Preg Cube DIRECT HCG PREGNANCY TEST Intended for the Qualitative Determination of Human Chorionic Gonadotropin in Urine and Serum    

SUMMARY AND EXPLANATION

Human chorionic gonadotropin (HCG) is a glycoprotein hormone secreted by the developing placenta shortly after fertilization. In normal pregnancy, HCG can be detected in serum as early as 7 days following conception (1-4). The concentration of HCG continues to rise rapidly, frequently exceeding 100 mlU/ml by the first missed menstrual period (2-5) and peaking in 30-200,000 mlU/ml range by 10-12 weeks into pregnancy. The appearance of HCG soon after conception and its subsequent rise in concentration during early gestational growth make it an excellent marker for the early detection of pregnancy.

Elevated serum HCG levels comparable to those observed in early pregnancy may also be associated with trophoblastic or nontrophoblastic neoplasms (6-7) such as hydatidiform mole, choriocarcinoma, therefore, the possibility of such diseases should be ruled out before a positive HCG result is considered diagnostic for pregnancy.

PRINCIPLE

The Preg Cube HCG test is a qualitative, sandwich enzyme immunoassay (8-9) for the determination of human chorionic gonadotropin (HCG) in urine and serum. The method employs a unique combination of monoclonal and polyclonal antibodies to selectivity identify HCG in test samples with a high degree of sensitivity. In less than 2 minutes, elevated levels of HCG as little as 25 mlU/ml can be detected.

The test sample is allowed to react with the antibodies on the solid phase (coated in a form of a vertical bar on the membrane) and the antibody-enzyme-conjugate, sequentially. In the presence of HCG, a specific antibody - antigen - antibody - enzyme complex will form on the membrane. After addition of substrate, the development of blue color on the vertical bar of the membrane indicates the presence of HCG. A horizontal bar will always appear on the membrane regardless of the presence of HCG. The visual formation of a (+) or (-) sign provides an easy readout for positive and negative specimens. The (-) sign also serves as an internal quality control. It will not appear when reagents are not active or are not added properly.

REAGENT: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set (Cat. No. 9100)  provides:

1. REACTION CUBES - (Cat. No. 9101)

REACTIVE INGREDIENTS: Polyclonal antibodies directed against HCG.

2. ENZYME CONJUGATE - (Cat. No. 9102)

REACTIVE INGREDIENTS: Mouse monoclonal antibody alkaline phosphatase enzyme conjugate in protein stabilizer, 0.1% sodium azide added as preservative.

3. SUBSTRATE REAGENT - (Cat. No. 9103)

REACTIVE INGREDIENTS: Substrate chromogen and stabilizer.

4. POSITIVE CONTROL - (Cat. No. 9104)

REACTIVE INGREDIENTS: HCG in a buffered protein solution. 0.1% sodium azide added as a preservative.

5. DISPOSABLE DROPPERS for dispensing

PRECAUTIONS:

1. Certain reagents in this test set contain sodium azide. Dispose with large amounts of water to prevent azide reacting with plumbing.

2. Do not mix reagents from a different lot.

3. Avoid exposure of substrate reagent to strong light.

4. Do not ingest. Toxicity has not been established.

STORAGE AND STABILITY:

Store at 2 - 8ºC. Stable until expiration date if protected from contamination. May be stored at room temperature for 90 days, at the end of which ALL reagents must be considered expired regardless of dating.

DETERIORATION:

Failure to visualize a horizontal bar on the membrane after reagent additions indicates reagent deterioration or improper reagent additions.

SPECIMEN COLLECTION

URINE (0.5 mL) - The sample must be collected in a clean, dry container, either plastic or glass, without preservatives. Specimens collected at anytime may be used, however, the first morning urine generally contains the highest concentration of hormone. Urine specimens may be refrigerated (2-8° C) and stored up to 72 hours prior to assaying. If samples are refrigerated, they must be equilibrated to room temperature before testing. Urine samples exhibiting visible precipitates should be filtered, centrifuged or allowed to settle and clear aliquots obtained for testing.

SERUM (0.5 mL) - No special preparation of the patient specimen is required. Plasma sample prepared from EDTA can be used in lieu of serum. Serum not to be assayed immediately must be stored in a refrigerator or a freezer. Bring these specimens to room temperature prior to testing. Do not freeze and thaw repeatedly.

PROCEDURE

MATERIALS PROVIDED:

REACTION CUBES (Cat. No. 9101), ENZYME CONJUGATE (Cat. No. 9102), SUBSTRATE SOLUTION (Cat. No. 9103), POSITIVE CONTROL (Cat. No. 9104) and DROPPERS.

MATERIALS REQUIRED BUT NOT PROVIDED:

1. Specimen collection container

2. Timer

PROCEDURAL INSTRUCTIONS:

All reagents including patients samples, controls and reference material should be brought to room temperature.

URINE:

1. Remove the reaction pack from its protective package. Label the packs with patient or control identifications. DO NOT REMOVE PREFILTER FROM THE PACK.

2. Draw urine sample with the provided dropper. Dispense 5 drops of the urine sample to the center of the prefilter. Wait for complete drainage of sample through the prefilter prior to reagent additon.

3. Dispense three drops from Bottle A (ENZYME CONJUGATE) onto the center of the prefilter. Allow the solution to soak through. Wait an additional 60 seconds to ensure complete drainage and antibody reaction.

4. Remove and discard the prefilter.

5. Dispense seven drops from Bottle B (SUBSTRATE SOLUTION) onto the center of reaction pack. Wait for two minutes for result to develop.

6. Observe the symbol development at the center of the reaction pack. Depending on the concentration of HCG, positive results can be observed in less than 20 seconds. However, to confirm negative results, the complete 2 minute incubation time is required. DO NOT INTERPRET RESULTS AFTER 2 MINUTES.

7. After complete incubation, the color reaction can be stopped by adding 0.5 mL of 0.2 N HCL solution of water to the pack to prevent further development of the background color.

SERUM:

1. Label test tubes for each patient and control samples. Using the disposable dropper, dispense 3 drops of serum or reference sample into each test tube.

2. Dispence three drops from Bottle A (ENZYME CONJUGATE) into each test tube; wait2 minutes to allow antibody reactions to occur.

3. Remove the reaction pack from its protective package. Label the packs with patient or control identifications. DO NOT REMOVE PREFILTER FROM THE PACK.

4. Pour the entire contents of the test tube onto the prefilter cup. Allow the solution to soak through. Wait an additional 30 seconds to ensure complete drainage and antibody reaction.

5. Remove and discard the prefilter.

6. Dispense seven drops from Bottle B (SUBSTRATE SOLUTION) onto the center of reaction pack. Wait for two minutes for result to develop.

7. Observe the symbol development at the center of the reaction pack. Depending on the concentration of HCG, positive results can be observed in less than 20 seconds. However, to confirm negative results, the complete 2 minute incubation time is required.

8. After complete incubation, the color reaction can be stopped by adding 0.5 mL of 0.2 N HCL solution of water to the pack to prevent further development of the background color.

INTERPRETATION OF TEST

1. NEGATIVE: A (-) symbol on the center of the membrane indicates

the absence of detectable HCG. This horizontal color bar will

always appear if the test is carried out correctly and all reagents

are active.

2. POSITIVE: A (+) symbol on the center of the membrane indicates

the presence of HCG. When a clearly recognizable vertical color

bar developes, even though its intensity is less than that of the

horizontal bar, the test should be considered positive. The

vertical bar indicates HCG concentrations greater than 50

mlU/mL.

3. VOID: When the reaction pack does not result in either (+) or (-)

the test should be voided since improper additon or deterioration

of reagents probably occured.

QUALITY CONTROL:

The procedural control on each test membrane shows that reagents were applied in correct sequence and were reactive. A horizontal bar appearing on the membrane indicates proper performance. Good laboratory practices include the use of control specimens to ensure proper kit performance. Negative and positive controls containing HCG at different levels of activities are available commercially.

LIMITATIONS

1. Individuals taking a variety of drugs have shown false positive and negative results. It is advisable to test specimens from patients who are not taking drugs.

2. In certain pathological conditions, such as choriocarcinomand chorioepithelioma, patients may excrete HCG in urine. In these patients, a positive test may not be an indication of pregnancy.

3. Extremely low concentrations of HCG at the time of a missed menses may cause a negative result; in these cases, a second specimen should be tested 5 - 7 days later.

4. If a urine specimen is too dilute (i.e. low specific gravity) it may not contain representative levels of HCG. If pregnancy is suspected, a first morning urine should be obtained from the patient 48 - 72 hours later and tested.

5. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the results of a single test, but should only be made by the physician after all clinical and laboratory findings have been evaluated.

EXPECTED VALUES

Healthy men and healthy non-pregnant women do not have detectable HCG. HCG levels of 25 mlU/ml can be reached on the day of the first missed menstrual period. HCG levels peak about 8 weeks after the last menstrual period and then decline to lower values for the remainder of the pregnancy. Following delivery, HCG levels rapidly decrease and usually return to normal within days after parturition.

PERFORMANCE CHARACTERISTICS

SENSITIVITY AND SPECIFICITY:

The Preg Cube HCG Pregnancy Test detects Urinary HCG concentrations greater than 25 mlU/ml (calibrated to the second international standard) as indicated by the development of a (+) symbol on the center of the membrane. Additionally, when the samples contain less than 25 mlU/ml HCG, the vertical bar may also demonstrate lighter intensity purple-blue than that found on 25 mlU/ml positive reference pattern.

Specificity of the Preg Cube HCG test was determined from cross reaction studies with known amounts of Leteinizing Hormone (HLH) Follicle Stimulating Hormone (HFSH), and Thyroid Stimulating Hormone (HTSH) - 500 mlU/ml HLH, 1000 mlU/ml HFSH and 1000 mlU/ml HTSH all gave negative results.

ACCURACY:

1. Correlation with Qualitative Visual Tests - Urine: 78 randomly selected urine samples were analyzed by the Preg Cube HCG test procedure in parrallel with a commercially available visual method. Of these patients, 75 showed complete agreement (41 negative and 34 positive for a 96% correlation). The three specimens which yielded positive results in Preg Cube HCG test but negative in the competitive test were further tested by a quantitative RIA method. The results indicated that the samples contained between 25 and 52 mlU/mL HCG activities.

2. Correlation with Qualitative Visual Tests - Serum: 65 randomly selected serumsamples were analyzed by the Preg Cube HCG test procedure in parrallel with a commercially available visual method. Of these patients, 63 showed complete agreement (36 negative and 27 positive for a 97% correlation). The two specimens which yielded positive results in Preg Cube HCG test but negative in the competitive test were further tested by a quantitative RIA method. The results indicated that the samples contained between 20 and 24 mlU/mL HCG activities.

INTERFERING SUBSTANCES:

The following substances were added in HCG free and 25 ml/U/ml HCG spiked urine samples. None of the substances at concentration tested interfered in the assay.

Acetaminophen 20 mg/dl

Acetylsalicylic Acid 20 mg/dl

Ascorbic Acid 20 mg/dl

Atropine 20 mg/dl

Caffeine 20 mg/dl

  Gentesic Acid 20 mg/dl

Glucose 2   g/dl

Hemoglobin 1 mg/dl

REFERENCES

1. Batzer, F.R., Fertility and Sterility, Vol. 34, 1, 1980.

2. Catt, K.J., Dufan, M.L., and Vaitukaitis, J.L.J. Clin. Endocrinol Metab., Vol. 40,537.1975.

3. Braunstein, G.D., Rastor, J., Adler, D., Danzer, H., Wade. M.E., Am. J. Obstet. Gynecol, Vol. 126, 678, 1976.

4. Lenton, E.A., Neal, L.M., Sulaiman, R., Fertility and Sterility, Vol. 37, 773, 1982.

5. Batzer, F.R., Fertility and Sterility, Vol. 34, 1, 1980.

6. Dawood, M.Y., Saxeba, B.B., and Landesman, R. Ob. Gyn. Vol. 12 6, 678, 1976.

7. Braunstein, G.D., et al Ann. Inter, Med. Vol. 78, pp. 39 - 45, 1973.

8. Engvall, E., Method in Enzymology, Vol. 60, pp. 419 - 439, 1980.

9. Uotila, M., Rusoslahti, E., and Engvall, E.J., Immunol Methods, Vol.42, 11, 1981,