School of Biological Sciences
BI2BC45 Cells and Immunity
Practical 2
Pregnancy Testing
BI2BC45 Cells and Immunity: Practical 2
Introduction
The ability of the adaptive immune system to produce an antibody response when challenged
by a foreign antigen has long been exploited by scientists and clinicians to provide a source of
antibodies that specifically bind to a molecule of interest. The basic procedure involves
injecting a laboratory antibody on a repeated monthly basis with a small amount of the
desired molecule and subsequently bleeding a suitable volume of blood from the animal
around a fortnight later. The blood is allowed to clot and the serum collected, which contains
the antibodies of interest, can be used for laboratory or clinical purposes - one such
application is for the generation of immunoassays.
Immunoassays
An immunoassay is a biochemical test that uses the specific interactions of antibodies with
their antigens to measure the presence of a specific protein in a biological sample, typically
serum or urine. Immunoassays can measure the presence of an antigen (e.g. a hormone such
as in this practical) or an antibody (e.g. a HIV test detects the presence of antibodies to HIV in
the serum of a patient).
Different types of immunoassays are used in clinical laboratories. The most common
differences between these assay types stem from; (i) what is being detected by the assay
(antibody or antigen) and, if an antigen is being detected, (ii) what antibodies are available to
this protein and (iii) how the interaction between the antibody and the antigen is detected.
Examples of different assay types are illustrated in Figure 3, which shows the difference
between an indirect ELISA, a format commonly used to detect antibodies to a specific antigen
in a patient’s serum (i.e. diagnostic tests for HIV infection or allergy, and a “sandwich” ELISA.
Methods of detection of antibody-antigen interactions include enzymatic reactions, as is
shown in Figure 1 and will be used in today’s Enzyme-Linked ImmunoSorbant Assay (ELISA),
radioactive labeling (radioimmunoassay), fluorescent dyes or latex beads (home pregnancy
tests).
Figure 1: Indirect and sandwich ELISAs. The principles underlying these ELISA methods are
illustrated. (A) The indirect ELISA method allows the detection of antibody in the serum of a
patient (e.g. HIV test) (B) A sandwich ELISA uses two distict antibodies to detect a target
protein in a patient sample (e.g. pregnancy test). Figure modified from Berg et al 2007.
The immunoassay that will be performed in this practical class is an example of a “sandwich”
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ELISA. The “sandwich” ELISA is versatile and highly sensitive immunoassay that utilizes two
distinct antibodies to detect the protein in a biological sample. The first “capture” antibody is
bound to the surface of a 96 well ELISA plate. The patient’s sample then is applied and any
antigen present allowed to bind to the capture antibody. Unbound proteins then are washed
away and the second “detection” antibody is added. If the antigen has bound to the capture
antibody, the detection antibody will bind to the complex, forming a “sandwich” (Figure 3).
The assay is then washed again to remove any unbound detection antibody. In many modern
assays, the detection antibody is conjugated directly to an enzyme that catalyses a colour
change when the enzyme’s substrate is added. When a chemical substrate for the enzyme
used is applied to the assay, any enzyme present will catalyse a change in the substrate for the
enzyme is applied, and catalysis by the enzyme leads to a color change proportional to the
amount of detection antibody bound, and hence, is proportional to how much antigen is
present in the sample. By also measuring the colour changes associated with known
concentrations of antigen (standard curve), levels of antigen present in a sample can be
quantitated.
The Pregnancy Test
The pregnancy test is among the most widely used clinical assays and is unique in that it is
performed both by healthcare professionals and the public. Most pregnancy tests work by
detecting the hormone human chorionic gonadotrophin (hCG) in the patient’s urine, blood or
serum. Home pregnancy tests are very simple to use and provide an easy to interpret
qualitative result, pregnant or not pregnant, in a matter of minutes. Laboratory tests can
quantitate hCG levels in patient samples but these testing methods are often more technically
demanding. In this practical class, you will perform both quantitative and qualitative
pregnancy tests.
Background
Human Chorionic Gonadotrophin (hCG)
hCG is a member of the glycoprotein hormone (GPH) family, which also includes luteinizing
hormone (LH), follicle-stimulating hormone (FSH) and thyrotrophin-stimulating hormone
(TSH). Each of the GPH members consists of two non-covalently linked polypeptide subunits,
an alpha-subunit and a beta-subunit. The alpha-subunit is common to all GPH family members
whereas the beta subunits, which confer each hormone’s distinct biological activities, display
various degrees of sequence homology (Figure 2). For example, the beta subunits of hCG and
LH are approximately 80% identical, differing only in the presence of a 24 amino acid
extension in hCG. Specific diagnostic tests that measure levels of hCG, LH, and FSH, therefore,
must recognise divergent sequences in the beta-subunits of these hormones.
hCG and normal pregnancy
hCG is a reliable biomarker of pregnancy as hCG levels in the serum and urine of healthy non-
pregnant women and in healthy men usually are low (Table 1).
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Figure 2: Diagrammatic illustration of the heterodimeric structures of LH, hCG and FSH. Each
shares a common alpha subunit. The divergent beta subunits confer the distinct biological
activities of the these hormones.
Table 1: Upper reference limits for urine and serum concentrations of hCG in non-pregnant
women and in men (Stenman et al., 2006).
Upper reference limit for hCG (mIU/ml)
Non-pregnant women Men
<50years >50years <50years >50years
Urine 3.1 4.0 1.0 2.9
Serum 3.0 5.4 0.7 2.1
In a pregnant woman, however, hCG is produced by trophoblastic cells in the developing
placenta and is secreted into the maternal circulation from the time of blastocyst implantation
(approx. 6-7 days post-conception). Although the absolute levels of hCG produced will vary
from woman to woman, the levels of hCG in the maternal circulation and urine rise rapidly
during a normal pregnancy, doubling every 2 days over the first 6 weeks of the pregnancy and
peak at 8-10 weeks before declining (Figure 3 and Table 2). Thus, the appearance of hCG in
the maternal circulation and/or urine shortly after conception is a reliable indicator of early
pregnancy.
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BI2BC45 Cells and Immunity: Practical 2
Figure 3: Changes in hCG, oestrogen and progesterone levels during normal pregnancy.
Figure modified from Guyton and Hall (2006).
Table 2: Reference limits for hCG concentrations in normal pregnancies. Reference values
taken from Frye (2007).
Weeks after last Days post-conception hCG levels for single baby
menstrual period (mIU/mL)
3 7 0-5
4 14 5 to 425
5 21 18 to 7340
6 28 1080 to 56500
7-8 35-42 7650 to 229000
9-12 49-70 25700 to 288000
13-16 77-98 13300 to 254000
17-24 105-154 4060 to 165400
25-birth 161- 3640 to 117000
4-6 weeks after birth <5
hCG and the complications of pregnancy
The changing levels of hCG can be used to monitor abnormal pregnancies, including ectopic,
molar and multiple pregnancies, and miscarriage. For instance, in an ectopic pregnancy, hCG
levels increase and remain at lower levels than in normal pregnancy. In the case of a
miscarriage, hCG levels also might not rise at the rate expected if the pregnancy was
progressing normally, although hCG might be detected in the maternal circulation for as long
as 4-6 weeks following a miscarriage. High levels of hCG also can indicate multiple
pregnancies. Levels of hCG also can be used as prenatal screening test for trisomy 21 (Down's
syndrome), since high levels of hCG often are found in Down's syndrome pregnancies.
Elevated levels of hCG also are seen in patients with neoplasm such as molar pregnancies
(hydatiform mole) and certain cancers, including ovarian carcinoma and testicular carcinoma.
Although indicative that problems might be occurring during a pregnancy, hCG levels alone
cannot be used to diagnose these conditions, other confirmatory tests must be performed.
High or low levels of hCG merely might indicate that a pregnant women has miscalculated the
dates of her pregnancy.
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Measuring hCG – Past and Present
The first biological assay for the measurement of hCG, the Ascheim-Zondek test, was
described in 1927 (reviewed in Chaud (1992)). This assay assessed the effect that patient’s
urine had upon the ovaries of juvenile white mice. Variations on this first biological assay
were developed subsequently using other animal species, including toads, frogs, rabbits and
other rodents. Despite demonstrating that these assays could be made to be sensitive and
accurate, the protocols were expensive, technically demanding and time consuming to
perform. The introduction of immunoassays in the 1960s was a huge development in the field
of clinical biochemistry and rapid and simple immunoassays that detected hCG quickly
replaced the cumbersome early bioassays. The pregnancy tests sold over the counter today
are modified versions of a “sandwich” ELISA (Figure 3B).
The immunoassays used in home pregnancy tests are robust, sensitive and allow a result of
pregnant or not pregnant to be obtained rapidly. Most current home pregnancy tests can
detect the presence of hCG in urine in minutes with a sensitivity of 20-25mIU/ml. Although
the exact levels of hCG produced vary dramatically from individual to individual (Table 2),
many pregnancy tests can detect hCG before the woman’s first missed menstrual period is due
with reasonable accuracy. The sensitivity of each pregnancy test does vary and it is important
to understand their limitations.
References
Berg, Tymoczko and Stryer (2007) Biochemistry. 6th Ed. WH Freeman ISBN 071676766X
Chaud (1992) Pregnancy Tests: A Review. Human Reproduction 7: 701-10
Frye (1997) Understanding Diagnostic Tests in the Childbearing Year. 6th Ed..
Labrys Pr. ISBN 1891145509.
Guyton and Hall (2006) Textbook of Medical Physiology. 11th Ed. Elsevier ISBN 0721602401
Stenman, Tiitinen, Alfthan and Valmu (2006). The classification, functions and clinical use of different isoforms of
hCG. Human Reproduction Update 12: 769-84
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The aims of this practical: To measure hCG in a urine sample (provided) using quantitative
ELISA and a qualitative cassette-based pregnancy test and to develop an appreciation of the
advantages and limitations offered by each different assay format.
Safety: Laboratory coats and gloves must be worn at all times during this practical.
Special Hazards: This practical involves the manipulation of a sample containing proteins
purified from human urine, extra care must be taken. Appropriate eye protection must be
worn whenever removing liquid from the ELISA plate. Eye and skin protection also should be
worn when using the TMB COLOUR DEVELOPMENT solution and STOP solution provided. See
risk assessment for further information.
Materials:
Each PAIR of students will be provided with the following:
• 1 ELISA plate, pre-coated with anti-hCG antibody (columns 1-6 coated only)
• hCG solution (500mIU/ml) (STANDARD)
• Dilution Buffer (DILUTION BUFFER)
• FOUR 1mL TEST aliquots of URINE samples for ELISA ONLY (Labelled A, B, C or
D)
• WASH solution (1X phosphate-buffered saline (PBS) + 0.1% (v/v) Tween-20
detergent)
• ANTIBODY solution
• TMB colour reagent solution (TMB SOLUTION)
• 0.5M hydrochloric acid (STOP SOLUTION)
• 7 x 1.5ml microcentrifuge tubes (Eppendorf tubes)
• Paper towels
• Disposable plastic transfer pipettes
• Micropipettes for serial dilution of hCG standard
• Pipette tips
In the lab you will be provided with the following:
• 5mL urine samples (URINE A, B, C or D) for cassette pregnancy tests
• InstAlert immuno-hCG detector pregnancy tests
Procedures:
The immunoassay you are performing in this practical is a modified form of a research ELISA.
Incubation times have been shortened to allow us to complete the assay during the practical
class. You will use this modified assay to determine the amount of hCG in a urine sample. Each
student pair will test for the presence or absence of hCG in URINE sample (A-D).
(A) Dilution of hCG standard for assay standard curve
You are supplied with 1mL of 500 mIU/mL standard solution of purified hCG (labelled
STANDARD). You will need to serially dilute this solution to generate a hCG standard curve for
your assay.
1. Label the caps of seven 1.5mL plastic microcentrifuge tubes, S1, S2, S3, S4, S5, S6 and S7,
respectively, and arrange in numerical order (S1-S7) in a rack.
2. Using a P1000 pipette and disposable tip, transfer 500 microlitres (500µl) of DILUTION
BUFFER into each of the labelled microcentrifuge tubes.
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3. Take the tube containing the hCG standard solution (STANDARD) and label the cap of this
tube, S8, and place it in the rack with the other tubes.
This will be the top concentration of your assay’s standard curve, representing 500mIU/mL.
In subsequent steps, you will progressively dilute this standard to generate a series of diluted
hCG standard solutions.
5. Dilute the S8 standard solution 2-fold by transferring 500 microlitres (500µl) of the S8
solution to the microcentrifuge tube labelled S7 using a P1000 pipette and tip. Discard the
pipette tip. Replace cap on tube S8 and place to one side.
6. Ensuring that the lid of the tube containing the S7 standard is securely fastened, mix the
diluted standard solution using a bench top vortex for 5 seconds. This tube should contain a
total of 1mL of diluted hCG standard (S7).
5. Dilute the S7 standard solution 2-fold by transferring 500 microlitres (500µl) of the S7
solution to the S6 tube using a P1000 pipette and tip. Discard pipette tip. Replace cap on tube
S7 and place to one side. Replace cap on tube S6 and vortex to mix.
6. Repeat this process to make successive 2-fold dilutions of the hCG standard (see Figure 4).
Remember to discard the pipette tip after each use and vortex each diluted sample before
proceeding to dilute it further.
Figure 4: Diagrammatic illustration of the serial dilution of the hCG standard solution.
(B) Quantitative hCG immunoassay – STEP 1
You have now serially diluted a solution of known concentration of purified hCG. These
solutions will form the standard curve for your assay. You have been provided with an ELISA
plate where the first 6 columns have been pre-coated with a capture antibody that recognises
hCG. Each sample, standard, test or control will be assayed in triplicate. A suggested layout of
the assay is described and illustrated below (Figure 5). It is important to use the wells
indicated so you know where each sample is on the plate and to discard your pipette tip
in between each new solution/sample.
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Figure 5: Suggested layout of samples for immunoassay plate
1. Remove the liquid from the antibody-coated wells of the supplied 96 well ELISA plate by
carefully flicking the solution into a sink (as shown by the demonstrator). Tap the plate
upside down on paper towel to remove any remaining liquid.
2. Using a P200 pipette, transfer 100 microlitres (100µl) of DILUTION BUFFER into wells A1,
A2 and A3. Discard the pipette tip. This is your assay BLANK (B).
3. Using a P200 pipette, transfer 100 microlitres (100µl) of S1 standard solution into wells B1,
B2 and B3. Discard the pipette tip. This is your assay standard 1 (S1).
4. Using a P200 pipette, transfer 100 microlitres (100µl) of S2 standard solution into wells C1,
C2 and C3. Discard the pipette tip. This is your assay standard 2 (S2).
5. Using a P200 pipette, transfer 100 microlitres (100µl) of S3 standard solution into wells D1,
D2 and D3. Discard the pipette tip. This is your assay standard 3 (S3).
6. Using a P200 pipette, transfer 100 microlitres (100µl) of S4 standard solution into wells E1,
E2 and E3. Discard the pipette tip. This is your assay standard 4 (S4).
7. Using a P200 pipette, transfer 100 microlitres (100µl) of S5 standard solution into wells F1,
F2 and F3. Discard the pipette tip. This is your assay standard 5 (S5).
8. Using a P200 pipette, transfer 100 microlitres (100µl) of S6 standard solution into wells G1,
G2 and G3. Discard the pipette tip. This is your assay standard 5 (S6).
9. Using a P200 pipette, transfer 100 microlitres (100µl) of S7 standard solution into wells H1,
H2 and H3. Discard the pipette tip. This is your assay standard 5 (S7).
10. Using a P200 pipette, transfer 100 microlitres (100µl) of S8 standard solution into wells
A4, A5 and A6. Discard the pipette tip. This is your assay standard 5 (S8).
11. Using a P200 pipette, transfer 100 microlitres (100µl) of URINE SAMPLE A into wells B4,
B5 and B6. Discard the pipette tip. This is your TEST sample A (TA).
12. Using a P200 pipette, transfer 100 microlitres (100µl) of URINE SAMPLE B into wells C4,
C5 and C6. Discard the pipette tip. This is your TEST sample B (TB).
13. Using a P200 pipette, transfer 100 microlitres (100µl) of URINE SAMPLE C into wells D4,
D5 and D6. Discard the pipette tip. This is your TEST sample C (TC).
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14. Using a P200 pipette, transfer 100 microlitres (100µl) of URINE SAMPLE D into wells E4,
E5 and E6. Discard the pipette tip. This is your TEST sample D (TD).
15. Incubate your assay at room temperature for 30 minutes to allow hCG to bind to the
capture antibody that coats the 96 well plate.
While you are waiting, we will show you the pregnancy test animation on the screen.
Ensure you understand how it works – if in doubt ask!
(C) Quantitative hCG immunoassay – STEP 2
After an incubation of 30 minutes, any hCG present in any of the solutions should have bound
to the capture antibody. In the next section of the protocol we will add a second antibody that
will specifically detect any hCG that has been captured. This will be achieved through the
addition of a detection antibody (ANTIBODY) that has been chemically linked to the Horse
Radish Peroxidase (HRP) enzyme.
1. Any unbound proteins must be removed before proceeding adding the detection antibody.
Remove the liquid from each well of the ELISA plate by carefully flicking the solution into a
sink. Tap the assay plate upside down on paper towel to remove any residual liquid.
2. Using a disposable 3 mL transfer pipette, add 3-4 drops of WASH solution to each used
assay well.
3. Remove wash solution as performed previously.
4. Using a P200 pipette, transfer 100 microlitres (100µl) of the detection antibody
(ANTIBODY) into all of the assay wells used. Be careful not to contaminate the pipette tip by
touching the side or base of the wells during the addition of antibody solution. If you think
you have contaminated the pipette tip, discard it and use a new one.
4. Incubate your assay at room temperature for 20 minutes to allow detection antibody to
bind to the hCG-capture antibody complexes.
While you are waiting, please read the instructions of the immuno-hCG detector cassette
and use a supplied cassette pregnancy test to detect the presence or absence of hCG in
ONE of the FOUR urine samples. Ensure that each group in your bay tests a different
sample so you have a full set of results! Make sure you record the results for your
randomly selected urine sample and those of the other pairs.
(D) Quantitative hCG immunoassay – STEP 3
After an incubation of 20 minutes, the detection antibody should have bound to any hCG-
capture antibody complexes present. In this section of the protocol you will add HRP
substrate (TMB COLOUR REAGENT) to detect the presence of bound detection antibody. Be
very careful when handling the TMB colour reagent solution as it is an irritant.
1. Any unbound detection antibody must be removed before proceeding adding the enzyme
substrate. Remove most of the antibody solution by flicking the assay plate over a sink and tap
the assay plate upside down on paper towel three times to remove any remaining liquid.
2. Using a disposable 3 mL transfer pipette, add 3-4 drops of WASH solution to each assay
well used. Remove wash solution as performed previously.
3. Using a P200 pipette, transfer 200 microlitres (200µl) of the HRP substrate (TMB COLOUR
REAGENT) into ALL of the assay wells used. Incubate the assay for 5 minutes to allow the
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colour change to develop. Ideally you will have a clear gradient of increasing colour for your
standard curve.
4. Check with a demonstrator to confirm that the colour is sufficiently developed for analysis.
If sufficient colour development has been achieved, add 100 microlitres (100µL) of STOP
SOLUTION to each well.
5. Measure the colour development at 450 nm using a spectrophotometer plate reader –
consult a demonstrator. Print out 2 copies of your assay results to allow each member of the
pair to include a copy in their report.
6. Plot the average absorbance readings obtained for the hCG standard curve against hCG
concentration on a graph (using graph paper provided), and estimate the concentration of
hCG in each urine sample (TA,TB, TC and TD) tested.
Write Up
Each student should complete the practical report form available on Blackboard and submit
this electronically as one file by the deadline on Blackboard. The assessment is worth 15% of
the module mark.
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