Testosterone ELISA Kit -sample data-see actual kit insert for batch specific information

Testosterone ELISA Kit -sample data-see actual kit insert for batch specific information

 Catalogue No   : RDI-RE52151  
 Product group  : Gynaecology                         12 x 8      
 Product name   : Testosterone   **510K**       ELISA   96
 Method         : ELISA                                                       
 Incubation time: 1 h, 15 min                                                 
 Standard curve : 0.2-16 ng/ml                                                
 Sample/Prep.   : 50 µl Serum, Plasma                                         
 Isotope/Substr.: TMB, 450 nm


Contents of the Kit
Principle of the Test
Test Procedure
Performance Characteristics
Expected Values
Alternative Applications
(More) Clinical Background
Sales Arguments
Product Literature



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  The testosterone assay is an Enzyme-Imunoassay (EIA) for the quantitative  
  determination of testosterone in human serum or plasma.                    
  Testosterone (17ß-Hydroxy-4-Androstene-3-on) is a C19 steroide hormone     
  with a molecular weight of 288 Dalton.                                     
  Testosterone is one of the most important male sex hormones. In man it is  
  mainly synthesized by the testes, in women both the ovaries and by the     
  adrenal cortex; it is secreted into circulation. Testosterone is           
  transported in the plasma by a beta-globulin, called testosterone binding  
  globulin. It is estimated that about 98 % of the circulating testosterone  
  is bound. The remainder, present as free testosterone, is assumed to be    
  the metabolic active portion. In the target organ, it is transformed by    
  enzymatic reduction into the physiologically effective androgen            
  In man the determination of testosterone is used as an indicator for the   
  function of the testes: low hormone levels are found in cases with         
  Klinefelter's syndrome, cryptorchism or anorchia. Male or female patients  
  with an androgen producing tumor (ovaries, adrenal cortex, testes) show    
  increased values. Measurement of testosterone is used to confirm hirsutism 
  in woman. The determination of free or not specifically protein-bound      
  testosterone can be helpful in cases of hyperprolactinemic women or        


Contents of the Kit

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  Do components contain < 250 µl solution, please care that all solution is  
  on the bottom of the vial.                                                 
  1.  Microtiter Strips                                    12 x 8 wells      
      break apart strips coated with polyclonal                              
      anti testosterone antiserum (rabbit, polyclonal)                       
      in foilbag with desiccant.                                             
  2.  Enzyme conjugate                                        1 vial         
      22 ml, ready to use,                                                   
      Testosterone conjugated to the enzyme horseradish                      
      peroxidase supplemented by enzyme stabilizers                          
      and 0.3 % procline.                                                    
  3.  Standard A (Zero Standard)                              1 vial         
      0.5 ml, ready to use                                                   
      proteinmatrix with 0.1 % thimerosal                                    
      and 0.3 % procline.                                                    
  4.  Standards (B - G)                                      6 vials         
      0.25 ml, ready to use,                                                 
      proteinmatrix with 0.1 % thimerosal                                    
      and 0.3 % procline.                                                    
      Concentration in ng/ml    0.2   0.5   1   2   6  16                 
  5.  TMB Substrate Solution                                  1 vial        
      22 ml, ready to use,                                                   
      containing a solution of tetramethylbenzidine (TMB)                    
      with hydrogen peroxide in buffer supplemented with                     
  6.  TMB Stop Solution                                       1 vial        
      11 ml, ready to use, 0.5 M sulphuric acid (H2SO4).                     
      Avoid contact with stop solution it may                                
      cause skin irritations and burns.                                      
  7.  Wash Buffer, concentrate (20 x)                         1 vial         
      25 ml, concentrate, in phosphat-buffered saline,                       
      with nonionic detergent, dilute 1 to 20 with                           
      distilled water.                                                       


Principle of the Test

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  This Testosterone ELISA KIT is based on the competition principle and the  
  microtiterplate separation. An unknown amount of antigen present in the    
  sample and a fixed amount of enzyme labelled antigen compete for the       
  binding sites of the antibodies coated onto the wells. After an incubation 
  the microtiterplate is washed to stop the competition reaction. Having     
  added the substrate solution the concentration of antigen is inversely     
  proportional to the optical density measured. The measured ODs of the      
  standards are used to construct a calibration curve against which the      
  unknown samples are calculated.                                            


Test Procedure

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  1. Summary:                                                                
  1.1.  Secure the desired number of coated strips in the holder.            
  1.2.  Pipet 25 µl of standards, controls or samples.                       
  1.3.  Incubate 5 minutes at room temperature.                              
  1.4.  Add 200 µl of enzyme conjugate.                                      
  1.5.  Thoroughly mix the plate for 10 seconds.                             
  1.6.  Incubate for 60 minutes at room temperature.                         
  1.7.  Rinse the wells 3 times with diluted wash buffer.                    
  1.8.  Add 200 µl of TMB substrate solution.                                
  1.9.  Incubate for 15 minutes at room temperature.                         
  1.10. Add 100 µl of TMB stop solution.                                     
  1.11. Determine the absorbance at 450 ñ 10 nm.                             
  2. Detailed Instructions for use                                           
  2.1.  Secure the desired number of coated strips in the holder.            
  2.2.  Dispense 25 µl of standards into appropriate wells.                  
  2.3.  Dispense 25 µl of sample into selected wells. Time between           
        distribution of first standard and last sample can be up to 10       
        minutes without affecting the results.                               
  2.4.  Incubate 5 minutes at room temperature.                              
  2.5.  Dispense 200 µl of enzyme  conjugate into each well.                 
  2.6.  Thoroughly mix the plate for 10 seconds. It is important to have     
        complete mixing in this step.                                        
  2.7.  Incubate for 60 minutes at room temperature ( 18 - 24 °C) without    
        covering the plate.                                                  
  2.8.  Briskly shake out the contents of the wells.                         
  2.9.  Rinse the wells 3x  with diluted wash buffer (300 µl per             
        well). Strike the wells sharply on absorbent paper to remove         
        residual droplets.                                                   
  2.10. Add 200 µl of TMB substrate solution to each well, at timed          
  2.11. Incubate for 15 minutes at room temperature (18 - 24 °C).            
  2.12. Stop the enzymatic reaction by adding 100 µl of TMB stop solution to 
        each well at the same timed intervals as in step 10 and determine    
        the absorbance of each well at 450 ñ 10 nm, within 30 minutes        
        following step 12.                                                   
  Storage and Stability                                                      
  When stored at 2 - 8 °C unbroken reagents will retain reactivity until     
  expiration date. Do not use reagents beyond this date.                     
  Enzyme conjugate, standards, substrate solution, wash buffer must be       
  stored at 2 - 8°C.                                                         
  Microtiter strips must be stored at 2 - 8 °C. Once the foilbag has been    
  broken care should be taken to close it tightly again. The                 
  immuno-reactivity of the coated microtiter wells is stable for approx. 6   
  weeks in the broken but tightly closed plastic zip pouch containing the    
  Specimen collection and preparation for analysis                           
  Serum and plasma                                                           
  Serum or EDTA plasma should be used in the assay. No special pretreatment  
  of sample is necessary. The specimen may be stored at 2 - 8° C for up to   
  24 hours, and should be frozen at - 10° C or lower for longer periods. Do  
  not use grossly hemolyzed or grossly lipemic specimens.                    
  Samples suspected to contain testosterone concentration higher than        
  16 ng/ml are to be diluted with zero standard.                             
  Attention: Samples containing sodium azide should not be used in this      
  PREPARATION OF SAMPLES AND REAGENTS                                        
  Dilute the wash buffer concentrate with distilled water 1 to 20. Ready to  
  use wash buffer is stable for 2 weeks when stored at room temperature.     
  GENERAL REMARKS:                                                           
  All reagents and specimens must be allowed to come to room temperature     
  before use. All reagents must be mixed without foaming.                    
  Once the test has been started, all steps should be completed without      
  Use new disposable plastic pipette tips for each reagent, standard or      
  specimen in order to avoid cross contamination. For the dispensing of the  
  substrate  solution and the stop  solution avoid pipettes with metal       
  Pipette standards and samples onto the bottom of the well. For pipetting   
  of enzyme conjugate and stop solution it is recommended to hold the        
  pipette in a vertical position above the well and dispense the             
  correspondent solution into the centre of the well so that a complete      
  mixing of enzyme conjugate with sample or standard and of the stop         
  solution with the substrate solution is achieved.                          
  Before starting the assay, it is recommended that all reagents be ready,   
  caps removed, all needed wells secured in holder, etc. This will ensure    
  equal elapsed time for each pipetting step without interruption.           
  As a general rule the enzymatic reaction is linearly proportional to time  
  and temperature. This makes interpolation possible for fixed               
  physico-chemical conditions. If in a test run the absorbance of zero       
  standard is lower than 1.0 or above the upper performance limit of your    
  microtiterplate spectrophotomter you can extend or reduce the incubation   
  time of the final enzymatic formation of color to 30 or 10 minutes         
  accordingly. Since calibrators are assayed in each run, absorbance         
  fluctuations do not affect the result.                                     
  The substrate solution should be colourless or slightly blue or green. If  
  the solution is dark blue the reagent is unusable and must be discarded.   
  During incubation with substrate solution avoid direct sunlight on the     
  microtiter plate.                                                          


Performance Characteristics

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  1.  Specificity                                                            
  The specificity of the testosterone assay was assessed according to        
  Abraham's method.                                                          
                  Steroid                         Cross-reactivity (%)       
                  Testosterone                              100              
                  Androstendione                            0.9              
                  Androsterone                              0.9              
                  5à- Dihydrotestosterone                 < 0.05             
                  17-ß-Estradiol                          < 0.05             
                  Estron                                  < 0.05            
                  Estriol                                 < 0.05            
                  Epitestosterone                         < 0.05            
                  17-OH-Progesterone                      < 0.05            
                  Progesterone                            < 0.05            
                  Cortisol                                < 0.05            
                  DHEA-SO4                                < 0.05           
  2.  Sensitivity                                                           
  The lowest detectable level of testosterone - defined as the testosterone  
  concentration given by the mean absorbance of  the zero  standrad minus 2  
  standard deviations was assessed to be approx. 0.1 ng/ml.                  
  3. Precision                                                               
  Intra Assay Variation                                                      
  Within run variation was determined by replicate determination of three    
  different control sera in one assay. The within assay variability is shown 
     Mean concentration   n           CV (%)                                 
            0.83         20           3.9                                    
            5.54         20           4.0                                    
            9.91         20           5.8                                    
  Inter Assay Variation                                                      
  Between run variation was determined by replicate measurements of three    
  different control sera in several differebt assays. The between assay      
  variability is shown below:                                                
     Mean concentration   n           CV (%)                                 
            0.82         11           6.4                                    
            5.44         21           5.5                                    
            13.1         11           8.7                                    
  4. Linearity                                                               
  Three patient samples were serially diluted with zero standard in a        
  linearity study.                                                           
    Sample    Dilution   Measured conc.  Expected conc.    Recovery          
                           (ng/ml)          (ng/ml)          (%)             
       1        neat        2.42             --              --              
                1:2         1.32             1.21            109             
                1:4         0.62             0.60            102             
                1:8         0.31             0.30            102             
       2        neat        5.13             --              --              
                1:2         2.63             2.57            103             
                1:4         1.29             1.28            101             
                1:8         0.69             0.64            108             
                1:16        0.36             0.32            112             
       3        neat        12.0             --              --              
                1:2         6.31             6.00            105             
                1:4         3.21             3.00            107             
                1:8         1.73             1.50            115             
                1:16        0.79             0.75            105             
  5. Recovery                                                                
  Spiked serum samples were prepared by adding varying levels of             
  testosterone to two different serum samples.                               
   Serum    Added Testosterone   Measured Testosterone     Recovery          
              (ng/ml)                   (ng/ml)              (%)             
     1           0                        3.0                 --             
                 1                        3.6                 90             
                 2                        4.9                 98             
                 4                        7.1                101             
     2           0                        0.6                 --             
                 1                        1.3                 82             
                 2                        2.1                 81             
                 4                        4.2                 92             
                 8                        8.0                 93             


Expected Values

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          Females       0.1 - 1.2 ng/ml                                      
          Males         2.4 - 12  ng/ml                                      
          Conversion factor: 1 ng/ml = 3.47 nmol/l                           


Alternative Applications

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  Determination of Steroids in Saliva                                        
  Preparation of Saliva Samples                                              
  The saliva samples have to be extracted prior to use. Patients should      
  rinse their mouth with tap water 15 min before taking the sample. It is    
  recommended to  freeze the sample. After thawing, the sample should be     
  centrifuged to separate mucines.                                           
  The following extraction procedures are suitable for the determination of  
  DHEA-S, progesterone, 17-OH-progesterone, estradiol, testosterone and      
  cortisol in saliva.                                                        
  The supernatant should be used for extraction. In general concentrations   
  of steroids in saliva are about 10 times lower compared to serum.          
  Therefore we recommend to reconstitute the extract in a lower volume (e.g. 
  20 µl zero standard if the original volume was 100 µl saliva) of zero      
  To compensate possible loss due to extraction, standards should be treated 
  in the same way.                                                           
  The following extraction methods are recommended:                          
  Method 1:                                                                  
  1.  Mix 100 µl saliva with 2 ml ether (*).                                 
  2.  Shake for 20 minutes at room temperature.                              
  3.  Freeze-out organic phase at -20 °C (*).                                
  4.  Pipette the supernatant into a test tube and evaporate (e.g. with      
      nitrogen) in a water bath at 35 °C.                                    
  5.  Reconstitute the residue with 100 µl zero standard.                    
  6.  This extract may be used as described in the assay procedure.          
  (*) If H2O saturated ether is used, the separation of the phases may be    
      performed without the freezing step (3).                               
  Method 2:                                                                  
  1.  Mix 150 µl saliva with 1.5 ml Methylenchloride.                        
  2.  Shake for 20 minutes at room temperature.                              
      Note: If the sample is cloudy, centrifuge 10 minutes at 500 x g.       
  3.  Aspirate 1 ml of the lower organic phase and evaporate (e.g. with      
      nitrogen) in a water bath at 40 °C.                                    
  4.  Reconstitute the residue with 100 µl zero standard.                    
  5.  This extract may be used as described in the assay procedure.          


(More) Clinical Background

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  Monitoring of androgen suppressing drugs:                                  
  Testosterone measurements may also be utilized in women for the adjustment 
  of androgen suppressing drugs and their dosages. Common oral               
  contraceptives drugs and drugs containing cyproterone acetate (CPA) are    
  very effective in the suppression of testosterone concentrations.          
  Factors affecting normal values:                                           
  1. Combination oral contraceptives:                                        
     Testosterone concentrations are suppressed by administration of oral    
  2. Corticosteroids:                                                        
     Testosterone concentrations are suppressed in patients taking adrenal   
     corticosteroids even with very small doses.                             
  3. GnRH analogous:                                                         
     Testosterone concentrations are suppressed during pituitary down        
     regulation with analogous.                                              
  4. Danazol:                                                                
     Testosterone concentrations will be artifactually high in women taking  
     danazol. Although danazol (17à-pregna 2,4-dien-20-yno-(2,3-d)-isoxazol  
     -17-0l ) shows negligible cross reactivity, its metabolic by-products   
     may display a high cross-reactivity. There is rarely, if ever, an       
     indication to determine testosterone concentrations in serum of women   
     taking danazol.                                                         
  Testosterone concentrations are relatively unchanged over the three