amount of ligand analog bound to the receptor, being dependent on the amount of ligand present in the medium. By following the change in electron spin resonance spectrum of the ligand analog and comparing it to the change in spectrum which would be obtained in the absence of any ligand, the amount of ligand can be determined.
An immunoassay by which a plurality of kinds of antigens or antibodies in one test sample may be simultaneously quantified. Each of a plurality of kinds of microcapsules is first provided for each kind of a plurality of antigens or antibodies to be quantified such that the microcapsules bind an antibody or antigen specific to one kind of the plurality of antigens or antibodies to be quantified. The microcapsules are formed of a membrane capable of being lysed by the complement activity, and each kind of microcapsules contains therein a substance that is quantifiable and does not interact with another quantifiable substance contained in other kinds of microcapsules. The plurality of kinds of microcapsules are mixed with a test sample and complement, and the quantifiable substances are released from the microcapsules upon lysis of the microcapsules by the complement activity. The quantifiable substances are then quantified.
In a homogeneous assay, binder supported on a solid support, having a sac lysing agent conjugated thereto, is contacted with analyte and tracer comprised of sacs containing a detectable marker. The sacs of the bound tracer portion are lysed to release marker to determine analyte in the sample.
