Hypothesis: Qualitative analysis of  water samples taken from five different sites located within the Stony Brook/Millstone Watershed of Princeton New Jersey will detect a presence of 2,4 D.
 
Materials in Kit:                                                               Other Materials  (not in kit)
 
20 antibody-coated test tubes                                                        1 Marking pen for test tubes
  1 vial of Negative Control                                                            1 Disposable-tip pipette which will measure
  1 vial of 2.0 ppb 2,4-D Calibrator                                                  160 micro liters
  1 vial of 50 ppb 2,4-D Calibrator                                                 1 Stopwatch or wristwatch with second hand
  1 vial of 2,4-D Enzyme Conjugate                                                1 Pipette which will measure 0.5 milliliters (mL)
  1 vial of Substrate                                                                           (for photometric interpretation only)
  1 vial of Chromogen                                                                     1 Calculator
  1 vial of Stop Solution                                                                  1 Spectrophotometer
  1 test tube rack

** The EnviroGard, 2,4-D Test kit used in data analysis from Millipore is based on the use of polyclonal antibodies which bind either 2,4,-D residues or 2,4-D-enzyme conjugate.  The 2,4-D from the sample competes with 2,4-D-enzyme conjugate for a limited number of antibody binding sites.  Antibodies that bind 2,4-D are immobilized to the walls of the test tubes.
    Since there are the same number of antibody binding sites on every test tube, and each test tube receives the same number of 2,4-D-enzyme conjugate molecules, a sample which contains a low concentration of 2,4-D enables the antibody to bind many 2,4-D-enzyme conjugate molecules.  Therefore, a low concentration of 2,4-D will allow fewer 2,4-D-enzyme conjugate molecules to be bound by the antibodies. 

Procedure:

    1.    Test tubes coated with antibody were marked as follows:

                Tube Marking                            Tube Contents

                          "-"                                        negative control
                          "+"                                       2.0 or 50 ppb calibrator
                          S1                                        sample 1
                          S2                                        sample 2
                          S3                                        sample 3
                          S4                                        sample 4
                          S5                                        sample 5
 
    2.    Add 4 drops (160 ul) of sample or negative control or 50 ppb calibrator depending on test tube
    3.  Add 4 drops (160 ul) of enzyme conjugate to each test tube.
    4.  Gently mix and let stand for 10 minutes.
    5. Fill the test tubes to overflowing with tap or distilled water, decant  and vigorously shake out the water.
    6.  Repeat rinse 4 times.
    7.  Add 4 drops of substrate to each test tube.
    8.  Immediately add 4 drops of chromogen to test tubes.
    9.  Mix by shaking and let sit for 10 minutes.
    10.  Add 1 drop of STOP solution to each test tube. - Solution will turn yellow.
    11.  Add 500 ul of distilled water prior to reading on the Spectrophotometer 20.
    12.  Measure and record the absorbence at 450nm of the negative control, each sample, and the calibrator.

 
 Calculations:

    Compare the OD of each sample to the OD of the calibrator.  If the OD of the sample is less than the OD of the calibrator, the sample contains a concentration greater than the concentration of that calibrator.  Conversely, if the OD of the sample is greater than the OD of a calibrator, the sample contains a concentration that is lower than the concentration found in the calibrator.

                                    Example:                        OD                    Interpretation

                                    Neg. Ctrl.                                 1.298
                                                2.0 ppb                                    1.068
                                                50 ppb                                     0.439
                                                Sample                                    0.755                            >2.0 ppb; <50 ppb
 
 
 
 
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