TUNG WAH COLLEGE

MED3035 Clinical Immunology (2023/2024)

Laboratory 03 – Serological Diagnosis of Infectious Diseases

Learning objective:
To understand how infectious diseases can be diagnosed using different serological techniques
through case studies

Grouping:
2 students per group

Patient 1
Aim of experiment:
To serologically diagnose whether case 1 is infected with hepatitis B virus

Case:
Miss White is a healthcare worker and one month ago unfortunately she had an accidental
needlestick injury. Although she has taken post-exposure prophylaxis immediately, she has still
been worried that she may have got infected with hepatitis B virus. In view of this her doctor
took a blood sample from her and sent it to your laboratory for testing this morning.

Antigen (HBs-Ag) detection

Materials:
1. ELISA strip coated with anti-hepatitis B surface antigen (anti-HBs Ag) antibody (8
wells) (to be shared by two groups)
2. Washing solution
3. Negative control I
4. Negative control II
5. Positive control
6. Serum sample from Miss White
7. Peroxidase-conjugated anti-HBs Ag antibody

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 8. H2O2-containing buffer
9. Tetramethyl benzidine (TMB)
10. 1 N sulphuric acid
11. Micropipettes and pipette tips
12. 37°C incubator
13. Microplate reader
14. Absorbent papers
15. Adhesive tapes

Procedures:
1. Carefully establish the sample distribution and identification plan as follow:
a. Well 1: Negative control I
b. Well 2: Negative control II
c. Well 3: Positive control
d. Well 4: Miss White’s serum sample
2. From Well 1 to Well 4 of the ELISA strip, add the corresponding samples (100 μL each).
3. Add 50 μL of peroxidase-conjugated anti-HBs Ag antibody to each of the wells.
Homogenise the reaction mixture.
4. Cover the strip with adhesive tape and incubate at 37°C for 1.5 h.
5. Remove the adhesive tape, empty all wells by aspiration and wash each well with
washing solution for 5 times (200 μL each time). Blot try the wells after washing by
turning them upside down on absorbent paper.
6. Mix 50 μL of TMB with 500 μL of H2O2-containing buffer to give the working enzyme
development solution.
7. Dispense 100 μL of the working enzyme development solution into each of the wells.
Incubate the wells in the dark for 30 min at room temperature. Do not use adhesive tape
during this incubation.
8. After incubation, add 100 μL of 1 N sulphuric acid into each of the wells using the same
sequence and rate of distribution as for the working enzyme development solution.
Homogenise the reaction mixture.
9. Carefully wipe the bottom of the strip with Kimwipes. Wait for at least 4 min after
sulphuric acid addition before reading of absorbance at 450/620 nm using a microplate
reader.
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 10. Interpret the results through the following calculations:

Cut-off value = Mean OD of negative controls + 0.050

Ratio value = OD of sample / Cut-off value

Samples with ratio values <1 are considered HBs Ag-negative and samples with ratio
values ≥1 are considered HBs Ag-positive.

Antibody (anti-HBc) detection (see demo results)

Materials:
1. ELISA strip coated with hepatitis B core antigen (HBc) (8 wells)
2. Washing solution
3. Negative control
4. Positive control
5. Serum sample from Miss White
6. Sample diluent
7. Peroxidase-conjugated anti-human IgG secondary antibody
8. H2O2-containing buffer
9. Tetramethyl benzidine (TMB)
10. 1 N sulphuric acid
11. Micropipettes and pipette tips
12. 37°C incubator
13. Microplate reader
14. Absorbent papers
15. Adhesive tapes

Procedures:
1. Carefully establish the sample distribution and identification plan as follow:
a. Well 1: Negative control (duplicate)
b. Well 2: Negative control (duplicate)
c. Well 3: Positive control (triplicate)
d. Well 4: Positive control (triplicate)
e. Well 5: Positive control (triplicate)
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 f. Well 6: Miss White’s serum sample
2. Add 200 μL of sample diluent into each of Well 1 to Well 6 of the ELISA strip.
3. From Well 1 to Well 6, add the corresponding samples (20 μL each). Homogenise by
pipetting up and down for at least 3 times.
4. Cover the strip with adhesive tape and incubate at 37°C for 30 min.
5. Remove the adhesive tape, empty all wells by aspiration and wash each well with
washing solution for 4 times (370 μL each time). Blot try the wells after washing by
turning them upside down on absorbent paper.
6. Add 200 μL of peroxidase-conjugated anti-human IgG secondary antibody to each of
the wells. Homogenise the reaction mixture.
7. Cover the strip with adhesive tape and incubate at 37°C for 1 h.
8. Remove the adhesive tape, empty all wells by aspiration and wash each well with
washing solution for 4 times (370 μL each time). Blot try the wells after washing by
turning them upside down on absorbent paper.
9. Mix 60 μL of TMB with 600 μL of H2O2-containing buffer to give the working enzyme
development solution.
10. Dispense 100 μL of the working enzyme development solution into each of the wells.
Incubate the wells in the dark for 30 min at room temperature. Do not use adhesive tape
during this incubation.
11. After incubation, add 100 μL of 1 N sulphuric acid into each of the wells using the same
sequence and rate of distribution as for the working enzyme development solution.
Homogenise the reaction mixture.
12. Carefully wipe the bottom of the strip with Kimwipes. Wait for at least 4 min after
sulphuric acid addition before reading of absorbance at 450/620 nm using a microplate
reader.
13. Interpret the results through the following calculations:

Cut-off value = Mean OD of positive controls / 5

Samples with ODs < cut-off value are considered anti-HBc-negative and samples with
ODs ≥ cut-off value are considered anti-HBc-positive.

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Patient 2
Aim of experiment:
To serologically diagnose whether case 2 is infected with Cryptococcus neoformans
(2 groups work together)

Case:
A 59-year-old male patient was admitted to a hospital because of cough, shortness of breath
and fatigue for around 2 weeks. He had been treated with cephalosporin and azithromycin for
10 days prior to admission but unfortunately the symptoms continued.

Computed tomography (CT) revealed multiple consolidations and ground-glass shadows of

bilateral lung fields. Laboratory tests revealed elevated C-reactive protein level (27.1 mg/L)
and erythrocyte sedimentation rate (44 mm/h), as well as positive result to anti-HIV antibodies.
Other findings were unremarkable. The patient reported that he was exposed to bird droppings
recently. His physician suspected that the patient is having cryptococcosis and therefore, he
took a blood sample from the patient and sent it to your laboratory for Cryptococcus antigen
testing.

Materials:
1. Cryptococcus test latex (mix well before use)
2. High positive control
3. Low positive control
4. Negative control
5. Serum sample from case 2
6. Specimen diluent
7. Protease solution
8. Reaction cards (6 wells)
9. Micropipette and pipette tips
10. 1.5 mL-microcentrifuge tubes, 2 mL-screw cap tube & microcentrifuge
11. 100°C dry bath
12. Vortex mixer

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Procedures:
1. Dispense 30 μL of case 2 patient serum in a 2 mL-screw cap tube and add an equal
volume of protease solution. Mix by vortexing.
2. Short-spin the mixture using a microcentrifuge and then boil for 10 min.
3. Centrifuge the boiled mixture at 3,000 rpm for 10 min.
4. During centrifugation, label 3 new 1.5 mL-microcentrifuge tubes (Tube 1 – Tube 3);
and transfer 40 μL of specimen diluent into each of the tubes.
5. After centrifugation, aliquot 40 μL of the supernatant into Tube 1.
6. After mixing, transfer 40 μL of Tube 1 content to Tube 2. After further mixing, transfer
40 μL of Tube 2 content to Tube 3. This produces a serial, doubling dilutions of the
specimen from Tube 1 through Tube 3. The prepared serum dilution will be 1:4 to 1:16
for Tube 1 to Tube 3, respectively.
7. Label the circles on the reaction card as follow:
a. High positive control
b. Low positive control
c. Negative control
d. Tube 1
e. Tube 2
f. Tube 3
8. Dispense 36 μL of the test latex (mix well before use) into each of the labelled circle.
9. Dispense the samples (36 μL each) to the circles according to their labels. Gently mix
the solutions using a pipette tip after liquid transfer.
10. Place the reaction cards on a rotator and rotate at ~50 rpm for 15 min.
11. Examine each circle for agglutination. Compare the reaction of each test sample to the
controls and interpret your results.

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Experimental results (5 marks):
Case 1
OD for OD for
antigen antibody
detection detection
Negative control I Negative control (duplicate)
Negative control II Negative control (duplicate)
Positive control Positive control (triplicate)
Miss White’s serum sample Positive control (triplicate)
Positive control (triplicate)
Miss White’s serum sample

Briefly discuss if there was any abnormality found.

Case 2
Present a photo showing the results of your latex agglutination test, with proper labelling.
Briefly discuss if there was any abnormality found.

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Questions (20 marks):
1. Which type(s) of ELISAs were used for serological diagnosis in case 1 (Miss White)?
(2 marks)

2. Is Miss White (case 1) infected with hepatitis B virus? Briefly explain your answer. (6
marks)

3. Vaccination can help prevent hepatitis B infection. What is hepatitis B vaccine made up
of? Unfortunately, Miss White (case 1) forgets whether she has already received
hepatitis B vaccination before. Based on the clinical information and your experimental
results, could you determine whether Miss White is at risk of future hepatitis B virus
infection? Briefly explain your answer. (3 marks)

4. What additional test should be performed to determine whether Miss White needs to
receive hepatitis B vaccination now? And why? Briefly explain your answer. (3 marks)

5. Suggest three possible reasons why case 2 was suspected to have cryptococcosis. (3
marks)

6. Is case 2 infected with Cryptococcus? If yes, what was the antigen titre? Briefly explain
your interpretation. (2 marks)

7. Cryptococcus very often can lead to meningitis. Suggest another type of specimen
which can be obtained from case 2 for latex agglutination testing. (1 mark)

Present your experimental results, answer the above questions and submit your assignment at
or before 17:00 on 28th March, 2024. Each student should submit his/her own lab assignment.
Indicate the course name, lab title, your name and your student ID on the front page clearly. A
hard copy should be submitted to the drop box outside 19/F School of Medical and Health
Sciences Office and an identical PDF copy should be submitted to Blackboard before the
submission deadline.