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TSH ELISA KIT by dmm14830

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									                                                                                                  Substrate Tablets                 MATERIALS AND
                                                                                                  1 vial      REF # EST4            EQUIPMENT REQUIRED
                                                                                                  4 x 4 mg tablets of ortho-        BUT NOT PROVIDED
                                                                                                  phenylenediamine (OPD) with       * Distilled or deionised
                                                                                                  inactive ingredients.                water
                                                                                                  TSH: Calibrators                  * 1M H2SO4
                                                                                                  6 vials     REF # ETSS1-6         * Precision pipettes
                                                                                                  1.0 mL each in bovine serum.      * Repeating pipette
                                                                                                  Contains sodium azide,            * 1L measuring cylinder

                  ELEGANCE                                                                        0.1% w/v. Lyophilized.
                                                                                                  PRECAUTIONS AND
                                                                                                                                    * Absorbent tissue
                                                                                                                                       (lint-free)
                                                                                                                                    * Timer
                TSH ELISA KIT                                                                     WARNINGS TO USERS
                                                                                                  Handling of specimens and kit
                                                                                                                                    * Vortex mixer
                                                                                                                                    * Microtitre plate shaker
                                                                                                  components, their use, storage    * Microtitre plate washer
                                                                                                  and disposal should be in         * Microplate reader system.
                                                                                                  accordance with any local or
                                      40 460096                                                   national laboratory safety        PROCEDURAL NOTES
                                                                                                  procedures or regulations.        Bring all reagents and
                                      96                                                          Specimens and Calibrators         specimens to room tempera-
                                                                                                  The source material of the        ture (20-25°C) and mix by
WARRANTY                                                                                          calibrators has been tested by    gentle inversion prior to use.
The manufacturer makes no express warranty other than the diagnostic kit will measure the         an approved accredited            Duplicates are recommended.
designated analyte when used in accordance with the manufacturer's printed instructions.          method for the presence of        Contamination of reagents
The use of the diagnostic kit for any other purpose is outside the intended use of this product
and is done at the user's own risk.                                                               hepatitis B surface antigen,      will lead to poor performance.
The manufacturer disclaims any and all implied warranties of merchantibility, fitness for use     antibody to hepatitis C and       A calibrator curve should be
or implied utility for any other purposes. Any and all damages for failure of the diagnostic      antibody to HIV - 1/2 (AIDS)      run with each assay. Speci-
kit to perform according to its instructions are limited to the replacement value of the kit.
The sole liability of Bioclone Australia Pty. Limited and its distributors is limited to either   and has been found to be non-     mens suspected of having
replacement of the product or refund of the purchase price. Bioclone Australia Pty. Limited       reactive for all. However it is   concentrations above the top
is not liable for property damage, personal injury or economic loss caused by the products.       recommended that all samples      calibrator should be diluted in
                                                                                                  be handled as if capable of       zero calibrator before assay.
                      Manufactured by Bioclone Australia Pty Limited                              transmitting infectious           All assay steps should be
              (a subsidiary of Hitachi Chemical Co., Ltd) ABN 14 002 036 071                      disease.                          performed without interrup-
                71-73 Railway Parade Marrickville NSW AUSTRALIA 2204                              Preservatives                     tion, but if the wells cannot be
          Tel +61 (0) 2 9517 1966 Fax +61 (0) 2 9517 2990 Freecall 1800 251 138
                  Email sales@bioclone.com.au Web www.bioclone.com.au                             The kit contains sodium azide,    filled with Amplification
                                                                                                  thiomersal and Bronidox L as      Reagent or substrate solution
                                                                                                  preservatives. As reagents        immediately after washing,
            Hitachi Chemical Diagnostics Inc.
                                                                                                  contain potentially toxic         then the microwells may be
            Hitachi Europe Limited, Whitebrook Park, Lower Cookham Road                           preservatives, care should be     left upside down on absorbent
             Maidenhead, Berkshire, SL6 8YA, UK. Tel: +44 1628 585 591                            taken in handling, to avoid       lint-free tissue for a maximum
                                                                                                  ingestion or skin contact.        of 15 minutes.
INTENDED USE                                      TSH: Coated Microwells                          Sodium azide may react with       Reagents are matched in each
The ELEGANCE TSH ELISA                            96 wells REF # TSA96                            lead and copper plumbing to       kit and therefore reagents
has been designed for the                         Frame containing microwells                     form potentially explosive        from different lot numbers
quantitative in vitro diagnostic                  coated with anti-TSH                            azides.                           should not be mixed.
measurement of human                              antibody. Ready to use.                         Substrate                         The photometer and all
thyroid stimulating hormone                       TSH: Antibody Reagent                           Avoid any skin contact.           pipettes used should be
(TSH) in serum or plasma.                         1 vial       REF # TSB96                                                          calibrated appropriately
                                                                                                  SPECIMEN COLLECTION               before use.
                                                  5 mL biotinylated anti-TSH
PRINCIPLES OF THE                                                                                 AND HANDLING                      Washing
                                                  antibody in a buffered solution
ELEGANCE ELISA                                                                                    No special patient preparation    The efficiency of the wash
                                                  containing bovine serum
The ELEGANCE ELISA is an                                                                          is required. Specimens can be     step is vital for good preci-
                                                  albumin, non-immune animal
enzyme-linked immunoassay.                                                                        either serum or plasma            sion. Microwells are washed
                                                  sera and a blue dye. Contains
The sample antigen is                                                                             collected in a manner             using an automatic plate
                                                  sodium azide, 0.2% w/v and
"sandwiched" between the                                                                          appropriate for laboratory        washer. Avoid overflows
                                                  thiomersal, 0.01% w/v.
antibody bound to the                                                                             testing. Serum is preferred       from one well to another.
                                                  Ready to use.
microwell and the biotinylated                                                                    however, the anticoagulants       Quality Control
                                                  TSH:
antibody reagent. The                                                                             heparin or EDTA can be            Control specimens should be
                                                  Amplification Reagent
microwells are washed to                                                                          employed without sacrificing      run in every assay to ensure
                                                  1 vial       REF # TSP96
remove any unbound material.                                                                      accuracy.                         correct procedure. Control
                                                  10 mL streptavidin-peroxidase
Streptavidin-peroxidase                                                                           Avoid grossly haemolytic,         values should lie within
                                                  (streptavidin from S. avidinii)
(Amplification Reagent) is                                                                        lipaemic and turbid specimens.    laboratory ranges before assay
                                                  in a buffered solution contain-
added and binds to the                                                                            Specimens can be stored at        is approved.
                                                  ing bovine serum albumin and
biotinylated antibody at                                                                          2-8°C for up to 48 hours.
                                                  a violet dye. Contains
many sites. After washing,                                                                        Specimens held for longer         ASSAY PROCEDURE
                                                  Bronidox L, 0.2% v/v and
the substrate solution reacts                                                                     should be stored at or below      Preparation of Reagents
                                                  thiomersal, 0.02% w/v.
with any bound peroxidase to                                                                      -20°C. Specimens should not       Wash Solution
                                                  Ready to use.
produce colour in direct                                                                          be frozen and thawed              Dilute the wash concentrate
                                                  Wash Concentrate
proportion with the amount                                                                        repeatedly. Thawed speci-         1 in 15 with deionised water.
                                                  1 vial       REF # EWC96
of sample antigen, which can                                                                      mens should be checked for        The wash solution can be
                                                  50 mL of a 15 x concentrated
be calculated from the                                                                            flocculent matter and mixed       stored at room temperature
                                                  wash solution. Contains
calibrator curve.                                                                                 by inversion just prior to        (20-25°C) for 12 weeks.
                                                  thiomersal, 0.09% w/v.
                                                                                                  testing.                          Substrate Solution
ELEGANCE REAGENTS                                 To be diluted before use.
                                                                                                  Turbid specimens or speci-        It is recommended that this
PROVIDED, STABILITY                               Substrate Buffer
                                                                                                  mens containing particulate       reagent be made up just prior
AND STORAGE                                       1 vial       REF # ESB20
                                                                                                  matter should be centrifuged      to use. Place correct number
Kit size - 96 tests. The kit                      20 mL urea peroxide in a
                                                                                                  prior to use.                     of OPD tablets into the
and all its components,                           citrate-phosphate buffer.
                                                                                                                                    required amount of substrate
unopened or opened, should be                     Contains thiomersal,
                                                                                                                                    buffer. Add 1 tablet per 5 mL.
stored at 2-8°C until the listed                  0.01% w/v.
                                                                                                                                    After tablets have completely
expiry dates.                                                                                                                       dissolved (1-2 minutes) and no
                                                                                                                                    bubbles remain, replace
                                                                                                                                    stopper on bottle and mix by
inversion. Avoid strong light.    12. Pipette 50 µL of               PERFORMANCE CHARACTERISTICS
The substrate solution must be    1M H2SO4 into all wells in the     Intra-assay Precision
used within 30 minutes of         same timed sequence as for         Sample        n       Mean ± 2SD                         %CV
preparation.                      substrate solution addition.                               (mIU/L)
Calibrators                       13. Carry out an end-point         1           16         1.1 ± 0.09                            4.0
To reconstitute the lyophi-       reading at 490 nm and process      2           16        16.2 ± 1.60                            4.9
lized calibrators, add the        data as described in the           3           16        32.0 ± 4.40                            6.8
volume of deionized water         microplate reader user's           Inter-assay Precision
indicated on each vial label.     manual. This reading step          Sample       n*       Mean ± 2SD                         %CV
Allow the vials to sit undis-     should be carried out within                               (mIU/L)
turbed until completely           30 minutes of stopping the         1           21         1.2 ± 0.33                            7.0
dissolved (at least 30 minutes)   reaction.                          2           21        15.4 ± 2.20                            7.2
and then mix by gentle                                               3           21        26.4 ± 5.00                            9.5
inversion. Exact concentra-       CALCULATION OF                            * duplicate
tions determined lot-to-lot are   RESULTS
stated on a separate label        Calculation of results can be      Specificity
inside the kit. After reconsti-   carried out manually if there is   Analyte                 Concentration          Apparent TSH
tution, the calibrators should    no automatic data reduction.                                  Assayed             Result (mIU/L)
be stored at -20°C for up to      Determine the OD for each          hCG                     280000 IU/L             undetectable
4 weeks.                          well. Plot the calibrator curve    LH                          88 IU/L                 <0.2
Protocol                          using log-log graph paper with     FSH                         83 IU/L                 <0.1
1. Assemble the microwells        concentration of calibrators       Accuracy
in the frame according to the     on the x-axis and OD on the        Recovery was calculated by assaying before and after addition of
number of tests required. Bag     y-axis. The curve may be           exogenous analyte.
and return unused wells to        drawn point-to-point or a          Sample      TSH (mIU/L) TSH (mIU/L) % Recovery
2-8°C.                            curve-fitting routine, such as                   Observed         Expected
2. Pipette 150 µL of sample       spline interpolation, may be       1                  0.77            0.69         111.6
(calibrator, control, specimen)   used. Interpolate the sample       2                  5.30            5.00         106.0
in duplicate into the appropri-   values from OD measured            3                14.30           14.40            99.2
ate wells. Time taken to          from this calibrator curve.        4                27.90           27.40          102.2
dispense the samples should       Record the value for each          Dilution
not exceed 20 minutes.            sample in mIU/L TSH.               A sample was diluted in zero calibrator, assayed and recovery
3. Cover microwells with lid      The range of the ELEGANCE          calculated.
and incubate for 30 minutes       TSH ELISA is from 0 to             Sample      TSH (mIU/L) TSH (mIU/L) % Recovery
on a plate shaker at room         approx. 40 mIU/L, but the                        Observed          Expected
temperature (20-25°C).            maximum concentration that         Neat             13.6
4. Pipette 50 µL of TSH           can be reported is limited by      1/2               6.7               6.8            99.0
Antibody Reagent (blue) into      the linear performance             1/4               3.3               3.4            97.0
all wells.                        characteristics of the photom-     1/8               1.7               1.7           100.0
5. Cover microwells with lid      eter used.
                                  If the OD value of the highest     High-dose Hook Effect
and incubate for 30 minutes
                                  calibrator is above the range of   Due to the high-dose hook effect characteristic of the assay,
on the plate shaker at room
                                  the photometer, then this          samples greater than 36000 mIU/L may yield aberrant results,
temperature (20-25°C).
                                  calibrator must be omitted         less than that of the kit's highest calibrator. Those samples
6. After second incubation,
                                  from the plot of the calibrator    should be diluted with the zero calibrator and reassayed.
wash the microwells. Aspirate
the liquid and rinse each well    curve.                             Sensitivity
4 times with 250 µL wash          MODEL CALCULATIONS                 The sensitivity of the assay is typically less than 0.05 mIU/L.
solution. After the final wash,   Endpoint Data                      The sensitivity is defined as that concentration of analyte which
invert the microwells and tap     ID        Ave OD TSH               corresponds to the dose response variable (OD) that is two
firmly on absorbent tissue to                      (mIU/L)           standard deviations from the mean dose response variable of 16
remove any remaining wash         0          0.059                   replicate determinations of the zero calibrator run in three
solution. Ensure that no air      0.2        0.098                   different assays.
bubbles remain in the wells.      0.6        0.199                   Interference
7. Pipette 100 µL of TSH          2.5        0.564                   No interference with analyte recovery was observed for
Amplification Reagent             11.7       1.688                   concentrations of haemoglobin up to 250 mg/dL, bilirubin up to
(violet) into all wells.          42.5       2.964                   10 mg/dL and triglycerides up to 970 mg/dL.
8. Cover microwells with lid      Sample 1   0.290   1.04
and incubate for 10 minutes       Sample 2   1.541  10.31            ORDERING INFORMATION
on a plate shaker at room         Sample 3   2.820  35.90            The ELEGANCE TSH ELISA is manufactured by:
temperature (20-25°C).                                               Bioclone Australia Pty Limited,
9. After incubation, repeat       CALIBRATION
                                  The calibrators supplied in this   71-73 Railway Parade, Marrickville
wash step.                                                           NSW 2204, AUSTRALIA.
10. Pipette 100 µL of             kit are calibrated and labelled
                                  in mIU/L, referenced to the        Telephone       +61 (0) 2 9517 1966 Freecall 1800 251 138
prepared substrate solution                                          Fax             +61 (0) 2 9517 2990
into all wells. Timing of the     WHO 1983 2nd IRP 80/558.
                                                                     Email sales@bioclone.com.au        Web www.bioclone.com.au
incubation step is measured       LIMITATIONS
from the addition of substrate    Serum specimens showing            TECHNICAL SERVICE
solution to the first well.       gross haemolysis, gross            Full technical service is available by calling Bioclone on
11. Cover microwells with lid     lipaemia, or turbidity may give    +61 (0) 2 9517 1966 or Freecall 1800 251 138
and incubate for 5 minutes        false results.
stationary at room tempera-                                          PART No.: EKBTSH Ed. 5           Revision date: 14 January 2006
ture (20-25°C).

EXPECTED VALUES
It is recommended that each laboratory establish its own
reference range based on a representative sample population.
The following reference range was obtained by assaying serum
samples from healthy individuals and is given as a guide only:
                               n         TSH Range
                                          (mIU/L)
Healthy Males & Females       188          0.4 - 4.0

								
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