Patent Publication Number: US-2010112599-A1

Title: Method for the coupled enzyme immunochemical assay of analytes by means of endogenous calibrators

Description:
The invention relates to a method for assaying a plurality of analytes such as e.g. metabolites and antigens in biological and other liquid samples by means of analytical elements, especially lateral-flow test strips, flow-through membrane systems (flow-through tests), wells/cavities of microtitre plates or test tubes. Application areas of the invention are principally medical diagnostics, the pharmaceutical industry and protection of the environment. 
     Analytical elements, such as e.g. immuno-chromatographical test systems for assaying numerous analytes in biological liquids or for assaying pollutants in environmental samples have been known for years and have proved very successful in practice. They work mainly according to the sandwich or competition principle (in the case of small analytes). The use of affinity assays, e.g. immunoassays, receptor and DNA assays is becoming ever increasingly important for clinical and numerous other applications. In this case, very different analytes are assayed in different sample matrices (e.g. urine, saliva, tear fluid, sweat, liquor or blood). There is still one problem at the moment, that the dilution of at least part of the said matrices (e.g. urine) is not constant; therefore, in the course of the day, definite concentration changes occur, which influence the corresponding analyte measured value. However, endogenously formed substances are described for individual body matrices, with the help of which the corresponding dilution can be corrected; therefore, the system can be calibrated (Federal health sheet—health research—health protection 5 2005; Norpoth K, Heger M (1984), Creatinine as a reference variable for indicating substance concentrations in the urea. In: Hentschler D, Lehnert G (Hrsg) Biological Agent Tolerance Values (BAT values), occupational medical toxicological grounds, Vol. 1, Commission for the testing of health-hazardous biological agents of the DFG). Creatinine in the sample matrix urine is an example of these substances acting as endogenous calibrators. In the case of spontaneous urine samples of healthy test subjects, concentration fluctuations between 4 and 28 mmol of creatinine per litre of urine are quite normal (Fundamentals of Laboratory Testing: Urine, Roche Diagnostics GmbH, Mannheim, Germany). If these values are compared with the average value (15 mmol/l) of the 24 hour urine, it becomes clear that a dilution by the factor 3.5 is in the normal range, but the urine can also be concentrated by the factor 2. Based on an analyte to be determined in the sample matrix urine, this means: the measured value determined has to be corrected accordingly, so therefore a double determination (endogenous calibrator and analyte) followed by a calculation is necessary. In practice, this means carrying out two separate tests. In the case of urine, the creatinine concentration is determined first of all via an enzymatic cascade, and the volume of the analyte in the second stage (usually immuno-chemically). This approach is both time-consuming and labour-intensive. 
     Therefore, the object of the invention was to find new solutions for test systems that determine various analytes in sample matrices with concentrations that are not constant. 
     The invention is achieved according to the claims. It relates to a method for assaying a plurality of analytes such as e.g. metabolites and antigens in biological or other liquid samples by means of analytical elements, especially lateral-flow test strips, flow-through membrane systems (flow-through test), wells/cavities of microtitre plates or test tubes, the method being based on coupled enzyme and affinity reactions and implemented by means of endogenous calibrators. 
     The main idea consists of coupling enzyme and immunochemical methods and including the concentration of endogenous calibrators of the corresponding sample matrix in the total result, the analyte concentration being corrected automatically by the system achieved. With this combined enzymatic/immunological test system, body fluids are incubated with an enzyme mix (reaction 1), when the hydrogen peroxide (H 2 O 2 ) produced with incubation through conversion of the endogenous calibrator is used partly or completely in a second, now immune reaction (reaction 2) with the same body fluid for signal formation through a marker enzyme. This signal is picked up and compared or offset with the signal of the H 2 O 2  produced in the first reaction. 
     The two reactions of the method can be carried out simultaneously or sequentially and can also be carried out in one compartment or in 2 separate compartments. Preferably lateral-flow test strips, flow-through membrane systems (flow-through test), wells/cavities of microtitre plates or test tubes are used as compartments. According to the invention, the signal is picked up visually (naked eye), colourimetrically, through fluorescence or electrochemically. 
     The evaluation is made by means of nomogram, comparator (reference strip), reader or comparison with the naked eye, when, in the case of the latter, the number of signals generated enzymatically by means of a calibrator (e.g. test lines or test dots) is set in the ratio to the number of signals generated immunologically by means of analyte (e.g. test lines or test dots). 
     With the new method, body fluids, including urine, saliva, tear liquid, sweat, liquor or blood can be investigated. Creatinine, glucose, glucose-6-phosphate, lactate, glutamate, aspartate, cholesterol, pyruvate, urea and triglycerides in particular are used as endogenous calibrators. Marker enzymes are preferably peroxidases and oxidases. Preferably, an enzyme mix is used that reacts with the endogenous calibrator present in the body fluid concerned with the formation of H 2 O 2 . For example, in the case of the body fluid urine, when using creatinine as the endogenous calibrator, a mix of creatininase, creatinase and sarcosin oxidase is used. However, if glucose is used as the endogenous calibrator in the same sample matrix, glucose oxidase is used to generate the H 2 O 2 . 
     Surprisingly, it was found that with such a combined system in sample matrices with non-constant concentrations, a very wide range of analytes can be determined directly in a reliable manner, i.e. with the corresponding dilution correction. The invention is now explained in more detail below with the help of illustrations. 
    
    
     EXAMPLES 
     Example 1 
     Assay of Cardio-Specific Fatty Acid Binding Protein (FABP) with Simultaneous Correction by Means of an Endogenous Calibrator (Creatinine) with Lateral-Flow Test Systems in the Sample Matrix Urine 
     See FIGS.  1 ,  3 ,  4  and  6   
     The urine sample (200 μl) is divided into 2 equal portions (A and B). Part A is introduced into a test tube (see  FIG. 3 ), which contains the following enzyme mix in lyophilised form (per 20 μl): Creatininase (18.8 U/ml), Creatinase (7.5 U/ml) and Sarcosin Oxidase (11.3 U/ml). The test tube is agitated and incubated for 20 minutes at room temperature (20-25° C.). The endogenous creatinine present in the urine is converted by the enzyme mix reconstituted in this way to hydrogen peroxide (H 2 O 2 ) among other things (see  FIG. 1 ). Part B is diluted 1:4 first of all with a commercial protein stabiliser and 50 μl is then applied to the test strips ( FIG. 4 ; sample opening  1 ). The anti-FABP/horse-radish peroxidase conjugate immobilised under sample opening  1  is dissolved out of its matrix, forms a complex (FABP-Anti-FABP/horse-radish peroxidase) with the analyte and in this form flows over the test field, on which a further anti-FABP antibody is immobilised as a catcher. With the presence of FABP, the latter binds the complex mentioned as a function of the analyte concentration. 20 minutes after the application of part B, 100 μl of pre-incubated part A is introduced into sample opening  2 . Therefore, the immobilised H 2 O 2 -free and locally tightly limited precipitating peroxidase substrate on the test strip is dissolved and, together with the H 2 O 2  (resulting from the conversion of the creatinine), flows over the test field. If the position of the catcher line is reached, the liquid, colourless substrate is now converted into a blue/violet precipitate. The volume of the precipitate, i.e. the generated signal, is a function of the analyte concentration and the volume of endogenous calibrator, which is used to generate the H 2 O 2  necessary for the reaction. Highly endogenous calibrator (concentrated morning urine) generates a stronger signal than a sample with an average calibrator concentration (day-time urine) with the same analyte concentration. The test system described corrects this effect through the way it works and in this case, allows the true analyte concentration to be recorded with the aid of a comparator card (see  FIG. 6 ). 
     Example 2 
     Assaying FABP with Simultaneous Correction by Means of an Endogenous Calibrator (Glucose) with Microtitre Plate-Based Macrodot Assays in the Sample Matrix Urine 
     See FIGS.  2  and  5   
     Commercial membrane-coated microtitre plates (96 wells) are used for the macrodot assays (FIG.  5 / 1 ). Preferably the membrane material consists of PVDF or nitrocellulose. The dot model used depends on the application concerned; examples are illustrated in FIG.  5 / 2 . Preferably, 5 dots are used (FIG.  5 / 3 ), when the centre dot functions as a control dot as a rule. The outer dots can be used both for assaying different analytes and for the dilution corrections of individual analyte concentrations. The example to be described focuses on the assay of individual analytes (FABP) with simultaneous correction of the dilution of the sample matrix used (urine). 4 dots (see  FIG. 5 , centre section), which contain different concentrations of anti-FABP antibodies (catchers), were used. The urine sample (400 μl) is divided into 2 equal portions (A and B). Part A is introduced into a test tube containing 6.5 U glucose oxidase and 0.5 mM glucose. A small, fixed amount of glucose has to be added separately to produce a basic level of H 2 O 2 . The test tube is agitated and incubated for 60 minutes at room temperature (20-25° C.). The endogenous glucose present in the urine is converted to hydrogen peroxide (H 2 O 2 ) among other things (see  FIG. 2 ). Part B is diluted 1:10 first of all with a commercial protein stabiliser. The following reaction mixtures are made: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 1 
                 2 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 μl of the dilution (part B) 
                 199 
                 194 
               
               
                   
                 μl Anti-FABP antibody 
                 1 
                 1 
               
               
                   
                 horse-radish peroxidase conjugate 
               
               
                   
                 μl FABP from 4,000 ng/ml 
                 0 
                 5 
               
               
                   
                 solution 
               
               
                   
                 Corresponds to following 
                 0 
                 100 
               
               
                   
                 FABP final concentration in ng/ml 
                   
                   
               
               
                   
                 μl final volume 
                 200 
                 200 
               
               
                   
                   
               
            
           
         
       
     
     100 μl reaction mixture is added to each well and incubated at room temperature (20-25° C.) every 60 minutes. FABP is added to be able to illustrate the effects to be described more clearly. Only with the presence of FABP can the following complex form on the catcher antibody: Catcher antibody/FABP/Anti-FABP antibody horse-radish peroxidase conjugate. The plate is then washed 4 times (0.1 M Na—P buffer, pH 7.2) and 50 μl of the following mixture is added to each well: 
     1 part Part A
 
1 part H 2 O 2 -free, locally tightly limited precipitating peroxidase substrate.
 
     Incubation of at least 5 minutes at room temperature follows. In this case, at the positions at which the horse-radish peroxidase is bound, the liquid, colourless substrate is converted into a blue precipitate, when the volume of the precipitate, i.e. the signal generated, depends on the analyte concentration and the volume of endogenous calibrator (glucose) that is used to generate the H 2 O 2  necessary for the reaction. The plate is then again washed 4 times (as above). After drying, the evaluation is made using an imaging process, for example.  FIG. 5  shows both the RGB pictures without FABP addition ( 4 ) and with its addition ( 5 ) and also the grey stage pictures without FABP addition ( 6 ) and with it ( 7 ) used for the evaluation. Quantification took place on the basis of the latter pictures, whose data are given in Table 1. The effects of this self-calibrating assay become clear with the addition of 100 ng/ml FABP to both urine samples. It is evident from the last line that the morning urine used is concentrated 1.74 times compared with day-time urine. The signals generated through the model used with the catcher antibodies (1; 2; 4 and 8 ng/ml) are evaluated with reference to their dynamic. After strong signals for 1 ng/ml catcher antibody, from 2 ng/ml catcher antibody, a continuous signal rise to saturation takes place. With the same analyte concentration in the day-time urine, this saturation is reached very much earlier, because this non-concentrated sample contains less endogenous calibrator (glucose in this case) and therefore only a smaller amount of H 2 O 2  can be formed. Therefore, the saturation curves can be described mathematically for diagnostically relevant concentration ranges and implemented in the evaluation software. The corrected concentration determination of analytes in samples then takes place through the comparison of the saturation curves. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Quantification of the results shown in FIG. 5 
               
            
           
           
               
               
               
            
               
                   
                 without analyte addition 
                 with 100 ng/ml analyte addition 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Catcher Ng/ml 
                 1 
                 2 
                 4 
                 8 
                 1 
                 2 
                 4 
                 8 
               
            
           
           
               
            
               
                 Measured values correspond to the pixel average value of the range selected for evaluation 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Day-time urine 
                 2,830 
                 3,113 
                 1,582 
                 2,958 
                 5,623 
                 3,472 
                 4,042 
                 4,062 
               
               
                 Morning urine 
                 3,897 
                 2,278 
                 1,950 
                 3,215 
                 8,970 
                 5,117 
                 6,808 
                 9,011 
               
               
                 Morning urine − Day-time urine 
                 1,067 
                 −835 
                 368 
                 257 
                 3,347 
                 1,645 
                 2,766 
                 4,949 
               
               
                 Morning urine:Day-time urine 
                 1.38 
                 0.73 
                 1.23 
                 1.09 
                 1.60 
                 1.47 
                 1.68 
                 2.22 
               
            
           
           
               
               
               
            
               
                 Total Day-time urine 
                 10,483 
                 17,199 
               
               
                 Total Morning urine 
                 11,340 
                 29,906 
               
               
                 Total morning urine − Total day-time urine 
                 857 
                 12,707 
               
               
                 Total morning urine:Total day-time urine 
                 1.08 
                 1.74 
               
               
                   
               
            
           
         
       
     
     Example 3 
     Flow-Through Macro Dot Assays for Assaying Analytes with Simultaneous Correction by Means of Endogenous Calibrators for Various Sample Matrices 
     FIG.  7   
     Example 3 describes the application of flow-though macrodot assays for assaying analytes with simultaneous correction by means of endogenous calibrators for various sample matrices. In this case, 7 dots are applied to a flow-through membrane system first of all (cf.  FIG. 7 ). Dots ‘1 ’ to ‘4 ’ contain anti-analyte antibodies in increasing concentrations. Dot ‘K’ functions as a control dot and preferably contains anti-mouse immunoglobulin G or protein A. Dot ‘EK’ is used for assaying the corresponding endogenous calibrator and therefore preferably consists of individual enzymes or enzyme mixtures and also peroxidase. Dot ‘EK-Ü’ is optional and, when used, contains the corresponding calibrator (e.g. creatinine or glucose) in the optimum concentration for the application and also the components that are necessary for converting the calibrator into H 2 O 2 . A locally tightly limited precipitating peroxidase substrate is added to all the dots. 
     When this test system is used, the sample is pre-incubated first of all in a test tube that contains the necessary enzyme mix to convert the endogenous calibrator concerned or the corresponding individual enzyme and also horse-radish perioxidase-coupled anti-analyte antibodies. This reaction mix is then applied to the test field of the flow-through membrane system. If analyte is present in the sample, the analyte-horse-radish peroxidase-coupled anti-analyte antibody binds to the corresponding catcher antibodies (dots ‘1 ’ to ‘4 ’). Because of the different catcher antibody concentrations, a saturation takes place in the direction of dot ‘4 ’. The product formed through conversion of the endogenous calibrator in the reaction mix (preferably H 2 O 2 ) now initiates the conversion of the H 2 O 2 -free, locally tightly limited precipitating peroxidase substrate previously immobilised in each dot from its colourless preliminary stage to a blue/violet precipitate. In this case, the precipitate volume depends on the peroxidase present locally and the H 2 O 2  concentration, for example.