6. Introduction
• Most pregnancy tests used today, whether a home urine
test, a physician's office urine or blood test, or a clinical
laboratory blood test are "sandwich assays".
• Sandwich assays use two or more animal antibodies
raised against different sites on Human Chorionic
Gonadotropin (hCG).
• Usually a mouse monoclonal antibody against one site on
the hCG molecule, and a mouse monoclonal, or a sheep,
rabbit or a goat polyclonal antibody against a second
distant site on the hCG molecule.
7. SUMMARY AND EXPLANATION OF THE TEST
• Human chorionic gonadotropin (hCG) is a
glycopeptide hormone produced by the placenta
during pregnancy.
• The appearance and rapid rise in the
concentration of hCG in the woman's urine makes
it a good pregnancy marker.
• Usually, concentration of hCG in urine is at least
25 mIU/ml as early as seven to ten days after
conception.
• The concentration increases steadily and reaches
its maximum between the eighth and eleventh
weeks of pregnancy.
9. A level less than 5.0 mIU/ml is considered to be a negative pregnancy test.
3 Weeks: 5 to 50 mIU/ml
4 Weeks: 5 to 426 mIU/ml
5 Weeks: 18 to 7,340 mIU/ml
6 Weeks: 1,080 to 56,500 mIU/ml
7 to 8 Weeks: 7,650 to 229,000 mIU/ml
9 to 12 Weeks: 25,700 to 288,000 mIU/ml
13 to 16 Weeks: 13,300 to 254,000 mIU/ml
17 to 24 Weeks: 4,060 to 165,400 mIU/ml
25 to 40 Weeks: 3,640 to 117,000 mIU/ml
hCG is measured in milli-international units per milliliter. This is abbreviated as
mIU/ml.
10. PRINCIPLE OF THE TEST
• The pregnancy testing device contains a unique set of dye-conjugated and
immobilized antibodies used to produce a distinctive visual pattern indicating
elevated concentration of hCG (=25 mIU/ml) in the test sample.
• One antibody, the capture antibody, is in a solid phase permanently attached
to a tube, plate, membrane, or bead.
• Conjugate pad contains the label reagent, i.e. antibody labeled with either
red, gold or blue latex particles.
• Sample is applied and dissolves the label mixture and migrates to the zone
of immobilized antibody lines. If hCG is present, labeled antibody-dye
conjugate binds it, forming an antibody-antigen complex.
• Positive, that is hCG containing, sample causes the formation of a colored
test line, which indicates a positive test result.
• As the reaction mixture continues to flow along the test membrane, the
complex binds to the anti-hCG antibody in the test zone of the membrane,
and produces a color band.
• Unbound conjugate binds to the reagents immobilized in the control zone
producing a color band, demonstrating proper performance of the test.
11. Figure 1
• Device with solid phase capture antibody to one site on
hCG, and liquid phase tracer antibody (label shown by
red star) to second or distant site on hCG. In this way the
label becomes immobilized.
12. Figure 2
• Serum or urine containing hCG (shown as αβ) added to device. The hCG
forms a sandwich or bridge between capture and tracer antibody. After a
short incubation period the hCG binds both the solid phase and liquid
phase antibodies linking them.
13. Figure 3
• Excess tracer antibody is washed away. Amount of label or tracer
(red star) is measured. This is proportional to amount of hCG.
15. TEST PROCEDURE
• Bring test components and specimens to room
temperature prior to testing.
• Remove a Testing Device from the foil pouch by
tearing at the "notch" and place it on a level
surface.
• Holding a Sample Dropper vertically, add exactly
four drops of the urine specimen to the sample
well.
• Read results at time indicated in procedure.
16. Interpretation
• If two color bands are visible the test is
positive.
• The presence of a Control Band only
indicates a negative test.
18. Function of the Control Band
• The Control Band is used as a reference and
built in quality control check.
• If the Test Band is darker or similar to the
Control band, the test result is considered
positive.
• The Control Band is used for procedural control
to check whether the test reagents are working
properly and that a sufficient amount of urine
sample has been applied to the test area.
19. Invalid Tests
• If, after performing the test, no purple color
band is visible anywhere within the
Results Window, the result is considered
invalid.
• If a color appears in the test area but NO
color appears in the control area, the test
is invalid.
21. Causes of Invalid Results
• The directions may not have been
followed correctly.
• Inadequate amount of sample has been
exposed to the test system.
• The test may have deteriorated.
22. PRECAUTIONS
• Do not use test kit components after the
expiration dates.
• Dispose of all used test components in a proper
biohazard container.
• If specimens or test components have been
stored in a refrigerator, allow them to warm to
room temperature before performing the test.
• Human specimens should be handled as if
capable of transmitting infectious agents.
23. SPECIMEN COLLECTION AND STORAGE
• First morning urine usually contains the highest
concentration of hCG and is therefore the best sample when
performing the urine test. However, randomly collected urine
specimens may be used.
• Collect a urine specimen in a clean glass, plastic, or wax
coated container. Do not use preservatives.
• If the test is not run immediately following collection of the
sample specimen, but is to be run within 48 hours following
collection, the specimen should be refrigerated (2-8°C), and
brought back to room temperature (15-28°C) before testing.
•
• If testing is delayed more than forty-eight hours, the
specimen should be frozen. A frozen specimen should not
be used if stored more than two weeks.
• Prior to testing, the frozen specimen must be completely
thawed, thoroughly mixed, and brought to room
temperature.
