III.          PRINCIPLES  OF  THE METHOD

The Bio-Line FSH-ELISA is a solid phase Enzyme Amplified Sensitivity Immunoassay performed on microtiterplate.  The assay uses monoclonal antibodies (MAbs) directed against distinct epitopes of FSH.   Calibrators and samples react with the capture monoclonal antibody (MAb 1) coated on microtiter well and with a monoclonal antibody (MAb 2) labelled with horseradish peroxidase (HRP).  After an incubation period allowing the formation of a sandwich: coated MAb 1 – human FSH – MAb 2 – HRP, the microtiterplate is washed to remove unbound enzyme labelled antibody.   Bound enzyme-labelled antibody is measured through a chromogenic reaction.  Chromogenic solution (TMB – H₂O₂) is added and incubated.  The reaction is stopped with the addition of Stop Solution and the microtiterplate is then read at the appropriate wavelength.   The amount of substrate turnover is determined colourimetrically by measuring the absorbance, which is proportional to the FSH concentration. 

A calibration curve is plotted and FSH concentration in samples is determined by interpolation from the calibration curve.  The use of the ELISA reader (linearity up to 3 OD units) and a sophisticated data reduction method (polychromatic data reduction) result in a high sensitivity in the low range and in an extended calibration range.

  IV.                 REAGENTS PROVIDED

1. Microtiterplate with  96 anti FSH (monoclonal antibodies) coated wells

    Ready for use

2. Conjugate: HRP labelled anti-FSH (monoclonal antibodies) in TRIS-HCl buffer with bovine serum albumin and thymol

                1 vial - 6 ml

                Ready for use

3. Zero calibrator  in TRIS-HCl buffer with bovine serum albumin, benzamidin and thymol

          1 vial, lyophilized

    Add 2.0 ml distilled water

         4. Calibrator  N = 1 to 5

(see exact values on vial labels) in TRIS-HCl buffer with bovine serum albumin, benzamidin and thymol

                5 vials, lyophilized

    Add 0.5 ml distilled water

5. Wash Solution (Tris-HCl)

                1 vial, 10 ml

                Dilute 200 x with distilled water (use a magnetic stirrer).

         6. Controls - N = 1 or 2, in human serum with  thymol

                2 vials, lyophilised

                Add 0.5 ml distilled water

7. Chromogen TMB (Tetramethylbenzydine) in Dimethylformamide

          1 vial, 1 ml

                Dilute 0.2 ml into 1 vial of substrate buffer

8. Substrate buffer: H₂O₂ in acetate / citrate buffer

                3 vials, 21 ml

                Ready for use

9. Stop solution: H₂SO₄ 1.8N

          1 vial, 6 ml

                Ready for use

Note: 1.   Use the zero calibrator for sample dilutions.

          2.   1 mIU of the calibrator is equivalent to 1 mIU of the 1^st IS 92/510.

V.       SUPPLIES NOT PROVIDED

The following material is required but not provided in the kit:

1.           High quality distilled water

2.           Pipettes for delivery of: 50 μl, 200 μl, 500 µl and 2 ml (the use of accurate pipettes with disposable plastic tips is recommended)

3.           Vortex mixer

4.           Magnetic stirrer

5.           Horizontal microtiterplate shaker capable of 700 rpm ± 100 rpm

6.           Washer for Microtiterplates

7.           Microtiterplate reader capable of reading at 450 nm, 490 nm and 650 nm (in case of polychromatic reading) or capable of reading at 450 nm and 650 nm (monochromatic reading)

8.           Optional equipment: The ELISA-AID™ necessary to read the plate according to polychromatic reading (see paragraph XI.A.) can be purchased from Robert Maciels Associates, Inc. Mass. 0.2174 USA.

VI.     REAGENT PREPARATION

a.       Calibrators : Reconstitute the zero calibrator with 2.0 ml distilled water and other calibrators with 0.5 ml distilled water.

b.       Controls : Reconstitute the controls with 0.5 ml distilled water.

c.       Working Wash solution : Prepare an adequate volume of Working Wash solution by adding 199 volumes of distilled water to 1 volume of Wash Solution (200x). Use a magnetic stirrer to homogenize. Discard unused Working Wash solution at the end of the day. 

d.       Revelation Solution: pipette 0.2 ml of the chromogen TMB into one of the vials of substrate buffer (H₂O₂ in acetate/citrate buffer).  Extemporaneous preparation is recommended. 

VII.  STORAGE  AND  EXPIRATION  DATING  OF  REAGENTS

§             Before opening or reconstitution, all kits components are stable until the expiry date, indicated on the vial label, if kept at 2 to 8°C.

§             Unused strips must be stored, at 2-8°C, in a sealed bag containing a desiccant until expiration date.

§             After reconstitution, calibrators and controls are stable for 1 week at 2 to 8°C. For longer storage periods, aliquots should be made and kept at ‑20°C.  Avoid successive freeze thaw cycles.

§             The concentrated Wash Solution is stable at room temperature until expiration date.

§             Freshly prepared Working Wash solution should be used on the same day.

§             After its first use, the conjugate is stable until expiry date, if kept in the original well-closed vial at 2 to 8°C.

§             The freshly prepared revelation solution is stable, before use, for maximum 15 minutes at room temperature and must be discarded afterwards.

§             Alterations in physical appearance of kit reagents may indicate instability or deterioration.

VIII.   SPECIMEN  COLLECTION  AND  PREPARATION

§             Serum and plasma must be kept at 2 - 8°C.

§             If the test is not run within 24 hours, storage in aliquots at -20°C is recommended.  Avoid subsequent freeze thaw cycles.

§             Prior to use, all samples should be at room temperature.  It is recommended to vortex the samples before use.

§             Serum, heparinized plasma or EDTA plasma provides similar results.

Y(serum) = 1.21 x (EDTA plasma) - 0.85                r=0.994                n=34

Y(serum) = 1.04x (Heparin plasma) + 0.25      r=0.996      n=36

§             Do not use haemolysed samples.

IX.      PROCEDURE

A.       Handling notes

          Do not use the kit or components beyond expiry date. 

Do not mix materials from different kit lots. 

Bring all the reagents to room temperature prior to use.

          Thoroughly mix all reagents and samples by gentle agitation or swirling.

          Perform calibrators, controls and samples in duplicate.  Vertical alignment is recommended.

          Use a clean plastic container to prepare the Wash Solution.

          In order to avoid cross-contamination, use a clean disposable pipette tip for the addition of each reagent and sample.

          For the dispensing of the Revelation Solution and the Stop Solution avoid pipettes with metal parts.

          High precision pipettes or automated pipetting equipment will improve the precision. 

          Respect the incubation times. 

          To avoid drift, the time between pipetting of the first calibrator and the last sample must be no longer than 30 minutes.

          Prepare a calibration curve for each run, do not use data from previous runs.

The chromogenic solution should be colourless.   If a dark blue colour develops within a few minutes after preparation, this indicates that the preparation is unusable and must be discarded.

Dispense the Revelation Solution within 15 minutes following the washing of the microtiterplate. 

During incubation with Revelation Solution, avoid direct sunlight on the microtiterplate.

B.       Procedure

1.           Select the required number of strips for the run.  The unused strips should be resealed in the bag with a desiccant and stored at 2-8°C.

2.           Secure the strips into the holding frame. 

3.           Pipette 50 µl of each Calibrator, Control and Sample into the appropriate wells.

4.           Pipette 50 µl of anti-FSH-HRP conjugate into all the wells.

5.           Incubate for 30 minutes at room temperature on a horizontal shaker set at 700 rpm ± 100 rpm.

6.           Aspirate the liquid from each well.

7.           Wash the plate 3 times by:

§      Dispensing 0.4 ml of Wash Solution into each well

§      Aspirating the content of each well

8.           Pipette 200 µl of the freshly prepared revelation solution into each well within 15 minutes following the washing step.

9.           Incubate the microtiterplate for 15 minutes at room temperature on a horizontal shaker set at 700 rpm ± 100 rpm, avoid direct sunlight.

10.        Pipette 50 µl of Stop Solution into each well.

11.        Read the absorbencies at 450 nm and 490 nm (reference filter 630 nm or 650 nm) within 1 hour and calculate the results as described in section XI.

X.      CALCULATION  OF  RESULTS

A.      Polychromatic Reading:

1.           In this case, the ELISA-AID™ software will do the data processing.

2.           The plate is first read at 450 nm against a reference filter set at 650 nm (or 630 nm).

3.           A second reading is performed at 490 nm  against the same reference filter.

4.           The ELISA-AID™ Software will drive the reader automatically and will integrate both readings into a polychromatic model.  This technique can generate OD’s up to 10.

5.           The principle of polychromatic data processing is as follows:

§          Xi = OD at 450 nm

§          Yi = OD at 490 nm

§          Using a standard unweighted linear regression, the parameters A & B are calculated : Y = A*X – B

§          If Xi < 3 OD units, then X calculated = Xi

§          If Xi > 3 OD units, then X calculated = (Yi-B)/A

§          A 4-parameter logistic curve fitting is used to build up the calibration curve.

§          The FSH concentration in samples is determined by interpolation on the calibration curve.

B.       Bichromatic Reading

1.           Read the plate at 450 nm against a reference filter set at 650 nm (or 630 nm).

2.           Calculate the mean of duplicate determinations.

3.           On semi-logarithmic or linear graph paper plot the OD values (ordinate) for each calibrator against the corresponding concentration of FSH (abscissa) and draw a calibration curve through the calibrator points by connecting the plotted points with straight lines.

4.           Read the concentration for each control and sample by interpolation on the calibration curve. 

5.           Computer assisted data reduction will simplify these calculations.  If automatic result processing is used, a 4-parameter logistic function curve fitting is recommended.

XI.      TYPICAL DATA

The following data are for illustration only and should never be used instead of the real time calibration curve.

XII.          PERFORMANCE  AND  LIMITATIONS

A.       Detection Limit

Twenty zero calibrators were assayed along with a set of other calibrators. The detection limit, defined as the apparent concentration two standard deviations above the average OD at zero binding, was 0.15 mIU/ml.

B.       Specificity

Cross-reactive hormones (FSH, TSH & hCG) were added to the zero calibrator and to a high value calibrator (30 mIU/ml).  The apparent FSH response was measured.

C.      Precision

SD : Standard Deviation; CV: Coefficient of variation

D.       Accuracy     

       RECOVERY  TEST

Samples were diluted with zero calibrator.

E.       Time delay between last calibrator and sample dispensing

As shown hereafter, assay results remain accurate even when a sample is dispensed 40 minutes after the calibrators have been added to the coated wells.

F.       Hook effect

A sample spiked with FSH up to 2000 mIU/ml gives higher OD’s than the last calibrator point.

XIII.   INTERNAL  QUALITY  CONTROL 

§             If the results obtained for Control 1 and/or Control 2 are not within the range specified on the vial label, the results cannot be used unless a satisfactory explanation for the discrepancy has been given.

§             If desirable, each laboratory can make its own pools of control samples, which should be kept frozen in aliquots.  Controls that contain azide will interfere with the enzymatic reaction and cannot be used.

§             Acceptance criteria for the difference between the duplicate results of the samples should rely on Good Laboratory Practises

§             It is recommended that Controls be routinely assayed as unknown samples to measure assay variability.  The performance of the assay should be monitored with quality control charts of the controls.

§             It is good practise to check visually the curve fit selected by the computer.

  XIV.             REFERENCE  INTERVALS

These values are given only for guidance; each laboratory should establish its own normal range of values.

The range is expressed as 2.5% to 97.5% percentiles.

XV.          PRECAUTIONS  AND  WARNINGS

Safety

For in vitro diagnostic use only.

The human blood components included in this kit have been tested by European approved and/or FDA approved methods and found negative for HBsAg, anti-HCV, anti-HIV-1 and 2.  No known method can offer complete assurance that human blood derivatives will not transmit hepatitis, AIDS or other infections.  Therefore, handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.

All animal products and derivatives have been collected from healthy animals. Bovine components originate from countries where BSE has not been reported. Nevertheless, components containing animal substances should be treated as potentially infectious.

Avoid any skin contact with all reagents,  Stop Solution contains H₂SO₄, the chromogen contains TMB in Dimethylformamide, Substrate buffer contains H₂O₂.   In case of contact, wash thoroughly with water.

Do not smoke, drink, eat or apply cosmetics in the working area.  Do not pipette by mouth.  Use protective clothing and disposable gloves. 

XVI.          BIBLIOGRAPHY

1.       CHAPPEL S.C., ULLOA-AGUIRRE A., COUTIFARIS C.  (1983)

          Biosynthesis and secretion of follicle-stimulating hormone. 

          Endocrinol. Rev. 4:179.

2.       CROWLEY W.F., FOLICORI M., SPRAIT D.I., SANTORO N.F. (1985)

          They physiology of gonadotrophin-releasing hormone (GnRH) secretion in men and women.

          Recent Prog. Horm. Res; 41:473.

3.       WIDE L. (1987)

          Evidence for diverse structural variations of the forms of human FSH within and between pituitaries.

          Acta Endocrinol.  115:7.

4.       CLARKE  J.J.,  CUMMINS  J.T. (1987)

Pulsatility of reproductive hormones: physiological basis and clinical implications.

Baillière's Clin. Endocrinol. Metab., 1:1

5.       FILICORI  M.,  SANTORO N., MERRIAN G.R., CROWLEY W.F. Jr., (1986)

Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle.

J. Clin. Endocrinol. Metab., 62:1136

6.       MAIS V., CETEL N.S., MUSE K.N., QUIGLEY M.E., REID R.L., YEN S.S.C. (1987)

Hormonal dynamics during luteal-follicular transition.

J. Clin. Endocrinol. Metab., 64:1109

7.       LOUMAYE E., VANKRIEKEN L., DEPREESTER S., PSALTI I., de COOMAN S., THOMAS K. (1989)

Hormonal changes induced by short-term administration of a gonadotropin-releasing hormone agonist during ovarian hyperstimulation for in vitro fertilization and their consequences for embryo development.

Fertil. and Steril., 51:105

8.       VERMES I., BONTE H.A., SLUIS VEER G., SCHOEMAKER J. (1991)

Interpretations of five monoclonal immunoassays of Lutropin and Follitropin: effects of normalization with WHO standard.

Clin. Chem., 37:415

9.       MASSA G., de ZECHER F., VANDERSCHUREN-LODEWYK K. (1992)

Serum levels of immunoreactive Inhibine, FSH and LH in human infants at Preterm and Term Birth.

Biol. of the Neonat., 61:150

10.     DE HERTOGH R., VANKRIEKEN L., THOMAS K. de GASPARO M. (1992)

Circhoral fluctuations of serum total renin, inhibin and related hormones around the mid-cycle in normal human females.

Hum. Reprod., 7:337

XVII.            SUMMARY OF THE PROTOCOL