Process for determining one or more analytes in samples of biological origin having complex composition, and use thereof

Abstract

The present invention relates to a process for detecting one or more analytes in one or more samples of biological origin having complex composition. The present invention also relates to a microarray for quantitative determination of one or more analytes in samples of biological origin having complex composition which are immobilized in measurement ranges of microarray, and also to a quantitative detection method based thereon.

1 - 39 . (canceled) 
     
     
         40 . A microarray for quantitative determination of one or more analytes in one or more samples of biological origin and complex composition, which microarray comprises
 a solid support,   a first multiplicity of discrete measurement areas in which in each case small amounts of samples of biological origin and complex composition in diluted or undiluted form are immobilized either directly or by mediation via an adhesion-promoting layer,   
       characterized in that
 at least a second multiplicity of measurement areas is provided in said array on the solid support, in which measurement areas substances of the same kind as the analytes to be detected are immobilized in different concentrations which are suitable, by means of contacting said microarray with a first solution comprising one or more binding reagents as specific binding partners for the analytes to be detected and present in the first multiplicity of discrete measurement areas in the applied samples of biological origin and complex composition, and for the substances of the same kind as said analytes to be detected, which are present in said second multiplicity of discrete measurement areas, and, optionally if required, one or more detection reagents, it being possible for binding reagents and detection reagents to be applied simultaneously or sequentially, and subsequent space-resolved measurement of first optical signals which are emitting from discrete measurement areas of one or more arrays, which have been contacted with the first solution, and recording said first optical signals, for generating a calibration curve for said analytes to be detected quantitatively and present in the immobilized samples of complex composition. 
 
     
     
         41 . A microarray as claimed in  claim 40 , characterized in that a third multiplicity of measurement areas is provided in said array on the solid support, in which in each case small amounts of samples of biological origin and complex composition in diluted or undiluted form and, in addition, known amounts added thereto of substances of the same kind as the analytes to be detected are immobilized. 
     
     
         42 . A microarray as claimed in  claim 40 , characterized in that a fourth multiplicity of measurement areas is provided in said array on the solid support, in which substances are immobilized which are of a similar kind as substances present in the sample matrix of the samples applied to the first multiplicity of measurement areas. 
     
     
         43 . A microarray as claimed in  claim 40 , characterized in that the measurement areas of the second multiplicity of measurement areas comprise substances which are of a similar kind as substances present in the sample matrix of the samples applied to the first multiplicity of measurement areas. 
     
     
         44 . A microarray as claimed in  claim 42 , characterized in that the substances which are of a similar kind as substances present in the sample matrix of the samples applied to the first multiplicity of measurement areas are derived from the group comprising albumins, in particular bovine serum albumin, immunoglobulins or diluted serum. 
     
     
         45 . A microarray as claimed in  claim 40 , characterized in that a fifth multiplicity of measurement areas is provided in said array on the solid support, which is used for referencing purposes. 
     
     
         46 . A microarray as claimed in  claim 45 , characterized in that the measurement areas of the fifth multiplicity of measurement areas comprise substances which are selected from the group comprising mass labels and luminescent labels. 
     
     
         47 . A process for quantitative determination of one or more analytes in a sample of biological origin and complex composition, comprising the following steps:
 providing one or more samples of biological origin and complex composition,   providing a multiplicity of solutions of different concentrations of substances of the same kind as the analytes to be detected in the samples of complex composition,   providing at least one solid support,   generating a first multiplicity of discrete measurement areas as part of a microarray by applying small amounts of the samples of biological origin and complex composition in diluted or undiluted form to discrete sites, either directly on the solid support or, after prior application of an adhesion-promoting layer, on said adhesion-promoting layer on said solid support,   generating a second multiplicity of discrete measurement areas as part of said microarray by applying in each case small amounts of the multiplicity of solutions of different concentrations of substances of the same kind as the analytes to be detected in the samples of complex composition,   contacting the microarray with a first solution comprising one or more binding reagents as specific binding partners for the analytes to be detected and present in the first multiplicity of discrete measurement areas in the applied samples of biological origin and complex composition, and the substances of the same kind as said analytes to be detected, which substances are present in the second multiplicity of discrete measurement areas, and optionally if required, one or more detection reagents, it being possible for binding reagents and detection reagents to be applied simultaneously or sequentially,   measuring in a space-resolved manner first optical signals emitting from said first and second multiplicities of discrete measurement areas of the microarray, recording said first optical signals from said first multiplicity of discrete measurement areas as signals characteristic for the latter contacting the first solution, recording the first optical signals from the second multiplicity of discrete measurement areas as signals characteristic for the latter contacting the first solution as a function of the concentration of the substances of the same kind as the analytes present in the samples of complex composition, which substances are present in said measurement areas,   generating calibration curves for the analytes to be detected in the samples of complex composition from the recorded optical signals from the second multiplicity of discrete measurement areas, if required after prior subtraction of background signals and suitable referencing,   quantitatively determining the analytes to be detected and present in the samples of complex composition by comparing the recorded first optical signals from the first multiplicity of discrete measurement areas with the calibration curves for the particular analytes, if required after previous subtraction of background signals and suitable referencing.   
     
     
         48 . (canceled)