Labeled immunoassays (ELISA) and immunofluorescence

12 ASCP MLS practice questions on Labeled immunoassays (ELISA) and immunofluorescence, part of Immunology. The first 12 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Hard

   A 27-year-old man has had painless, enlarged posterior cervical lymph nodes for 3 weeks. Toxoplasma IgM is positive and Toxoplasma IgG is 160 IU/mL. An IgG avidity ELISA is run in two pairs of wells: after the serum binds, one pair is washed with buffer and the other with 6 mol/L urea before the enzyme conjugate is added. Mean absorbance: buffer-washed wells 1.60; urea-washed wells 0.32. The kit calculates the avidity index as urea OD divided by buffer OD, times 100, and reports below 30% as low, 30-40% as borderline and above 40% as high; high avidity excludes infection in the past 4 months. The avidity result and its interpretation are:

   1. A20%: high avidity, over 4 months ago
   2. B500%: index invalid, repeat test
   3. C20%: low avidity, may be recent
   4. D80%: high avidity, over 4 months ago

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   Answer: C · 20%: low avidity, may be recent

   The avidity index is 0.32 / 1.60 x 100 = 20%, which the kit calls low. Urea breaks the weaker bonds of low-avidity IgG made early in a primary response; as the response matures over months, IgG binds more tightly and stays bound through the urea wash. A low index fits a recent infection, matching the positive IgM and the lymphadenitis, but low avidity can persist for months, so it supports rather than proves recent infection; only high avidity is used to exclude it. The 80% answer comes from calculating the fraction washed off instead of the fraction left, and 500% comes from inverting the ratio.

   • A: A low index means low avidity, because most IgG washed off.
   • B: 500% comes from dividing the buffer OD by the urea OD.
   • D: 80% is the fraction removed by urea, not the fraction left bound.

2. Question 2 · Medium

   Refer to the ELISA plate readings and the kit's validity rules shown. What should the technologist do with this run?

   

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   1. AReport all three results as calculated
   2. BReject the run and retest all three patients
   3. CReport patient 1 as clearly positive
   4. DRepeat only the positive control well

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   Answer: B · Reject the run and retest all three patients

   The blank (0.048) and negative control (0.095) pass, but the positive control reads 0.720, below the required 1.000, so the run is invalid and no patient result may be reported. A weak positive control means the assay has lost sensitivity, for example from deteriorated conjugate or substrate, cold reagents or a short incubation. Weakly reactive samples, such as patient 3 just above the 0.420 cutoff, could then read falsely negative. Even patient 1's strong result cannot be released; the cause is found and all samples are retested with fresh controls.

   • A: The failed positive control invalidates every result in the run.
   • C: No result from an invalid run can be released, however strong it looks.
   • D: Repeating one control well does not validate the wells already run.

3. Question 3 · Easy

   In the sandwich ELISA standard curve shown, the dashed line marks the absorbance of a patient's sample. What is its ferritin concentration?

   

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   1. A40 ng/mL [40 µg/L]
   2. B60 ng/mL [60 µg/L]
   3. C20 ng/mL [20 µg/L]
   4. D80 ng/mL [80 µg/L]

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   Answer: A · 40 ng/mL [40 µg/L]

   Reading across from the sample's absorbance of 1.00 to the curve and down to the concentration axis gives 40 ng/mL [40 µg/L]. In a sandwich assay the signal rises with the amount of analyte, but not in a straight line: the curve flattens toward the top standard. Results are therefore read from the fitted curve, often a four-parameter logistic fit, not by simple proportion. A sample above the top standard is diluted and retested.

   • B: 60 ng/mL reads about 1.3 on this curve.
   • C: 20 ng/mL reads about 0.54 on this curve.
   • D: 80 ng/mL reads about 1.56; it comes from treating the curve as a straight line to the top standard.

4. Question 4 · Medium

   What is the measured signal in a chemiluminescent immunoassay?

   1. ALight scattered by immune complexes
   2. BLight emitted after laser excitation
   3. CLight absorbed by a colored product
   4. DLight given off by a chemical reaction

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   Answer: D · Light given off by a chemical reaction

   In chemiluminescence, a label such as acridinium ester, or an enzyme acting on a substrate such as luminol or a dioxetane, forms an excited molecule that releases light as it returns to its ground state. No excitation light is needed, so the background is very low and the assays are very sensitive. Fluorescence assays, by contrast, need an excitation light source. Most automated immunoassay analyzers now use chemiluminescent labels.

   • A: Light scattered by immune complexes describes nephelometry.
   • B: Light emitted after excitation describes fluorescence.
   • C: Light absorbed by a colored product describes a colorimetric ELISA.

5. Question 5 · Hard

   A woman with rheumatoid arthritis (RF 220 IU/mL, reference < 15) has a positive indirect ELISA for rubella IgM at 14 weeks of pregnancy. Her rubella IgG is high, with high avidity. What is the best next step?

   1. AReport the result as acute rubella infection
   2. BRepeat the IgM test on undiluted serum
   3. CHeat the serum at 56 °C for 30 minutes and repeat
   4. DRepeat the IgM test after removing IgG from the serum

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   Answer: D · Repeat the IgM test after removing IgG from the serum

   In an indirect IgM assay, the patient's IgG and IgM both bind to the antigen in the well. IgM rheumatoid factor can then attach to the bound rubella-specific IgG and be detected by the anti-IgM conjugate, giving a false-positive IgM. Removing IgG first, with an anti-IgG absorbent or protein G, removes the target that RF binds; IgM-capture assays avoid the problem in another way. High-avidity IgG also argues against a recent primary infection.

   • A: A positive IgM with high-avidity IgG and a high RF does not prove acute infection.
   • B: Undiluted serum would make the interference stronger.
   • C: Heating at 56 °C inactivates complement but does not remove RF.

6. Question 6 · Easy

   Which chromogenic substrate is most often used with horseradish peroxidase in ELISA?

   1. ABCIP with nitro blue tetrazolium
   2. Bo-Nitrophenyl galactoside (ONPG)
   3. Cp-Nitrophenyl phosphate (pNPP)
   4. DTetramethylbenzidine (TMB)

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7. Question 7 · Easy

   In a competitive ELISA, enzyme-labeled drug competes with drug in the sample for antibody fixed to the well. A sample with a very high drug level gives:

   1. AStrong color
   2. BColor only if the sample is diluted
   3. CWeak color
   4. DColor equal to the zero calibrator

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8. Question 8 · Medium

   A two-site (sandwich) immunoassay can measure an analyte only if the analyte:

   1. ACarries a fluorescent group
   2. BHas enzyme activity of its own
   3. CHas at least two separate epitopes
   4. DWeighs less than 1,000 daltons

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9. Question 9 · Hard

   In an ELISA run, the blank and negative control wells turn as dark as the positive control. Reagents are in date and the substrate was colorless when added. The most likely cause is:

   1. APoor washing after the conjugate step
   2. BSubstrate incubation that was too short
   3. CConjugate left out of all the wells
   4. DStop solution added too early

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10. Question 10 · Medium

    Why is indirect immunofluorescence usually more sensitive than direct immunofluorescence?

    1. AIt uses a brighter fluorochrome than the direct method
    2. BEach bound primary antibody binds several labeled antibodies
    3. CIt needs no washing between the reagent steps
    4. DIts antigen is fixed more firmly to the glass slide

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11. Question 11 · Medium

    An IgG ELISA reports a signal-to-cutoff ratio. The kit defines < 0.90 as negative, 0.90 to 1.09 as equivocal and 1.10 or more as positive. A sample gives 0.96. The best action is:

    1. ADilute the sample 1:10 and repeat the test
    2. BReport negative, as the ratio is under 1.00
    3. CReport equivocal; retest a new sample in 2 weeks
    4. DReport positive, as the ratio is close to 1.00

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12. Question 12 · Easy

    Direct immunofluorescence on a skin biopsy detects:

    1. AAntigen released into the patient's serum
    2. BAntibody or complement bound in the tissue
    3. CAntibodies circulating in the patient's serum
    4. DImmune complexes filtered into the urine

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