LABORATORY ACTIVITY #3

ASSAYS OF ANTIBODY

I. Desired learning outcomes

Immunoglobulins are glycoproteins produced in response to an immunogenic stimulation. Following a
certain incubation period, antibody titer can be measured in the serum using certain laboratory assays. The
results of this measurement are useful in the treatment and management of an infection.

After performing this activity, students should be able to:

a. observe how the different procedures in determining the presence of antibodies such as Anti-HBs, Anti-
HCV, Anti-HAV, and Anti-HIV 1 &2 are performed with accuracy and precision;
b. explain the result and correlate it to the clinical symptoms.

II. Material
1. Pipettor
2. Centrifuge
3. Antibody Test Kits
4. Fresh serum sample

III. Procedures
1. Hepatitis A
A. Aria HAV IgM Rapid Test (Serum/Plasma/Whole Blood)
1. Bring the specimen and test components to room temperature if refrigerated or frozen. Once thawed, mix
the specimen well before performing the assay.
2. When ready to test, open the pouch at the notch and remove the device. Place the test device on a clean, flat
surface.
3. Be sure to label the device with the specimen’s ID number.
4. Fill the capillary tube with a specimen not exceeding the specimen is approximately 5 µL. For maximum
precision, transfer the specimen using a pipette capable of delivering a volume of 5 µL.

Holding the capillary tube vertically tube vertically, dispense the entire specimen into the center of the sample
well (S well), making sure that there are no air bubbles.

Immediately add 2 drops (approximately 60-80 µL) of Sample Diluent into the buffer well (B well) with the
bottle positioned vertically.

5. Set up a timer.
6. Read the result in 15 minutes. Positive results may be visible as soon as 1 minute.
 Do not read the result after 20 minutes. To avoid confusion, discard the test device after interpreting the
result.

Interpretation of Assay Result

2. Hepatitis B
A. ONE STEP HBsAb TEST
For Test Cards:
1. Bring all reagents and specimens to room temperature.
2. Remove the test card from the foil pouch and place it on a clean dry surface.
 3. Identify the test card for each specimen or control
4. Dispense 100 µL (3 drops) of the specimen or control into the sample well on the card.
5. Interpret test results at 15 minutes.

For Test Strips:

1. Bring all reagents and specimens to room temperature.
2. Remove the test strip from the foil pouch and place it on a clean dry surface.
3. Identify the test strip for each specimen or control.
4. Apply at least 80 µL of the specimen to the sample pad behind the ( ) mark at the bottom of the test
strip.
5. Interpret test results at 15 minutes.

3. Hepatitis C
ADVANCED QUALITY HCV – Rapid Anti-HCV Test (Serum/Plasma)
For Test Cards:
1. Dispense 1 drop (10 µL) of serum or plasma specimen to the “S” well of the test card using the plastic
dropper provided according to the figure.
2. Add 2 drops of Sample Diluent to the “D” well immediately after the specimen is added.
3. Interpret test results at 15 minutes.
 For Test Strips:
1. Dispense 1 drop (10 µL) of serum or plasma to the upper edge of the sample pad of the test strip using the
plastic dropper provided according to the figure.
2. Add 2 drops of Sample Diluent to the lower edge of the sample pad after the specimen is added.
3. Interpret test results at 15 minutes.

4. HIV
A. ADVANCED QUALITY HIV [ONE STEP Anti-HIV (1 & 2) Test]
For Test Cards:
1. Bring all reagents and specimens to room temperature.
2. Remove the test card from the foil pouch and place it on a clean dry surface.
3. Identify the test card for each specimen or control.
4. Dispense 100 µL (3 drops) of the specimen or control into the sample well on the card.
5. Interpret test results at 15 minutes.
For Test Strips
1. Bring all reagents and specimens to room temperature.
2. Remove the test strip from the foil pouch and place it on a clean, dry surface.
3. Identify the test strip for each specimen or control.
4. Apply at least 80 µL of the specimen to the sample pad behind the ( ) mark at the bottom of the
test strip.
5. Interpret test results at 15 minutes.
 LABORATORY OUTPUT

TITLE OF LABORATORY ACTIVITY: LAB. ACT. #3 - ASSAYS OF ANTIBODY

I. Observation/Results
Instruction: Paste pictures of the possible results in the 4 test systems (Anti-HAV (Aria & Advin), Anti-HBs, Anti-
HCV, and Anti-HIV 1 & 2). Label/Read and interpret the results.
Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

HAV IgM (Aria) HAV IgG/IgM (Advin) Anti-HBs Anti-HCV Anti-HIV 1 & 2

Table 1

Anti-HAV (Advin), Anti-HBs, Anti-HCV, and Anti-HIV 1 & 2 Test Results

Antibody HAV IgM (Aria) HAV IgG/IgM Anti-HBs (Vaxpert) Anti-HCV Anti-HIV 1 & 2
Assays (Advin) (Advance Quality) (Itec)

Result The test results There is only Two separate colored Only the purplish The membrane
show that there one colored lines should be visible red control band displays both a
is no detectable line and it is —although faint—with has appeared. test band and a
anti-HAV IgM in in the control one appearing in the control band,
the specimen if line region. control region (C) and both appearing
only the C line the other less faint one in a purplish-red
develops. in the test region (T). hue.
Interpretation Non-reactive Non-reactive Reactive Non-reactive Non-reactive

II. Discussion

The four test instruments, Anti-HAV Aria and Anti-HAV Advin, Anti-HBs (vaxpert), Anti-HCV, and Anti-HIV
1 & 2, exhibit distinctions in various parameters according to their package inserts (Advin Biotech, 2020; CTK
Biotech, 2019; InTec, 2019; InTec, 2024; Vaxpert, 2016). Notably, Aria HAV IgM can utilize serum, plasma, and
 whole blood as its specimen, whereas Advin HAV IgG/IgM is limited to handling serum and plasma. Additionally,
similar to Advin HAV IgG/IgM, the Anti-HBs and Anti-HIV 1 & 2 tests also use serum/plasma as specimens. The
amount of specimen required varies across the tests: Aria HAV IgM needs 5 µL, Anti-HBs requires 75 µL (3
drops), Anti-HCV uses 1 drop (10 µL), and Anti-HIV 1 & 2 necessitates 100 µL (3 drops). Regarding sample
diluent, the Anti-HAVs (including Aria HAV IgM) and Anti-HCV require 2 drops, while Anti-HBs and Anti-HIV 1 & 2
do not use any diluent. All tests feature a sample well, with only Aria HAV IgM and Advin HAV IgG/IgM having a
B (buffer) well, and Anti-HCV featuring a D (diluent) well. The uniform 15-minute incubation period applies to all
tests. Positive results are consistently displayed with two distinct lines in the result window. In contrast, negative
results are indicated by one line on the control only, maintaining a standardized presentation across the
instruments.

In the laboratory, the Anti-HAV (IgG/IgM) test returned a non-reactive result, indicating the absence of
hepatitis A virus (HAV) exposure. However, the Anti-HBs test was reactive, suggesting immunity either from a
recent hepatitis B infection or vaccination. The reactivity in anti-HBs could be considered positive, especially
given the patient's confirmation of recent vaccination. In the case of anti-HAV, a positive result typically
indicates prior exposure but does not rule out acute or recent infection (Haldeman-Englert et al., 2024). The
presence of anti-HB antibodies, on the other hand, suggests immunity to hepatitis B, either through recovery
from a previous infection or vaccination as implicated by Daniel (2023). For Hepatitis C (Anti-HCV), a non-
reactive result implies no current infection, while a positive result may indicate an active or resolved infection
(Khatr, 2022). In the context of HIV 1 & 2 testing, a negative result for both antibodies usually signifies the
absence of infection. Still, in individuals with a reactive initial combined HIV-1/-2 antigen and antibody test,
further testing is needed to rule out acute or early HIV infection. Positive results for HIV-1 antibodies indicate
HIV-1 infection, and positive HIV-2 antibodies suggest HIV-2 infection. (CDC, 2018) Interpretation also considers
indeterminate results, which may occur in early infections or due to nonspecific cross-reactivity.

III. Guide Questions

1. What possible conditions or factors may display false positive results in all these test instruments?
The Aria HAV IgM Rapid Test may yield false-positive results if subjected to multiple freeze-thaw cycles,
and caution should be exercised with specimens showing lipemia, hemolysis, or turbidity (CTK Biotech, 2019).
Interference may arise with rheumatoid factor levels exceeding 21,000 IU/ml, necessitating confirmation
through alternative testing methods. Moreover, false positives in Anti-HAV Advin IgG/IgM can occur in
conditions with similar clinical presentations, such as infectious mononucleosis, CMV and EBV infections, HIV
 infection, rubella, measles, and hepatitis A or E viral infections (Tan & Chen, 2021). High titers of antibodies to
immunological components, streptavidin, or ruthenium can also lead to interference. Similarly, for anti-HBs,
interference due to extremely high titers of antibodies is a rare possibility. Anti-HCV tests may generate false
positives if triggered by antibodies from another infection or due to rare lab errors (Moorman et al., 2017). In
the case of anti-HIV 1 & 2 tests, Increased assay sensitivity leads to a decline in the specificity of anti-HIV 1 & 2
tests, resulting in false-positive results (Tsoi et al., 2022). Technical errors such as specimen mix-up,
contamination, mishandling, and result misinterpretation contribute to inaccuracies in HIV testing. Rare
scenarios, like the presence of HIV antibodies in individuals receiving HIV-1 trial vaccines or potential cross-
reactivity with influenza vaccines, can also lead to misleading results, with the timing of such cross-reactivity
remaining uncertain.

2. What are the limitations of each test instrument? You may give 2 limitations for each test instrument.
The Aria Anti-HAV IgM Rapid Test has limitations that include the necessity for strict adherence to the
assay procedure and interpretation sections to avoid inaccurate results (CTK Biotech, 2019). Additionally, it is
qualitatively limited, as the test line intensity does not correlate linearly with the antibody titer in the specimen.
A negative result may not rule out HAV exposure or infection, either due to low anti-HAV IgM levels or the
absence of antibodies during the disease stage. Furthermore, unusually high titers of heterophile antibodies or
rheumatoid factor can impact results, requiring interpretation alongside other diagnostic methods. The Advin
Anti-HAV (IgG/IgM) test's limitations encompass the inability to determine quantitative values or the rate of
increase in HAV antibody concentration, and a negative result does not guarantee freedom from HAV exposure
(Advin Biotech, 2020). The Anti-HBsAb Rapid Test Cassette has limitations, such as being unable to detect less
than 10mlU/ml of HBsAb in specimens, and is intended solely for in vitro diagnosis (Vaxpert, 2016). The Anti-
HCV test is restricted to clear, fluid samples, preferably fresh, with frozen samples permitted after proper
thawing (InTec, 2024). For Anti-HIV 1 & 2, only non-hemolyzed samples with good fluidity are suitable, while
refrigerated and frozen samples are acceptable (InTec, 2019). Additionally, sample agitation should be avoided,
and collection must be performed carefully with a pipette just below the sample surface.

IV. Conclusion
In conclusion, the objectives were successfully met as I have properly performed the procedures for
detecting antibodies (Anti-HBs, Anti-HCV, Anti-HAV, and Anti-HIV 1 & 2) and effectively correlated the results. In
the laboratory. The antibody assays, including the Anti-HAV Aria and Anti-HAV Advin, Anti-HBs (Vaxpert), Anti-
HCV (Advance Quality), and Anti-HIV 1 & 2 (Itec), demonstrated specific distinctions in their parameters, with
detailed interpretations revealing the absence of detectable anti-HAV IgM, immunity to hepatitis B, and non-
reactivity to hepatitis C and HIV in the laboratory setting, underscoring the importance of understanding test
limitations for accurate result interpretation. This activity have clarified my misconception that these tests have
100% specificity due to cross-reactivities and interference which should give vigilance to the technologists.

V. Reference/s

Advin Biotech, Inc. (2020, January 3). HAV IgG/IgM Rapid Test Cassette [Package Insert]. 10237 Flanders Ct., San Diego,
CA 92121, USA. Retrieved from https://fda.report/Company/Advin-Biotech-Inc

Centers for Disease Control and Prevention. (2018). Quick Reference Guide: Recommended laboratory HIV testing
algorithm for serum or plasma specimens. CDC; January 2018. Accessed June 15, 2020. Available at
https://stacks.cdc.gov/view/cdc/50872
CTK Biotech, Inc. (2019). HAV IgM Rapid Test (Cassette) [Package Insert]. Poway, CA 92064, USA. Retrieved from
https://medtek.com.ph/wp-content/uploads/2020/09/Aria-Catalogue-2020_compressed.pdf

Daniel, C. (2023, September 14). Hepatitis Causes & Diagnosis: The Hepatitis B Surface Antibody (HBsAb) Test. Verywell
Health. https://www.verywellhealth.com/hbsab-hepatitis-b-surface-antibody-1759935

Haldeman-Englert, C., Watson, L. R., & Novick, T. (2024). Hepatitis A Antibody. Health Encyclopedia. University of
Rochester Medical Center. Retrieved from https://www.urmc.rochester.edu/encyclopedia/content.aspx?
contenttypeid=167&contentid=hepatitis_a_antibody

InTec, Inc. (2019). ADVANCED QUALITY™ ONE STEP Anti-HIV (1&2) Test [Package Insert]. 332 Xinguang Road, Xinyang
Industrial Area, Haicang, 361022, Xiamen, Fujian P. R. China. Retrieved from
https://www.medlider24.com.ua/en/products/shvydkyj-test-dlya-vyznachennya-antytil-do-vil-1-2-advanced-
quality-tm/

InTec, Inc. (2024). Rapid Anti-HCV Test [Package Insert]. 332 Xinguang Road, Xinyang Industrial Area, Haicang, 361022,
Xiamen, Fujian P. R. China. Retrieved from https://www.intecasi.com/rapid-anti-hcv-test_p10.html

Khatr, M. (2022). Hepatitis C diagnosis and tests for HCV: Antibody, PCR, and more. WebMD.
https://www.webmd.com/hepatitis/diagnostic-tests-hepatitis-c-virus

Moorman, A. C., Drobenuic, J., & Saleem Kamili. (2017). Prevalence of false-positive hepatitis C antibody results,
National Health and Nutrition Examination Study (NHANES) 2007–2012. Journal of Clinical Virology, 89, 1–4.
https://doi.org/10.1016/j.jcv.2017.01.007

Tan, Y., & Chen, L. (2021). Sustained false-positive results for hepatitis A virus immunoglobulin M: A case report and
literature review. Open Medicine (Warsaw, Poland), 16(1), 1311–1317. https://doi.org/10.1515/med-2021-0336

Tsoi, B. W., Fine, S. M., McGowan, J. P., et al. (2022, May). HIV Testing. Johns Hopkins University. Box 3: Reasons for
False Positive, False Negative, or Indeterminate HIV Test Results. Retrieved from
https://www.ncbi.nlm.nih.gov/books/NBK581840/table/nycghivtest.T.box_3_reasons_for_false_po/

Vaxpert Inc. (2016). HBsAb Rapid Test Cassette (Serum/Plasma) [Package Insert]. Suite 355, Two South Biscayne Blvd.,
Miami, FL, U.S.A. Retrieved from https://www.trulaboratories.com/files/brochure/05/Hepatitis%20A.pdf