A manual anti-HCV ELISA is run on pre-employment samples. The kit insert states: the run is valid if every negative control (NC) OD is below 0.150 and the mean positive control OD is at least 0.800; the cut-off is the mean NC OD plus 0.250; samples with an OD at or above the cut-off are reactive; samples from 10% below the cut-off up to the cut-off are in the gray zone and must be retested in duplicate. Results: NC 0.058, 0.064 and 0.061; positive controls 1.420 and 1.510; sample 17 OD 0.292. How should sample 17 be handled?

Grey zone: retest it in duplicate

Nonreactive: its OD is below the cut-off

Invalid run: repeat the whole plate

Reactive: its OD is above 0.250

A 40-year-old has suspected atypical pneumonia. An IFA antibody titer on the acute serum was 32. The convalescent serum, taken 14 days later and tested in a different run, gives 64. What should the laboratory do?

Test both sera together in the same run

Retest only the convalescent serum

Report no infection, as both titers are low

Report a significant rise in antibody titer

A competitive ELISA for a drug has these calibrators (concentration: OD): 0 ng/mL: 2.10; 5 ng/mL: 1.50; 20 ng/mL: 0.80; 80 ng/mL: 0.30. An undiluted patient sample gives an OD of 0.18. What is the correct action?

Above 80 ng/mL: dilute the sample and retest

Between 20 and 80 ng/mL: report by interpolation

Report 80 ng/mL, the top calibrator

Below 5 ng/mL: report as not detected

ELISA, chemiluminescence and immunofluorescence

15 DOH Abu Dhabi (HAAD) lab licensing practice questions on ELISA, chemiluminescence and immunofluorescence, part of Immunology and Serology. The first 15 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
A manual anti-HCV ELISA is run on pre-employment samples. The kit insert states: the run is valid if every negative control (NC) OD is below 0.150 and the mean positive control OD is at least 0.800; the cut-off is the mean NC OD plus 0.250; samples with an OD at or above the cut-off are reactive; samples from 10% below the cut-off up to the cut-off are in the gray zone and must be retested in duplicate. Results: NC 0.058, 0.064 and 0.061; positive controls 1.420 and 1.510; sample 17 OD 0.292. How should sample 17 be handled?
1. ANonreactive: its OD is below the cut-off
2. BInvalid run: repeat the whole plate
3. CReactive: its OD is above 0.250
4. DGrey zone: retest it in duplicate
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Answer: D · Grey zone: retest it in duplicate
First check validity: every NC is below 0.150 and the mean positive control is 1.465, so the run is valid. The mean NC is (0.058 + 0.064 + 0.061) ÷ 3 = 0.061, giving a cut-off of 0.061 + 0.250 = 0.311 and a gray-zone lower limit of 0.9 × 0.311 = 0.280. Sample 17, at 0.292 (S/CO 0.94), falls in the gray zone, so it is retested in duplicate before any result is reported. Calling it nonreactive ignores the gray zone, and calling it reactive uses 0.250 as the cut-off without adding the NC mean. The gray zone exists because results just below the cut-off include some early or weak true positives.
- A: It is below the cut-off but inside the gray zone, which needs a duplicate retest.
- B: Every NC is below 0.150 and the positive mean is 1.465, so the run is valid.
- C: 0.250 is the constant added to the NC mean, not the cut-off itself.
Question 2 · Easy
The standard curve of a quantitative anti-HBs ELISA is shown, with the calibrators joined point to point. A patient's sample gives an OD of 1.20. Its concentration is about:
Figure: MedExams
1. A25 mIU/mL
2. B1.2 mIU/mL
3. C38 mIU/mL
4. D50 mIU/mL
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Answer: C · 38 mIU/mL
The sample's OD of 1.20 lies between the 25 mIU/mL calibrator (OD 0.88) and the 50 mIU/mL calibrator (OD 1.52). Reading across to the curve and down gives about 38 mIU/mL: 25 + (1.20 − 0.88) ÷ (1.52 − 0.88) × 25 = 37.5. In a sandwich-type assay the OD rises with concentration, as here. A result outside the calibrator range must not be read off the curve; the sample is diluted or reported as above or below the range.
- A: 25 mIU/mL is the calibrator below the sample, not the sample's value.
- B: 1.2 is the optical density, not the concentration.
- D: 50 mIU/mL is the calibrator above the sample.
Question 3 · Medium
A qualitative Toxoplasma IgG ELISA gives the plate results shown. The kit states: the run is valid if every negative control is below 0.100 and every positive control above 0.800; cut-off = mean negative control + 0.150; results within ±10% of the cut-off are equivocal. How should sample 2 be reported?
Figure: MedExams
1. AReactive: S/CO above 1.0
2. BEquivocal: retest the sample
3. CInvalid: repeat the whole run
4. DNonreactive: below the cut-off
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Answer: B · Equivocal: retest the sample
All three negative controls are below 0.100 and both positive controls are above 0.800, so the run is valid. The mean negative control is (0.048 + 0.052 + 0.050) ÷ 3 = 0.050, so the cut-off is 0.200 and the equivocal zone is 0.180-0.220. Sample 2, at 0.212 (S/CO 1.06), falls inside this zone and is reported as equivocal and retested, or a new sample is requested. Sample 1 is nonreactive and sample 3 is reactive.
- A: An S/CO of 1.06 lies inside the stated equivocal zone, so it is not reported as reactive.
- C: Every control meets the kit's validity rules.
- D: 0.212 is above the 0.200 cut-off, not below it.
Question 4 · Hard
A 40-year-old has suspected atypical pneumonia. An IFA antibody titer on the acute serum was 32. The convalescent serum, taken 14 days later and tested in a different run, gives 64. What should the laboratory do?
1. ARetest only the convalescent serum
2. BReport no infection, as both titers are low
3. CReport a significant rise in antibody titer
4. DTest both sera together in the same run
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Answer: D · Test both sera together in the same run
A rise in titer is significant only when it is at least fourfold (two dilutions), because a twofold difference is within the normal variation of a dilution titer. Paired sera should be tested side by side in the same run with the same reagents, which removes run-to-run variation. The acute serum is stored frozen for this purpose. A fourfold or greater rise on parallel testing supports recent infection.
- A: Retesting one serum alone does not remove run-to-run variation.
- B: Sera tested in separate runs cannot rule infection in or out.
- C: A twofold rise is within the error of a titer and is not significant.
Question 5 · Hard
A competitive ELISA for a drug has these calibrators (concentration: OD): 0 ng/mL: 2.10; 5 ng/mL: 1.50; 20 ng/mL: 0.80; 80 ng/mL: 0.30. An undiluted patient sample gives an OD of 0.18. What is the correct action?
1. ABetween 20 and 80 ng/mL: report by interpolation
2. BAbove 80 ng/mL: dilute the sample and retest
3. CReport 80 ng/mL, the top calibrator
4. DBelow 5 ng/mL: report as not detected
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Answer: B · Above 80 ng/mL: dilute the sample and retest
In a competitive assay, drug in the sample and labeled drug compete for a limited amount of antibody, so the signal falls as the concentration rises. An OD of 0.18 is below the OD of the 80 ng/mL calibrator, so the concentration is above the top of the curve. A result outside the calibrated range must not be extrapolated; the sample is diluted, retested and corrected for the dilution. Reading the curve as if signal rose with concentration, as in a sandwich assay, would wrongly call the sample negative.
- A: 0.18 is below the 80 ng/mL OD of 0.30, so it lies beyond this part of the curve.
- C: A result above the top calibrator must not be reported as that calibrator's value.
- D: In a competitive assay a low OD means a high concentration, not a low one.
Question 6 · Hard
On an automated chemiluminescence analyzer, a low-risk patient's HBsAg is weakly reactive (S/CO 1.8; reactive at 1.0 or more). It ran straight after a sample with S/CO above 5,000. A fresh aliquot run after a blank gives 0.3. The most likely explanation is:
1. AHeterophile antibody interference
2. BThe high-dose hook effect
3. CVery early acute infection
4. DCarryover from the previous sample
Answer and explanation in LearnFree account
Question 7 · Medium
A double-antigen sandwich assay uses antigen on the solid phase and labeled antigen as the detector. It detects:
1. AIgM antibody only
2. BIgG antibody only
3. CTotal antibody of any class
4. DFree antigen in the sample
Answer and explanation in LearnFree account
Question 8 · Easy
After acid stop solution is added to a peroxidase ELISA that uses TMB substrate, the wells are read at:
1. A405 nm
2. B620 nm
3. C340 nm
4. D450 nm
Answer and explanation in LearnFree account
Question 9 · Easy
In an IgM antibody-capture ELISA, the wells are coated with:
1. AAnti-human IgG
2. BThe specific antigen
3. CEnzyme-labeled antigen
4. DAnti-human IgM
Answer and explanation in LearnFree account
Question 10 · Medium
On a 96-well ELISA plate, the same control serum placed in every column gives higher optical densities in the outer wells than in the inner wells. What is the most likely cause?
1. AUneven temperature across the plate
2. BContaminated substrate solution
3. CConjugate left out of the outer wells
4. DThe wrong reading wavelength
Answer and explanation in LearnFree account
Question 11 · Easy
Under a fluorescence microscope, antibody labeled with fluorescein isothiocyanate (FITC) gives fluorescence that is:
1. AGolden yellow
2. BDeep blue
3. CApple-green
4. DOrange-red
Answer and explanation in LearnFree account
Question 12 · Easy
Which chemiluminescent label gives a rapid flash of light when alkaline hydrogen peroxide is added, without needing an enzyme?
1. AEuropium chelate
2. BHorseradish peroxidase
3. CFluorescein isothiocyanate
4. DAcridinium ester
Answer and explanation in LearnFree account
Question 13 · Medium
While an IFA slide is being examined, the green fluorescence fades within seconds of exposure to the excitation light. What is the best way to reduce this?
1. ACounterstain longer with Evans blue
2. BStore slides at room temperature in light
3. CMount in antifade medium; limit light exposure
4. DUse a higher conjugate dilution
Answer and explanation in LearnFree account
Question 14 · Medium
A screening immunoassay is initially reactive (S/CO 1.3; reactive at 1.0 or more). By the kit algorithm, it is retested in duplicate and is reactive if either repeat is reactive. The repeats give 0.4 and 0.5. The sample should be reported as:
1. ARepeatedly reactive
2. BNonreactive
3. CIndeterminate
4. DReactive at a low level
Answer and explanation in LearnFree account
Question 15 · Medium
In an indirect ELISA that detects a patient's antibodies, what is the enzyme-labeled reagent?
1. AAnti-human immunoglobulin
2. BThe antigen
3. CThe patient's antibody
4. DThe substrate
Answer and explanation in LearnFree account

ELISA, chemiluminescence and immunofluorescence

The standard curve of a quantitative anti-HBs ELISA is shown, with the calibrators joined point to point. A patient's sample gives an OD of 1.20. Its concentration is about:

A 40-year-old has suspected atypical pneumonia. An IFA antibody titer on the acute serum was 32. The convalescent serum, taken 14 days later and tested in a different run, gives 64. What should the laboratory do?

A competitive ELISA for a drug has these calibrators (concentration: OD): 0 ng/mL: 2.10; 5 ng/mL: 1.50; 20 ng/mL: 0.80; 80 ng/mL: 0.30. An undiluted patient sample gives an OD of 0.18. What is the correct action?

On an automated chemiluminescence analyzer, a low-risk patient's HBsAg is weakly reactive (S/CO 1.8; reactive at 1.0 or more). It ran straight after a sample with S/CO above 5,000. A fresh aliquot run after a blank gives 0.3. The most likely explanation is:

A double-antigen sandwich assay uses antigen on the solid phase and labeled antigen as the detector. It detects:

After acid stop solution is added to a peroxidase ELISA that uses TMB substrate, the wells are read at:

In an IgM antibody-capture ELISA, the wells are coated with:

On a 96-well ELISA plate, the same control serum placed in every column gives higher optical densities in the outer wells than in the inner wells. What is the most likely cause?

Under a fluorescence microscope, antibody labeled with fluorescein isothiocyanate (FITC) gives fluorescence that is:

Which chemiluminescent label gives a rapid flash of light when alkaline hydrogen peroxide is added, without needing an enzyme?

While an IFA slide is being examined, the green fluorescence fades within seconds of exposure to the excitation light. What is the best way to reduce this?

A screening immunoassay is initially reactive (S/CO 1.3; reactive at 1.0 or more). By the kit algorithm, it is retested in duplicate and is reactive if either repeat is reactive. The repeats give 0.4 and 0.5. The sample should be reported as:

In an indirect ELISA that detects a patient's antibodies, what is the enzyme-labeled reagent?