Jimma Health Institute

Serology of Human Chorionic Gonadotropin

Hormone
Mr. Estifanos
8/1/2022

Group members ID/NO

1. Gadise Tesfaye RU5086/12

2. Kalkidan Asaye RU0037/12
3. Mihret Genene RU0343/12

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 History
Throughout history methods involving urine have been a popular way to test for
pregnancy. Early ideas ranged from simply observing the color of a woman’s urine to the
notion that the urine of pregnant women contains special crystals or secretions. Indeed,
pregnancy testing can be traced back to 1350 BCE in Ancient Egypt. A written document
from the time describes a process in which a woman would urinate on wheat and barley
seeds over several days and, depending on which plant grew, both the woman’s
pregnancy status and the sex of the fetus could be determined. In 1905, British
physiologists Ernest Starling and William Bayliss were the first to isolate special hormone
markers found in the urine of pregnant women.

One specific hormone that is used today for pregnancy testing is human chorionic
gonadotropin, or hCG. This hormone appears during the first trimester of pregnancy, a
time characterized by rapid cell division and tissue differentiation in the developing
embryo. The blastocyst implants itself about eight to ten days after ovulation, and begins
to secrete hCG, whose concentration in the urine doubles every two to three days during
early pregnancy. The hormone becomes detectable in the blood and urine within seven to
nine days, reaching its highest concentration around the eighth week of pregnancy. Due
to its early secretion, hCG is quite useful for pregnancy detection. Specifically, hCG
encourages the corpus luteum to produce estrogen and progesterone, which are vital to
the preservation of a pregnancy. The corpus luteum is a temporary structure that
develops from the ovarian follicle following ovulation.

Human chorionic gonadotropin was first discovered in the 1920s, when German
scientists Selmar Aschheim and Bernhard Zondek observed that hCG stimulates ovary
development in rabbits and mice and also affects the formation of the corpus luteum in
humans. Human chorionic gonadotropin consists of an alpha subunit and a beta subunit.
The alpha subunit is similar in composition to follicle stimulating hormone (FSH),
luteinizing hormone (LH), and thyroid stimulating hormone (TSH). The beta subunit,
however, is unique to hCG, its terminal twenty-eight to thirty amino acids not occurring in
any other glycoprotein hormone. Because of its composition, many early tests for
pregnancy were largely unsuccessful because of their inability to distinguish the alpha
subunit from several other commonly occurring hormones not necessarily linked to
pregnancy.

In 1927, Aschheim and Zondek invented the A-Z test, specifically designed to detect
hCG in urine. In this test, the female’s urine was injected into a young mouse or rat. Based
on the assumption that hCG has the same effect in mice as it does in humans, if the
animal then underwent ovulation, it implied that the woman was pregnant. Bioassays
similar to the A-Z test burgeoned during the 1930s, but their reliability was low and their
costs remained high.

In 1960, the hemagglutination inhibition test, an immunoassay to test for pregnancy,

became available. Developed by Leif Wide and Carl Gemzell, this test uses a mixture of the
patient’s urine and hCG antibodies. The test was said to be positive if the cells clumped in
a specific pattern. Though more efficient, the test still lacked sensitivity. Agglutination
immunoassays were eventually replaced with enzyme immunoassays that were able to

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detect hCG in much smaller concentrations. In 1966 A. R. Midgeley introduced a
radioimmunoassay for hCG. Despite the new development, however, the
radioimmunoassay suffered from the same problem that plagued the first bioassays and
immunoassays it could not adequately distinguish the alpha subunit of hCG from other
commonly occurring hormones. Finally, in 1972 Judith L. Vaitukaitis, Glenn Braunstein,
and Griff Ross developed a more sophisticated radioimmunoassay that could distinguish
between the two substances. This radioimmunoassay was pivotal in that it displayed
sensitivity to the beta subunit of hCG and thus could be used only days after a missed
menstrual period. In 1976, the FDA approved the use of an immunoassay originally used
in the detection of hCG-secreting tumors for use in the first at-home pregnancy test kit in
the United States.

Modern at-home pregnancy tests rely on the use of antibodies in a test known as a
sandwich assay. The term “sandwich” assay refers to an assay in which two antibodies, a
capture antibody and a tracer antibody, sandwich an antigen in the form of an hCG
molecule. An at-home pregnancy test consists of a plastic device of three parts: a urine
well, an opening that displays the test results, and a plastic-shielded region containing the
tracer antibody. If hCG is present in the urine poured into the urine well, it migrates
toward and binds to the tracer antibody. This complex will then continue to flow to the
immobilized capture antibody, which will glow to indicating positive test.

Qualitative tests (yes/no or positive/negative results) look for the presence of the beta
subunit of human chorionic gonadotropin (hCG) in blood or urine. For a qualitative test
the thresholds for a positive test are generally determined by an hCG cut-off where at
least 95% of pregnant people would get a positive result on the day of their first missed
period. Qualitative urine pregnancy tests vary in sensitivity. High-sensitivity tests are more
common and typically detect hCG levels between 20 and 50 milli-international units/mL
(mIU/mL). Low-sensitivity tests detect hCG levels between 1500 and 2000 mIU/mL and
have unique clinical applications, including confirmation of medication abortion success.
Qualitative urine tests available for home use are typically designed as lateral flow tests.

Quantitative tests measure the exact amount of hCG in the sample. Blood tests can
detect hCG levels as low as 1 mIU/mL, and typically clinicians will diagnose a positive
pregnancy test at 5mIU/mL.

Table 1. Human chorionic gonadotropin (hCG) detection thresholds by test type and
sample type

Urine pregnany test Blood pregnancy test

Detection High-sensitivity; Qualitative test;

threshold
Qualitative test;20 to 50miu/ml 5 to 10 miu/ml, depending
depending on test on test

Low-sensitivity; Quantitative test;

Qualitative test; 1500-2000miu/l, 1 to 2 miu/mL, depending

depending on test.

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 There is a multilevel urine pregnancy test (MLPT) that measures hCG levels semi-
quantitatively. The hCG levels are measured at <25, 25 to 99, 100 to 499, 500 to 1999,
2000 to 9999, and >10,000 mIU/mL. This test has utility for determining the success of
medication abortion.

HCG to detect pregnancy

An egg is normally fertilized by a sperm cell in a fallopian tube . Within 9 days the
fertilized egg moves down the fallopian tube into the uterus. It then attaches (implants) to
the wall of the uterus. After the fertilized egg implants, the growing placenta starts
releasing hCG into your blood. Some hCG also gets passed in your urine. HCG can be
found in the blood before the first missed menstrual period. This can be as early as 6 days
after the egg implants.

HCG helps to keep your pregnancy going. It also affects the development of your baby
(fetus). Levels of hCG go up fast in the first 14 to 16 weeks after your last menstrual
period. They are the highest around the 14th week following your last period. They then
go down gradually. The amount that hCG goes up early in pregnancy can give information
about your pregnancy and the health of your baby. Soon after delivery, hCG can no longer
be found in your blood.

More hCG is released in a multiple pregnancy, such as twins or triplets, than in a

single pregnancy. Less hCG is released if the fertilized egg implants in a place other than
the uterus, such as in a fallopian tube. This is called an ectopic pregnancy .

This chart shows how HCG levels rise quickly and steadily in the first
trimester before declining
Weeks since last menstrual period
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HCG levels (mIU/ml)

5-50

HCG levels (mIU/ml)

5-426

HCG levels (mIU/ml)

187,340

HCG levels (mIU/ml)

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 1,080-56,500

7 to 8

HCG levels (mIU/ml)

7,650-229,000

9 to 12

HCG levels

25,700-288,000

13 to 16

13,300-254,000

17-24

4,060-165,400

24-40

3,640-117,000

HCG blood tests

The level of hCG in the blood is often used as part of a screening for birth defects in a
maternal serum triple or quadruple screening test. These tests are usually done between
15 and 20 weeks of pregnancy to check the levels of three or four substances in a
pregnant woman's blood. The triple screen checks hCG, alpha-fetoprotein (AFP), and a
type of estrogen (unconjugated estriol, or uE3). The quad screen checks these substances
and the level of the hormone inhibin A. The levels of these substances—along with a
woman's age and other factors—help the doctor figure out the chance that the baby may
have certain problems or birth defects.

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 Types of urine testing kits
1. Rapid latex slide test
2. Heamagglutination inhibition techniques
3. Rapid chromatographic immunoassay
4. Enzyme immunoassay
5. Chemiluminescent microparticle immunoassay

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 1. Rapid latex slide of the inhibition
a) Direct agglutination test
A direct agglutination latex particle slide test uses latex particles coated with anti-HCG
as the only reagent. It is convenient to use and requires only 2 minutes to perform. This
test may be used on serum or urine and there is no interference from significant amount
of hemoglobin or protein in the urine.

Material and Reagent required;

Clean slide

Pipette

Anti-HCG antibody

Specimen used; Urine (morning urine is preferred)

DIRECT HCG LATEX PREGNANCY KIT

A latex agglutination test for the detection of
-HCG in urine
Stored at 2-8

INTENDED USE
ATLAS direct hCG kit is a rapid test for detection of beta human Chorionic
Gonadotropin (hCG) in urine which has been widely used in the diagnosis of pregnancy.
INTRODUCTION & PRINCIPLES
Detectable levels of Chorionic Gonadotropin (hCG) in urine start at 5 mIU/ml during the
first week of gestation and rises to 100,000 mIU/ml at 2 to 3 months. The hCG level
doubles approximately every 2 days during the first trimester. Values decline from 10% to
15% of peak concentrations during 2nd and 3 rd trimesters. ATLAS Direct hCG Latex kit
contains one reagent of latex particles coated with monoclonal antibodies to hCG. The
reagent is mixed with the urine samples.

PRECAUTIONS

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 All reagents are for in vitro use only.Patient specimens may contain infectious agents
and should be handled and disposed of as potential bio hazards.
 Do not interchange reagents from different kit lot numbers.
 Allow kit components and specimens to reach the room temperature before use, as
cold reagents and/or specimens may decrease assay performances. 20-30 minutes
are recommended.
 Do not use kit components beyond labeled expiration date.
 In case of packaging breakage, the product can be used if no component has been
damage. In case of vials breakage, clean the kit carefully to not get hurt white the
glasses.All product used should be rejected according to the legislation in force.
SPECIMEN COLLECTION AND PREPARATION
Generally, the first morning urine contains the highest concentration of the hCG
hormone and therefore, it is more recommended for testing. However, urine collected at
other periods can also be used. The urine in this case should have been kept in 2-8C and
used within 72 hours from collection time.
PROCEDURES
1. Bring reagents to room temperature.
2. Place 100 μl of patient urine, one drop positive and one drop negative controls into
different circles of the slide.
3. Add one drop of latex reagent directly to each sample.
4. Mix using the supplied sticks and spread the mixture over the entire circle.
5. Gently rock the slide. Agglutination may be observed after two minutes. Direct light
source may help to observe the results.
READING THE RESULT
Presence of agglutination within two minutes indicates positive reaction. Lack of
agglutination within two minutes indicates negative reaction.

PROCEDURE LIMITATION
1. This test provides a presumptive diagnosis for pregnancy. Physicians should evaluate all
clinical and laboratory findings before making a definitive diagnosis.
2. Elevated levels of hCG may be found in trophoblastic disease, choriocarcinoma, and
embryonal cell carcinoma. Islet cell tumors may also produce hCG as well as other
carcinomas.
3. Ectopic pregnancies may produce very low levels of hCG. If this condition is suspected,
further testing using a quantitative assay may be desirable.
4. Detectable levels of hCG may remain several weeks following normal pregnancy,
delivery by cesarian section, spontaneous or therapeutic abortion.
5. Approximately one third of all conceptions end in natural termination. This may
produce positive results when testing early in the pregnancy followed by negative results

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after the natural termination. Low positive results may be confirmed by retesting with first
morning urine 48 hours later.
6. Urine from very early stages of pregnancy may contain hCG at low levels. This may
cause the reaction to occur more slowly and produce a degree of agglutination which is
less than that produced by the negative control. Such reactions should be considered
doubtful and the test should be repeated on a first morning urine collected 48 hours later.
SENSITIVITY
The sensitivity of ATLAS HCG kit is 0.2 mIU/ml HCG, based on the World Health
Organization (WHO) reference standard. In normal pregnancies a positive reaction is
therefore possible 2-4 days after a missed period. If the first test should prove negative, it
should be repeated a few days later, unless menstruation occurs.
SPECIFICITY
The use of monoclonal antibodies in the elaboration of ATLAS HCG kit assures its high
degree of specificity for HCG. The cross reaction with LH (human Luteinizing Hormone) is
so low that concentrations of 4.0 UI/ml or less do not produce agglutination (this is about
20-30 times higher than the maximum excretion rate of LH in the urine of menopausal
women). High level of HCG may occur in urine of patients suffering with chorionic
epithelioma or hydatid mole.

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 2. Heamagglutination inhibition technique
The principal reaction of the HI test is the detection of HCG in pregnancy urine,
determined by means of an antigen-antibody reaction. In the HI method the antigen HCG
is adsorbed on to the surface of sheep erythrocytes. These treated cells will then behave
as the specific antigen and are therefore agglutinated by the corresponding anti-body. The
agglutination pattern appears as a uniform, opaque, diffuse sediment at the bottom of the
glass tube. If the antigen is present in the urine this will combine with the antibody and
inhibit agglutination. The red cells then appear as a reddish-brown ring in the centre of
the bottom of the tube.

Materials and methods

(a) Test suspension: Formalin-preserved sheep erythrocytes sensitized with HCG. When
agglutinated by rabbit anti-HCG a completely agglutinated pattern develops when the
suspension is added to a simple diluent.
(b) Control suspension: Formalin-preserved sheep erythrocytes stabilized in normal rabbit
serum but not sensitized with HCG.
SPECIMEN COLLECTION AND PREPARATION
Generally, the first morning urine contains the highest concentration of the hCG
hormone and therefore, it is more recommended for testing. However, urine collected at
other periods can also be used. The urine in this case should have been kept in 2-8C and
used within 72 hours from collection time.
Procedure
The method was as follows: Early-morning specimens of urine were centrifuged and from
the supernatant 1: 5, 1 : 10 and 1 : 20 dilutions were made with the buffer.
1: 5 = 0·5 mI. supematant+2 rnI. buffer.
1 : 10 = 0·5 mI. supematant+4'5 rnI. buffer.
1 : 20 = 0·5 ml. supernatant+9'5 rnI. buffer.
Of each dilution, 1 rnI. was pipetted into 3 different tubes, preparing simultaneously a
parallel series. The tubes were 3XO·5 inches with round bottom. It is important that there
should be no flaws since they may interfere with the formation of the pattern. To all tubes

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in the first series 0·1 ml. of test suspension was added and to the second series 0·1 rnI. of
control suspension was added.
In addition, with each group of tests. two tubes each containing 1 rnI. of buffer only
were included as reagent controls to which 0·1 rnI. of test and control suspension were
added respectively. After gentle shaking the tubes were set up in a test tube rack.
For the purpose of this trial a special rack was designed, fitted with an adjustable
mirror to facilitate reading the results without disturbing the precipitate formed.
The tubes were left standing at room temperature (in cold, unheated rooms false
agglutination may occur owing to cold agglutinins in the urine), undisturbed by draughts
and vibration and not exposed to sunlight. A convenient time for setting up of the test was
found to be 2 p.m. so that results could be read early next morning. It was found that
positive results in some cases could be read after 4 hours; but before deciding on a
negative result, it was felt advisable to wait until the following morning.
Reading of Tests
Interpretation of the agglutination patterns is illustated as follows:

(a) Non-pregnant. Complete agglutination in all dilutions of test suspension with inhibition
of agglutination in all controls.

(b) Pregnant. If the test suspension is treated with urine containing HCG in sufficient
concentration, agglutinationis inhibited and a pattern develops comparable with that of
the unsensitized control suspension. Inhibition of agglutination in test suspensions may
occur in all 3 dilutions. This is the most frequently encountered pattern. Inhibition at 1:10
and/or 1: 5 dilutions would indicate reduced HCG levels.

(c) Pregnant. Occasionally complete agglutination occurs in test and control in the 1: 5
dilution with inhibition of the 1 : 10 and 1 : 20 dilutions of test and control suspensions
indicating pregnancy with non-specific agglutination at the 1: 5 level

Sensitivity
An extremely important observation made was the ability of the HI test to determine early
pregnancy at a time at which the HCG level in the urine was less than 3,000 IU /1.
Specificity
The presence of non-specific agglutinins in a urine specimen can result in the production
of an agglutination pattern at the I : 5 level in which case agglutination will also occur in
the control tube. But in such cases a positive result can be recorded by observing

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inhibition of agglutination in the higher dilutions of test and control suspensions. It will
therefore be understood that preparation of adequate controls is of vital importance.

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 3.Rapid chromatographic immunoassay
The hCG Card Pregnancy Test is a rapid test that qualitatively detects the presences of
hCG in urine specimen at the sensitivity of 25 mIU/mL. The test utilizes a combination of
monoclonal and polyclonal antibodies to selectively detect elevated levels of hCG in urine.
At the level of claimed sensitivity, the hCG Card Pregnancy Test shows no cross-reactivity
interference from the structurally related glycoprotein hormones hFSH, hLH and hTSH at
high physiological levels.

INTENDED USE
The hCG Card Pregnancy Test is a rapid chromatographic immunoassay for the qualitative
detection of human chorionic gonadotropin in urine to aid in the early detection of
pregnancy.

PRINCIPLE
The hCG Card Pregnancy Test is a rapid chromatographic immunoassay for the qualitative
detection of human chorionic gonadotropin in urine to aid in the early detection of
pregnancy. The test utilizes a combination of antibodies including a monoclonal hCG
antibody to selectively detect elevated levels of hCG. The assay is conducted by adding a
urine specimen to the specimen well of the test device and observing the formation of
colored lines. The specimen migrates via capillary action along the membrane to react
with the colored conjugate. Positive specimens react with the specific antibody-hCG-
colored conjugate to form a colored line at the test line region of the membrane. Absence
of this colored line suggests a negative result. To serve as a procedural control, a colored
line will always appear in the control line region indicating that proper volume of
specimen has been added and membrane wicking has occurred.

REAGENTS
The test card contains anti-hCG particles and anti-hCG coated on the membrane.

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PRECAUTIONS
·For professional in vitro diagnostic use only. Do not use after the expiration date.

·The test device should remain in the sealed pouch until use.

·All specimens should be considered potentially hazardous and handled in the same
manner as an infectious agent.

·The test device should be discarded in a proper biohazard container after testing.

STORAGE AND STABILITY

The kit can be stored at room temperature or refrigerated (2-30°C). The test card is stable
through the expiration date printed on the sealed pouch. The test card must remain in the
sealed pouch until use. DO NOT FREEZE. Do not use beyond the expiration date.

SPECIMEN COLLECTION AND PREPARATION

A urine specimen must be collected in a clean and dry container. A first morning urine
specimen is preferred since it generally contains the highest concentration of hCG;
however, urine specimens collected at any time of the day may be used. Urine specimens
exhibiting visible precipitates should be centrifuged, filtered, or allowed to settle to obtain
a clear specimen for testing.

SPECIMEN STORAGE
Urine specimens may be stored at 2-8°C for up to 48 hours prior to testing. For prolonged
storage, specimens may be frozen and stored below -20°C. Frozen specimens should be
thawed and mixed before testing.

MATERIALS

 Specimen collection container

 Timer

DIRECTION FOR USE

1. Allow the test device, urine specimen and/or controls to equilibrate to room
temperature (15-30°C) prior to testing.

2. Bring the pouch to room temperature before opening it. Remove the test device from
the sealed pouch and use it as soon as possible.

3. Place the test device on a clean and level surface. Hold the dropper vertically and
transfer 3 full drops of urine (approx. 180 µl) to the specimen well (S) of the test device,
and then start the timer. Avoid trapping air bubbles in the specimen well (S).

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4.The result should be interpretated between 3-5 minutes. Please confirm negative results
at 10 minutes. Do not interpretate result exceeding 10 minutes.

INTERPRETATION OF RESULTS
POSITIVE:* Two distinct red lines appear. One line should be in the control region and another line
should be in the test region .

*NOTE: The intensity of the red color in the test line region will vary depending on the
concentration of hCG present in the specimen. Therefore, any shade of red in the test region
should be considered positive.

NEGATIVE: One red line appears in the control region). No apparent red or pink line appears in the
test region.

INVALID: Control line fails to appear. Insufficient specimen volume or incorrect procedural
techniques are the most likely reasons for control line failure. Review the procedure and repeat
the test with a new test device.

QUALITY CONTROL
A procedural control is included in the test. A red line appearing in the control region is the
internal procedural control. It confirms sufficient specimen volume and correct procedural
technique.

It is recommended that a positive hCG control (containing 25-250 mIU/mL hCG) and a negative
hCG control (containing "0" mIU/mL hCG) be evaluated to verify proper test performance when a
new shipment of test devices are received.

LIMITATIONS
1. The hCG Card Pregnancy Test is a qualitative test, therefore, neither the quantitative value nor
the rate of increase in hCG can be determined by this test.

2. Very dilute urine specimens, as indicated by a low specific gravity, may not contain
representative levels of hCG. If pregnancy is still suspected, a first morning urine specimen should
be collected 48 hours later and tested.

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3. False negative results may occur when the levels of hCG are below the sensitivity level of the
test. When pregnancy is still suspected, a first morning urine specimen should be collected 48
hours later and tested.

4. Very low levels of hCG (less than 50 mIU/mL) are present in urine specimen shortly after
implantation. However, because a significant number of first trimester pregnancies terminate for
natural reasons, a test result that is weakly positive should be confirmed by retesting with a first
morning urine specimen collected 48 hours later.

5. A number of conditions other than pregnancy, including trophoblastic disease and certain non-
trophoblastic neoplasms including testicular tumors, prostate cancer, breast cancer, and lung
cancer, cause elevated levels of hCG.6,7 Therefore, the presence of hCG in urine specimen should
not be used to diagnose pregnancy unless these conditions have been ruled out.

6. This test provides a presumptive diagnosis for pregnancy. A confirmed pregnancy diagnosis
should only be made by a physician after all clinical and laboratory findings have been evaluated.

ACCURACY
A multi-center clinical evaluation was conducted comparing the results obtained using the hCG
Card Pregnancy Test to another commercially available urine membrane hCG test. The study
included 159 urine specimens: both assays identified 88 negative and 71 positive results. The
results demonstrated a 100% overall accuracy of the hCG Card Pregnancy Test when compared to
the other urine membrane hCG test.

SENSITIVITY AND SPECIFICITY

The hCG Card Pregnancy Test detects hCG at a concentration of 25 mIU/mL or greater. The test
has been standardized to the W.H.O. Third International Standard. The addition of LH (300
mIU/mL), FSH (1,000 mIU/mL), and TSH (1,000 µIU/mL) to negative (0 mIU/mL hCG) and positive
(25 mIU/mL hCG) specimens showed no cross-reactivity.

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 4.Enzyme immunoassay
INTENDED USE
For the quantitative determination of Human Chorionic Gonadotropin (hCG)
concentration in human serum.

Principle of the Test

The β-HCG ELISA TEST is based on simoultaneous binding of human b-HCG to two
monoclonal antibodies, one immobilized on microwell plates, the other conjugated with
horseradish peroxidase (HPR).

After incubation the bound/free separation is performed by a simple solid-phase washing,

then the substrate solution (TMB) is added. After an appropriate time has elapsed for
maximum color development, the enzyme reaction is stopped and the absorbance are
determinated.

The b-HCG concentration in the sample is calculated based on a series of standard.

The color intensity is proportional to the b-HCG concentration in the sample. The ELISA
test is performed as an indirect solid phase sandwich-type immunoassay. Microwells are
coated with anti-monoclonal β - HCG followed by blocking the unreacted sites to reduce
non-specific binding.

Step 1 β - hCG Antigens present in calibrators and patient samples bind to the coated
antibody.

Step 2 The Antigen-Antibody complex is reacted with enzyme (HRP) labeled anti-
monoclonal β - HCG conjugate resulting in the β - HCG antigen being sandwiched between
the solid phase antibody and the enzyme conjugate.

Step 3 The enzyme converts added substrate (TMB) to form a colored solution.

Step 4 The intensity of color change, which is proportional to the concentration of

Antibodies present in the samples is read by a microplatereader at 450 nm. Results are
expressed in mIU/ml.

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REAGENTS
Materials provided with the kit:

 Microwell plate. 12x8 well strips. Individually separable wells. Coated with anti-
monoclonal β –HCG., packaged in an aluminum bag with a drying agent.

 Standards. 5 Vials. Standard A – Standard E each 0.4 mL, ready to use. Concentrations
0, 5, 20, 75 and 200 mIU/mL

 Sample diluent. 1 vial of Sample diluent 10 mL.

 Enzyme Conjugate. anti-β–HCG HRP-horseradish peroxidase (HRP). Ready-to-use. 12

mL.

 TMB Substrate solution. H2O2-TMB 0.25 g/L (avoid any skin contact). 12 mL.

 Stop Solution. Sulphuric acid 0.15 mol/L (corrosive: avoid any skin contact). 12 mL.

 Washing buffer. 1 bottle 25 mL, 40 fold

 Multichannel pipettes and micropipettes (Precision >1.5%) and disposable tips.

 Microplate reader with a 450 nm filter. Reference filter of 620 or 655 nm is advisable.

 Manual or automated wash system.

 Absorbent paper of blotting the microplate wells.

 Distilled or deionised water.

 Timer.

STORAGE OF TEST KITS

The components will remain stable through the expiration date shown on the label if
stored between 2-8°C in dark. Do not frezee. Do not use reagents beyond the kit
expiration date. The bag containing the microplate should be brought to room
temperature before opening to avoid condensation in the wells. Once opened the bag,
microplate strips are stable for 1 month at 2-8°C in the plastic bag tightly sealed, with the
silicagel. Opened reagents are stable for 1 month at 2-8°C.

REAGENT PREPARATION
Coated microwell strips are for one time use only. Calibrators, Substrate Solution, Enzyme
Conjugate and Stop Solution are ready to use and need not to be diluted. Washing Buffer
is concentrated and need to be diluted.

PRECAUTIONS
• Instructions should be followed exactly as they appear in this kit insert to ensure valid
results.

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• Avoid contact with the TMB (3,3`,5,5`-Tetramethylbenzidine). If TMB comes into contact
with skin wash thoroughly with water and soap.

• The stop solution contains sulphuric acid. If it comes into contact with skin, wash
thoroughly with water and seek medical attention.

• Avoid contact between the buffered peroxide solution and easily oxidized materials;
extreme temperatures may initiate spontaneous combustion.

• Do not use beyond expiration date on the label.

• Do not use if reagent is not clear or if a precipitate is present.

• Do not interchange kit components with those from other sources other than the same
catalog number from LINEAR.

• Follow good laboratory practices to minimize microbial and cross contamination of

reagents when handling.

• All human derived components used have been tested for HBsAg, HCV, HIV-1 and 2 and
HTLV-I and found negative by FDA required tests. However, human blood derivatives and
patient specimens should be considered potentially infectious. Follow good laboratory
practices in storing, dispensing and disposing of these materials.

SPECIMEN COLLECTION AND PREPARATION

Only Serum or Plasma specimens should be used in this procedure. The patients need not
to be fasting, and no special preparations are necessary. Use fresh serum or plasma.
Samples can be stored at 2-8°C for 2 days. For longer periods, samples should be frozen (-
20°C). Avoid repeated freezing and thawing.Grossly hemolyzed, lipemic or microbially
contaminated specimens may interfere with the performance of the test and should not
be used. Neither Bilirubin nor Hemolysis have significant effect on the procedure.

Store specimens at 2°- 8°C for up to a maximum of 2 days. For longer storage, specimens
should be frozen. Avoid repeated freezing and thawing of samples. For sample with
concent Preparation of Reagents

Preparation of sample
Dilute samples with concentrations above 200 mIU/mL with sample Diluent. Dilute
samples from woman after the 4th pregnancy week 1:50 with sample Diluent.

Test Procedure
Label protocol sheet to indicate sample placement in the wells according to the following
figure. 5 calibrators(standards) (A-E) and 1 Blank should be included. The user has the
option to run Patient Samples (P) in duplicate.ration over 200 mIU/mL dilute the sample
with Sample Diluent.

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1. Remove the required microwells from pouch and return unused strips in the sealed pouch to
refrigerator. Securely place the microwells into the extra provided holder .

2. Pipette 25 µL of Calibrators and Patient Samples into the wells.

3. Seal the plate and incubate 20 minutes at 37° C.

4. Add 100 µL of Enzyme Conjugate to the wells except for Blank well seal the plate and incubate
60 minutes at 37°C.

5. Discard the contents of the microwells and wash the wells with 200-300 µL Washing buffer.
Repeat the washing procedure for 4 times.

6. Pipette 100 µL of Substrate Solution into each microwell in the same order and timing as for the
Enzyme Conjugate, Blank well included.

7. Incubate 12 minutes at room temperature.

8. Add 100 µL of Stop Solution into each microwell using the same order and timing as for the
addition of the Substrate Solution.

9. Read absorbance of each microwell at 450 nm against blank using a microplate reader.

Interpretation
1. Calculate the mean of the absorbances (Em) corresponding to the single points to the
standard curve and of each sample

2. Subtract the mean absorbance value of the zero standard from the mean absorbance
values of standards and samples.

3. Draw the standard curve on log-log graph paper by plotting absorbance values of
standard against appropriate β - hCG concentration.

4. Read off the β - hCG concentrations of the calibrators and samples.

LIMITATIONS
The assay should not be performed on grossly hemolyzed, microbially contaminated or lipemic
samples. This method should be used for testing human serum samples only.

Sensitivity

19
The minimal detectable concentration of β - hCG by this assay is estimated to be 1 mIU/ml.

Specificity
The cross reaction of the coated microplate calculated according are shown in the table:

Precision
a. Intra Assay variation; within-run precision was determined by replicate determination
of three different control in one assay. The within assay variability is shown below:

b. Inter Assay variation; between-run precision was determined by replicate

determination of three different controls in one assay.

Limitations of the procedure

In this assay, no hook effect is observed up to 10.000 mIU/mL of β - HCG.

20
 5.Chemiluminescent microparticle immunoassay
INTENDED USE
The ARCHITECT Total β-hCG assay is a Chemiluminescent Microparticle Immunoassay
(CMIA) for the quantitative and qualitative determination of beta human chorionic
gonadotropin (β-hCG) in human serum and plasma for the early detection of pregnancy.

PRINCIPLES OF THE PROCEDURE

The ARCHITECT Total β-hCG assay is a two-step immunoassay to determine the presence
of β-hCG in human serum and plasma using Chemiluminescent Microparticle
Immunoassay (CMIA) technology with flexible assay protocols, referred to as Chemiflex. In
the first step, sample, and anti-β-hCG coated paramagnetic microparticles are combined.
β-hCG present in the sample binds to the anti-β-hCG coated microparticles. After washing,
anti-β-hCG acridinium-labeled conjugate is added in the second step. Pre-Trigger and
Trigger Solutions are then added to the reaction mixture; the resulting chemiluminescent
reaction is measured as relative light units (RLUs). A direct relationship exists between the
amount of β-hCG in the sample and the RLUs detected by the ARCHITECT i optical system.

Reagents
• 1 or 4 Bottle(s) (6.6 mL/27.0 mL) anti-β-hCG (mouse, monoclonal) coated
Microparticles in TRIS buffer with protein (bovine) stabilizers.

• 1 or 4 Bottle(s) (2.1 mL/7.4 mL) anti-β-hCG (mouse, monoclonal) acridinium-labeled

Conjugate in MES buffer with protein (bovine) stabilizers. Minimum concentration: 2.9
µg/mL.

 Manual Diluent containing phosphate buffered saline solution.

• Pre-Trigger Solution containing 1.32% (w/v) hydrogen peroxide.

• Trigger Solution containing 0.35N sodium hydroxide.

21
• Wash Buffer containing phosphate buffered saline solution.

WARNINGS AND PRECAUTIONS

• For In Vitro Diagnostic Use

Safety Precautions
CAUTION: This product requires the handling of human specimens. It is recommended
that all human sourced materials be considered potentially infectious and handled in
accordance with the OSHA Standard on Bloodborne Pathogens.12 Biosafety Level 213 or
other appropriate biosafety practices14,15 should be used for materials that contain or
are suspected of containing infectious agents.

• Do not use reagent kits beyond the expiration date.

• Do not mix reagents from different reagent kits.

• the microparticle bottle requires mixing to resuspend microparticles that have settled
during shipment

• Septa MUST be used to prevent reagent evaporation and contamination and to

ensure reagent integrity.

• To avoid contamination, wear clean gloves when placing a septum on an uncapped

reagent bottle.

• Once a septum has been placed on an open reagent bottle, do not invert the bottle
as this will result in reagent leakage and may compromise assay results.

• Over time, residual liquids may dry on the septum surface. These are typically dried
salts which have no effect on assay efficacy.

Storage Instructions
• Reagent Kit must be stored at 2-8°C and may be used immediately after removal
from 2-8°C storage.

• When stored and handled as directed, reagents are stable until the expiration date.

• Reagent Kit may be stored on-board the for a maximum of 30 days. After 30 days, the
reagent kit must be discarded.

SPECIMEN COLLECTION AND PREPARATION FOR ANALYSIS

• Human serum (including serum collected in serum separator tubes) or plasma
collected in lithium heparin, sodium heparin, or potassium EDTA may be used. .

• Inadequate centrifugation or the presence of fibrin or particulate matter in the

sample may cause an erroneous result.

22
• Abbott Laboratories recommends the use of plasma for STAT testing. Since plasma
does not require the clotting time of serum, it has a decreased likelihood of the presence
of fibrin or other particulates.

• Use caution when handling patient specimens to prevent cross contamination. Use of
disposable pipettes or pipette tips is recommended.

• For optimal results, inspect all samples for bubbles. Remove bubbles with an
applicator stick prior to analysis. Use a new applicator stick for each sample to prevent
cross contamination.

• For optimal results, serum and plasma specimens should be free of fibrin, red blood
cells or other particulate matter.

• Ensure that complete clot formation in serum specimens has taken place prior to
centrifugation. Some specimens, especially those from patients receiving anticoagulant or
thrombolytic therapy may exhibit increased clotting time. If the specimen is centrifuged
before a complete clot forms, the presence of fibrin may cause erroneous results.

• If testing will be delayed more than 24 hours, remove serum or plasma from the clot,
serum separator or red blood cells. Specimens may be stored for up to 7 days at 2-8°C
prior to being tested. If testing will be delayed more than 7 days, specimens should be
frozen at -10°C or colder. Specimens stored frozen at -10°C or colder for 12 months
showed no performance difference.

• Multiple freeze-thaw cycles of specimens should be avoided. Specimens must be

mixed THOROUGHLY after thawing, by LOW speed vortexing or by gently inverting, and
centrifuged prior to use to remove red blood cells or particulate matter to ensure
consistency in the results.

• When shipped, specimens must be packaged and labeled in compliance with

applicable state, federal and international regulations covering the transport of clinical
specimens and infectious substances. Specimens may be shipped under thermally
controlled refrigerated conditions or frozen (dry ice). Prior to shipment, it is
recommended that specimens be removed from the clot, serum separator or red blood
cells.

PROCEDURE
• The microparticle bottle requires mixing to resuspend microparticles that have
settled during shipment:

• Invert the microparticle bottle 30 times.

• Visually inspect the bottle to ensure microparticles are resuspended. If microparticles

are still adhered to the bottle, continue to invert the bottle until the microparticles have
been completely resuspended.

• Once the microparticles have been resuspended, remove and discard the cap.
Wearing clean gloves, remove a septum from the bag. Carefully snap the septum onto the
top of the bottle.

23
• If the microparticles do not resuspend, DO NOT USE.

• Load the Reagent Kit on to the System. Verify that all necessary assay reagents are
present. Ensure that septa are present on all reagent bottles.

• The minimum sample cup volume is calculated by the system and is printed on the
Orderlist report. No more than 10 replicates may be sampled from the same sample cup.
To minimize the effects of evaporation verify adequate sample cup volume is present
prior to running the test.

• Priority: 75 µL for the first Total β-hCG test plus 25 µL for each additional Total β-hCG
test from the same sample cup

• ≤ 3 hours onboard: 150 µL for the first Total β-hCG test plus 25 µL for each additional
Total β-hCG test from the same sample cup

• > 3 hours onboard: additional sample volume is required.

• If using primary or aliquot tubes, use the sample gauge to ensure sufficient patient
specimen is present.

• To obtain the recommended volume requirements, hold the bottles vertically and
dispense 4 drops of each calibrator or 3 drops of each control into each respective sample
cup.

• Load samples

• Press RUN.

The System performs the following functions:

• Moves the sample to the aspiration point

• Loads a reaction vessel (RV) into the process path

• Aspirates and transfers sample into the RV

• Advances the RV one position and transfers microparticles into the RV

• Mixes, incubates and washes the reaction mixture

• Adds conjugate to the RV

• Mixes, incubates and washes the reaction mixture

• Adds Pre-Trigger and Trigger Solutions

• Measures chemiluminescent emission to detect the presence and quantity of β-hCG

in the sample

• Aspirates contents of RV to liquid waste and unloads RV to solid waste

• Calculates the result

24
Specimen Dilution Procedures
Specimens with a Total β-hCG value exceeding 15,000.00 mIU/mL are flagged with the
code “ >15,000.00” and may be diluted with either the Automated Dilution Protocol or the
Manual Dilution Procedure.

• If using the Automated Dilution Protocol, the system performs a 1:15 dilution of the
specimen and automatically calculates the concentration of the diluted specimen and
reports the result.

• The suggested dilution for Total β-hCG is 1:15. It is recommended dilutions not
exceed 1:75.

• For a 1:15 dilution, add 20 µL of the patient specimen to 280 µL of ARCHITECT i Multi-
Assay Manual Diluent (7D82-50).

• The operator must enter the dilution factor in the patient or control order screen.
The system will use this dilution factor to automatically calculate the concentration of the
sample before dilution. This will be the reported result. The result (before dilution factor is
applied) should be greater than 467.00 mIU/mL.

• If the operator does not enter the dilution factor, the reported result will be that of
the diluted sample. This result (before dilution factor is applied) should be greater than
467.00 mIU/mL.

• NOTE: A printed or displayed result of < 7000.00 mIU/mL (1:15 Automated Dilution
Protocol) indicates the need to retest the sample at a lower dilution or undiluted. The
result and interpretation should not be reported.

• To perform an ARCHITECT Total β-hCG calibration, test Calibrators A, B, C, D, E, and F

in duplicate. A single sample of all levels of Total β-hCG controls must be tested to
evaluate the assay calibration. Ensure that assay control values are within the
concentration ranges specified in the package insert. Calibrators should be priority loaded.

• Calibrator Range: 0.00 - 15,000.00 mIU/mL.

• Routine and STAT protocols require separate calibrations but require only one
reagent kit.

• Once an ARCHITECT Total β-hCG calibration is accepted and stored, all subsequent
samples may be tested without further calibration unless:

• A reagent kit with a new lot number is used

• Controls are out of range

• For detailed information on how to perform an assay calibration, refer to the

ARCHITECT System Operations Manual, Section 6.

25
QUALITY CONTROL PROCEDURES
The recommended control requirement for the ARCHITECT Total β-hCG assay is a single
sample of all control levels tested once every 24 hours each day of use. If the quality
control procedures in your laboratory require more frequent use of controls to verify test
results, follow your laboratory specific procedure. Due to variation in analyte composition
and/or matrices, external quality control materials and proficiency survey samples, may
not elicit identical results across all hCG assays. Non-Abbott Quality Control and
proficiency testing material may contain high levels of free beta-subunit hCG molecules.
Non-Abbott Quality Control and proficiency testing material may generate different
results when comparing a whole molecule hCG assay to a total β-hCG assay. Each
laboratory needs to determine the suitability of each control material for specific
immunoassays and validate the material prior to use.

Alternate Result Units

• The default result unit for the ARCHITECT Total β-hCG assay is mIU/mL. When the
alternate result unit, IU/L, is selected, the conversion factor used by the system is 1.

• Conversion Formula: (Concentration in mIU/mL) x (1) = IU/LFlags

• Some results may contain information in the Flags field.

Interpretation of Results
For qualitative interpretation of the ARCHITECT Total β-hCG test results, specimens with
β-hCG levels less than or equal to 5.00 mIU/mL will be reported in the INTERPRETATION
field on the test results screen or printout as “NEGATIVE”. Specimens with β-hCG levels
greater than or equal to 25.00 mIU/mL will be reported as “POSITIVE”. Specimens with β-
hCG levels greater than 5.00 mIU/mL and less than 25.00 mIU/mL will be reported with
concentrations only. No interpretation will be reported for these results.

LIMITATIONS OF THE PROCEDURE

• This assay is capable of detecting whole molecule (intact) hCG as well as free β-hCG
subunits.

• For diagnostic purposes, hCG results should always be used in conjunction with other
data; e.g., patient’s medical history, symptoms, results of other tests, clinical impressions,
etc. Ectopic pregnancy cannot be distinguished from normal pregnancy by hCG
measurements alone.10,11 The results from this or any other diagnostic kit should be
used and interpreted only in the context of the overall clinical picture.

• If the hCG level is inconsistent with, or unsupported by, clinical evidence, results
should be confirmed by an alternate hCG method. This may include the qualitative testing
of urine.29 The absence of urinary hCG may suggest a falsely elevated serum result.
Results may also be confirmed by performing serial dilutions of the sample. Usually, but
not always, samples that contain interfering substances exhibit nonlinear results when
diluted.

26
• The ARCHITECT Total β-hCG assay is cleared for use in the early detection of
pregnancy only. It is not approved for any other uses such as tumor marker screening,
tumor marker monitoring, etc. and it should not be performed for any other uses.

• Infrequently, hCG levels may appear consistently elevated and could be due to, but
not limited to, the presence of the following:20,21,28,29,31,32

- heterophilic antibodies

- nonspecific protein binding

- hCG-like substances

- trophoblastic or nontrophoblastic neoplasms

• As with any immunochemical reaction, unknown interferences from medications or

endogenous substances may affect results.

• Elevated hCG levels have been associated with some abnormal physiological states
(e.g., trophoblastic and nontrophoblastic neoplasms)16,17 and the results of this test
should not be used in the diagnosis of these abnormal states. There have been reports of
people receiving unnecessary medical treatment and surgery, including chemotherapy
and hysterectomy, when hCG results were used in the diagnosis of abnormal states.

• Interfering substances (such as heterophilic antibodies, non-specific proteins, or hCG-

like substances) may falsely depress or falsely elevate results. These interfering
substances may cause false results over the entire range of the assay, not just at low
levels, and may indicate the presence of hCG when there is none.

• Detection of very low levels of hCG does not rule out pregnancy.21Low levels of hCG
can occur in apparently healthy, nonpregnant subjects.26,27 Because hCG values double
approximately every 48 hours in a normal pregnancy,21 patients with very low levels of
hCG should be resampled and retested after 48 hours.

• Post menopausal specimens may elicit weak positive results due to low hCG levels
unrelated to pregnancy. With a weak positive result, it is good laboratory practice to
resample and retest after 48 hours, or to test with an alternate hCG method.

• Because of the high degree of sensitivity of the assay, specimens tested as positive
during initial days after conception may later be negative due to natural termination of
the pregnancy. Natural termination occurs in 22% of clinically unrecognized pregnancies
and 31% of pregnancies overall.30 It is good laboratory practice to resample and retest
weak positive results after an additional 48 hours.

• Specimens from patients who have received preparations of Mouse Monoclonal

Antibodies for diagnosis or therapy may contain Human Anti-Mouse Antibodies (HAMA).
Such specimens may demonstrate either falsely elevated or falsely depressed results
when tested with assay kits which employ Mouse Monoclonal Antibodies.18,19 These
specimens should not be tested with the Abbott ARCHITECT Total β-hCG assay.

27
• Heterophilic antibodies in human serum can react with reagent immunoglobulins,
interfering with in vitro immunoassays.20,21,32Patients routinely exposed to animals or
to animal serum products, can be prone to this interference and anomalous values may
be observed. Additional information may be required for diagnosis.

Precision
The ARCHITECT Total β-hCG assay is designed to have a precision of < 10% (total CV). A
study based on guidance from Clinical and Laboratory Standards Institute (CLSI, formerly
NCCLS) document EP5-T2 24 was performed for the ARCHITECT Total β-hCG assay. A three
member processed human serum based panel was assayed, using two lots of reagents, in
replicates of two at two separate times per day for 20 days. Data from this study are
summarized in the following table.

Analytical Sensitivity
The ARCHITECT Total β-hCG assay is designed to have an analytical sensitivity of ≤ 1.2 mIU/mL.
Analytical Sensitivity is defined as the concentration at two standard deviations from the mean
RLU value of the ARCHITECT Total β-hCG Calibrator A (0.00 mIU/mL) and represents the lowest
measurable concentration of β-hCG that can be distinguished from zero.

Specificity
The ARCHITECT Total β-hCG assay is designed to have a mean analytical specificity of < 10% cross
reactivity with FSH, LH, and TSH. Aliquots of human serum containing β-hCG were supplemented
with 150 mIU/mL FSH, 250 mIU/mL LH, and 100 µIU/mL TSH and assayed for β-hCG. The cross
reactivity was calculated as a percent interference and was shown to be less than 10% for each
cross reactant.

Carryover
Carryover from a sample containing 1,000,000 mIU/mL β-hCG to an adjacent 0 mIU/mL β-hCG
sample was less than 7.5 mIU/mL β-hCG.NOTE: Please be aware that individual samples may
exhibit elevated concentration due to build up of proteins on the sample pipettor probe. For
further troubleshooting information, refer to the ARCHITECT System Operations Manual, Section
10.

28
References;
 Ethiopian public health training initiative, serology lecture notes, 2008E.C

 pubmed.ncbi.nlm.nih.gov

 en.m.wikipedia.org

 www.abnova.com

 PREGNANCY DIAGNOSIS - HAEMAGGLUTINATION INHIBITION METHOD(pREPUERIN)

COMPARED WITH THE XENOPUS LAEVIS TESTW. M. POLITZER. M.D., South African
Institute for Medical Research, Johannesburg

 ARCHITECT System Total Beta-hCG kit manual

 DIA source hCG Card Pregnancy Test (human chorionic gonadotropin) RAPU01C040

 www.linear.es

29