II.       PRINCIPLES  OF  THE METHOD

hCG + β-Elisa is a solid phase Enzyme Amplified Sensitivity Immunoassay performed on microtiter plate.  It is based on the oligoclonal systeme in which several monoclonal antibodies (Mabs) directed against distinct epitopes of hCG are used.  The use of several distincts Mabs avoids hyperspecificity ans allows high sensitive assays with extended standards range ans short incubation time.

Standards or samples containing hCG react with capture antibodies (Mabs 1) coated on a plastic well and with monoclonal antibodies (mabs 2) labelled with horseradish peroxydase (HRP).

After an incubation period allowing the formation of a sandwich : coated Mabs1 - hCG - Mabs2 - HRP, the microtiter plate is washed to remove unbound enzyme labelled antibodies.

The revelation solution (tetramethylbenzydine (TMB) is added and incubated.  The reaction is stopped with Stop Solution and the microtiter plate is read at the appropriate wavelengh.  The amount of substrate turnover is determined by interpolation from the standard curve.

III.      REAGENTS  PROVIDED

Note : Use the content of the zero standard for dilution of samples.

IV.          PRECAUTIONS  AND  WARNINGS

1.        The human blood components included in this kit have been tested by European approved and USA FDA approved methods and found negative for HBsAg, anti-HCV and 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.

2.        Avoid any skin contact with Stop Solution and Chromogenic Solution (TMB).  In case of contact wash thoroughly with water.

3.        Do not eat, drink, smoke or apply cosmetics where kit reagents are used.

4.        Do not pipette liquids by mouth.

V.       SUPPLIES NOT PROVIDED

1.       High quality distilled water.

2.       Precision pipette : 50 μl, 100 μl, 0.5 ml and 2 ml.

3.       Vortex mixer and magnetic stirrer.

4.       Horizontal  microtiter plate shaker capable of 700 rpm ± 100 rpm, microtiter plate reader capable of reading at 450 nm and at any wavelength from 600 to 750 nm, microtiter plate washer.

VI       REAGENTS  PREPARATION

1.       Standards and Controls : Reconstitute the lyophilized Standards and controls to the volume specified on the vial label with distilled water.  Allow the vials to remain undisturbed until completely dissolved, then mix well by gentle inversion.

2.       Wash working solution : dilute 2 ml in 400 ml distilled water or the vial content in 2000 ml distilled water (use a magnetic stirrer)

VII      STORAGE  AND  SHELF  LIFE  OF  REAGENTS

A.      Unopened vials

Store the unopened kit at 2°C to 8°C. All kit components are  stable until the expiry date printed on the labels.

B.       Opened vials

1.       The Conjugate vial must be stored at 2°C to 8°C.

2.       The reconstituted Standards and Controls are stable for 3 days at 2°C to 8°C. Aliquots held for longer periods of time should be frozen, a maximum of two times, at -20°C (maximum 2 months) or at -70°C for longer storage (until expiration date).

3.       Store the unused strips at 2°C to 8°C in the sealed bag containing the desiccant until expiration date.

4.       The Wash Solution Concentrate is stable at room temperature until expiration date. In order to avoid washer obstructions, it is recommended to prepare a fresh diluted Wash Solution each day.

VII     SPECIMEN  COLLECTION,  PREPARATION,  STORAGE  AND  DILUTION

A.      Specimen collection and preparation

-         Prior to use, all the samples should be at room temperature.  It’s recommended to vortex the samples before use.  Hemolysis has to be avoided.

-         Serum, heparinized plasma or EDTA plasma provide similar results and may be used.

Y(serum) = 0.97 x (EDTA plasma) + 0.21  R = 0.99 n = 34

Y(serum) = 0.96 x (hepar. plasma) + 0.79  R = 0.99 n = 34

B.       Storage

Serum/plasma samples must be kept at 2°C to 8°C for maximum  hours, and for longer storage (maximum one year) at ‑20°C.

C.      Sample dilution

If a specimen is expected or known to have a concentration above the highest standard it has to be diluted with the zero standard to fall within the measuring interval.

IX.      PROCEDURE

The instructions of the assay procedure must be followed to obtain reliable results.

A.       Procedural notes

1.       Allow the samples and reagents to equilibrate to room temperature (18°C to 25°C) before commencing the assay. Thoroughly mix the reagents and samples before use by gentle agitation or swirling.

2.       Do not use kit components beyond the expiration date.

3.       Do not mix materials from different kit lots.

4.       Do not mix strips from different plates.

5.       Perform Standards,Controls and Unknowns in duplicate. Vertical alignment is recommended.

6.       A standard curve should be run with each assay run or each plate run.

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

           Use a clean disposable pipette tip for the addition of each reagent, standard, control or specimen in order to avoid cross contamination.

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

10.     Use a clean plastic container to prepare the Wash Solution.

11.     The Chromogenic Solution should be colourless. If a blue colour develops before use, this indicates that the reagent is unusable, and must be discarded.

12.     During incubation with Chromogenic Solution, avoid direct sunlight on the microtiter plate.

13.     Respect the incubation times described in the assay procedure.

B.       Assay procedure

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

2.       Secure the strips into the holding frame.

3.       Pipette 50 μl of each Standards, Controls or Samples into the appropriate wells.  Time between distribution of first standard and last sample can be up to 30 minutes without affecting the results.  Vertical alignment is recommended

4.       Pipette 50 μl of conjugate into each well.

5.       Incubate for 30 minutes at room temperature (20°C - 27°C) on a horizontal shaker set at 700 rpm ± 100 rpm.

6.       Aspirate the liquid from each well ;

7.       Wash the plate three times by :

a)                dispensing of 0.3 ml of BioSource Wash Solution into each well ;

b)  aspirating the content of each well.

8.       Pipette 100 μl of the Chromogenic Solution into each well within 15 min. following the washing step.

9.       Incubate the plate for 15 min. at room temperature on an horizontal shaker set at 700 ±   100 rpm, avoiding direct sunlight.

10.     Pipette 100 μl of Stop Solution into each well.

11.     Read absorbances at 450 nm (reference filter : 650 nm) within 1 hour  and calculate the results as described in section XI.

X      CALCULATION  OF  ANALYTICAL  RESULTS

.         Read the microtiter plate at 450 nm (reference filter : 650 nm).

.         Plot the OD on the ordinate against the standard concentrations on the abscissa using either linear or semi-log graph paper and draw the curve by connecting the plotted points with straight lines.

.         Determine the hCG + β concentration of the samples from the standard curve.

Example of a typical standard curve

The following data are for demonstration purpose only and can not be used in place of data generated at the time of assay.

XI       QUALITY  CONTROL

-         The two Controls provided in the kit can be used as internal laboratory controls.

Note :      Other controls which contain azide will interfere with the enzymatic reaction and cannot be used.

-         Serum or heparin plasma pools can be collected and frozen immediately in aliquot to serve as controls. Repeated freezing and thawing are not permitted.

-         Record keeping : it is good laboratory practice to record the kit lot numbers and date of reconstitution for the reagents in use.

-         Controls : it is recommended that Controls be routinely assayed as unknown samples to measure assay variability. It is recommended that quality controls charts be maintained to monitor the performance of the kits. Control ranges are indicated on vial labels. Out of range control results indicate the assay must be repeated. Repeat patient samples may also be used to measure interassay precision.

-         Sample handling : strictly adhere to the instruction for handling and storage of samples. Standards, Controls, and Unknowns should be run in duplicate. A clean disposable tip should always be used to avoid carryover contamination.

-         Data reduction : it is good practice to construct a standard curve for each run to check visually the curve fit selected by the computer program.

XII      EXPECTED VALUES

These are provided for guidance only.  Each lab should establish its own normal range.

XIII   PERFORMANCE  CHARACTERISTICS

1.       Minimum Detectable Concentration (MDC).

The MDC is estimated to be 0.6 mIU/ml and is defined as the hCG + β concentration corresponding to the average OD of 20 replicates of the zero standard - 2 standard deviations.

2.       Precision

3.       Specificity

The cross-reactivity of FSH, LH, TSH, αhCG, βhCG was determined by addition of each analyte to serum samples respectively containing 36.6 and 156.4 mIU/ml of hCG and measuring the apparent hCG concentration.  As shown below, the cross-reactions with hLH, hFSH, TSH and αhCG are insignificant while βhCG totally cross-reacts with hCG.

(*)       Conversion assuming a MW for hCG of 37900 and 8.6 mIU/ng (3^rd IRP 75/537)

4.       Accuracy

5.       High dose hook-effect

A sample spicked with hCG up to 250 000 mIU/ml gives a result higher than the last standard point.

XIV    LITERATURE  REFERENCES

1.       BRAUNSTEIN G.D., RASOR J., ADLER D., DANZER H., WADE H.E., (1976)

Serum human vhorionic gonadotropin levels throughout normal pregnancy.

Am. J. Obstet. Gynecol., 126:678.

2.       CHIEN F., SETSUKO G., YOSHIHITO F., YUTAKA T., (1987)

Radioimmunoassay of the serum free beta-subunit of human chorionic gonadotropin in trophoblastic disease.

J. Clin. Endocrinol. Metab., 64:313.

3.       DAWOOD M.Y., SASCENA B.B., LANDESMAN R., (1977)

Human chorionic gonadotropin and its subunit in hydatidiform mole and choriocarcinoma.

Obstet. Gynecol., 50:172.

4.       HAY D.L., (1982)

Chorionic gonadotropin.

Clin. Biochem. Rev., 3:35.

5.       PIERCE J.B., PARSONS T.F., (1981)

Glycoprotein hormones : structure and function.

Annu. Rev. Biocham., 50:465.

XV     SUMMARY OF ASSAY PROCEDURE