A Punjabi family's samples show a variant band in the HbS zone at alkaline pH, but the sickle solubility test is negative. Which further electrophoresis distinguishes HbD from HbS?

Paper electrophoresis at pH 8.6

At pH 8.0, a protein with an isoelectric point of 5.5 is applied to ion-exchange columns. It will bind to:

An anion exchanger such as DEAE-cellulose

A cation exchanger such as CM-cellulose

Both exchangers equally, being uncharged

Neither exchanger, being positively charged

Electrophoresis, chromatography and immunochemical methods

27 Kerala MSc MLT Entrance practice questions on Electrophoresis, chromatography and immunochemical methods, part of Biochemistry. The first 25 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 healthy 24-year-old woman from Assam has antenatal screening: Hb 11.8 g/dL, MCV 71 fL, MCH 23 pg, RBC 5.2 million/µL, serum ferritin normal. Cation-exchange HPLC: HbA 69%, HbF 0.6%, and 28% in the HbA2 window (HbA2 in beta-thalassaemia trait is usually 3.5-7%). The most likely interpretation is:
1. ASickle cell trait
2. BHaemoglobin E trait
3. CHaemoglobin D Punjab trait
4. DBeta-thalassaemia trait
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Answer: B · Haemoglobin E trait
On the HPLC systems used for screening, HbE elutes in the same window as HbA2, so a value of 25-30% in that window is far too high to be HbA2 and indicates HbE trait. HbE is common in north-east India and gives mild microcytosis with a normal or near-normal haemoglobin. Beta-thalassaemia trait raises true HbA2 only to about 3.5-7%. HbD Punjab and HbS each elute in their own windows, not in the A2 window.
- A: HbS elutes in its own S window, not the A2 window.
- C: HbD Punjab elutes in a separate D window on HPLC.
- D: Beta-thalassaemia trait gives HbA2 of about 3.5-7%, not 28%.
Question 2 · Hard
A 34-year-old man who has never smoked has breathlessness from emphysema of the lower lobes and a mildly raised ALT. Serum protein electrophoresis (g/dL, with reference): albumin 4.3 (3.5-5.0), alpha-1 0.05 (0.1-0.3), alpha-2 0.8 (0.6-1.0), beta 0.9 (0.7-1.1), gamma 1.2 (0.8-1.6). Which condition best fits?
1. AAlpha-1 antitrypsin deficiency
2. BNephrotic syndrome
3. CAcute inflammatory response
4. DChronic liver cirrhosis
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Answer: A · Alpha-1 antitrypsin deficiency
The only abnormality is an almost absent alpha-1 band, and about 90% of that band is alpha-1 antitrypsin. Deficiency lets neutrophil elastase destroy alveolar walls, giving early basal emphysema in non-smokers, and the abnormal Z protein can injure the liver. Nephrotic syndrome lowers albumin and raises alpha-2; acute inflammation raises alpha-1 and alpha-2; cirrhosis lowers albumin and gives beta-gamma bridging. The diagnosis is confirmed by serum alpha-1 antitrypsin and phenotyping by isoelectric focusing.
- B: Nephrotic syndrome gives low albumin and a high alpha-2 band.
- C: An acute phase response raises the alpha-1 band rather than lowering it.
- D: Cirrhosis lowers albumin and raises gamma with beta-gamma bridging.
Question 3 · Medium
A Punjabi family's samples show a variant band in the HbS zone at alkaline pH, but the sickle solubility test is negative. Which further electrophoresis distinguishes HbD from HbS?
1. ACitrate agar at pH 6.0-6.2
2. BPaper electrophoresis at pH 8.6
3. CCellulose acetate at pH 9.2
4. DStarch gel at pH 8.6
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Answer: A · Citrate agar at pH 6.0-6.2
On alkaline electrophoresis HbS, HbD and HbG carry the same net charge and move together. On acid citrate agar, separation also depends on interaction with the agar, so HbS moves away from the HbA position while HbD stays with HbA. HbD Punjab is common in north-west India, and its distinction from HbS matters for counselling. HbD is also negative in the sickle solubility test.
- B: Paper at pH 8.6 separates by the same charge difference as cellulose acetate.
- C: A slightly higher alkaline pH does not change the shared charge of S and D.
- D: Starch gel at alkaline pH still separates mainly by charge, so S and D stay together.
Question 4 · Easy
By which property does isoelectric focusing separate proteins?
1. AAffinity for a ligand
2. BSolubility in salt
3. CMolecular mass
4. DIsoelectric point
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Answer: D · Isoelectric point
In isoelectric focusing, carrier ampholytes set up a stable pH gradient across the gel. Each protein migrates until it reaches the pH equal to its isoelectric point, where its net charge is zero and it stops as a sharp band. The method resolves proteins that differ only slightly in charge, and is used for CSF oligoclonal bands, alpha-1 antitrypsin phenotypes and haemoglobin variants.
- A: Affinity for a fixed ligand is the basis of affinity chromatography.
- B: Salt solubility is the basis of salting-out precipitation.
- C: Separation by molecular mass is the basis of SDS-PAGE and gel filtration.
Question 5 · Medium
At pH 8.0, a protein with an isoelectric point of 5.5 is applied to ion-exchange columns. It will bind to:
1. AAn anion exchanger such as DEAE-cellulose
2. BA cation exchanger such as CM-cellulose
3. CBoth exchangers equally, being uncharged
4. DNeither exchanger, being positively charged
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Answer: A · An anion exchanger such as DEAE-cellulose
Above its isoelectric point a protein carries a net negative charge. At pH 8.0 this protein (pI 5.5) is therefore an anion and binds to an anion exchanger, which carries positive groups such as diethylaminoethyl (DEAE). It can then be eluted by raising the salt concentration or lowering the pH. Ion-exchange chromatography is used for amino acids, haemoglobin variants and HbA1c.
- B: A cation exchanger has negative groups and binds proteins below their pI.
- C: The protein is uncharged only at pH 5.5, its isoelectric point.
- D: The protein is negative, not positive, at a pH above its pI.
Question 6 · Medium
After the wells of an ELISA plate are coated with antigen, a protein such as bovine serum albumin is added before the patient's serum. Its purpose is to:
1. ACover unused plastic sites to stop nonspecific binding
2. BStabilise the enzyme label during incubation
3. CSupply complement for the antigen-antibody reaction
4. DAct as the chromogenic substrate for the enzyme
Answer and explanation in LearnFree account
Question 7 · Medium
In capillary zone electrophoresis of serum proteins, the proteins reach the detector at the cathode end although they are negatively charged. This is due to:
1. AElectro-osmotic flow of the buffer
2. BGravity acting along the capillary
3. CPressure from the injection pump
4. DAdsorption of proteins to the capillary wall
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Question 8 · Easy
On what principle do boronate columns that retain all glycated haemoglobins work?
1. APartition between two liquid phases
2. BSieving by molecular size in porous gel
3. CSpecific binding to a fixed ligand (affinity)
4. DExchange of ions on charged resin
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Question 9 · Easy
DEAE-cellulose acts as an anion exchanger because its diethylaminoethyl groups are:
1. AUncharged and strongly hydrophobic
2. BBound to a specific antibody
3. CPositively charged at working pH
4. DNegatively charged at working pH
Answer and explanation in LearnFree account
Question 10 · Medium
In a competitive radioimmunoassay, a higher concentration of the analyte in the patient's sample gives:
1. ANo change in the bound labelled antigen
2. BLess labelled antigen bound to antibody
3. CMore labelled antigen bound to antibody
4. DMore antibody in the free fraction
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Question 11 · Medium
In reverse-phase HPLC with a C18 column and a water-methanol mobile phase, which compounds elute first?
1. AThe least polar compounds
2. BThe largest molecules
3. CThe most positively charged
4. DThe most polar compounds
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Question 12 · Easy
Which radioisotope is most often used to label antigens in radioimmunoassay?
1. ATechnetium-99m
2. BIodine-125
3. CPhosphorus-32
4. DCarbon-14
Answer and explanation in LearnFree account
Question 13 · Medium
On serum protein electrophoresis, a broad, diffuse rise of the whole gamma region usually indicates:
1. AA monoclonal protein from a plasma cell tumour
2. BA sample taken as plasma instead of serum
3. CA polyclonal increase from chronic inflammation
4. DA loss of immunoglobulins in nephrotic syndrome
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Question 14 · Medium
Affinity chromatography separates a protein from a mixture mainly by its:
1. ASize relative to the pores of the beads
2. BPolarity compared with the mobile phase
3. CNet charge at the pH of the buffer
4. DSpecific binding to a ligand on the matrix
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Question 15 · Medium
In nephrotic syndrome, serum protein electrophoresis typically shows a low albumin with a marked rise in:
1. AAlpha-2 globulin
2. BGamma globulin
3. CAlpha-1 globulin
4. DPrealbumin
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Question 16 · Hard
In an indirect ELISA for antibodies, every well on a plate, including the blank and the negative controls, turns deep blue after TMB substrate is added. The positive control reads as expected. What is the most likely error?
1. AInadequate washing after the conjugate step
2. BToo little coating antigen on the plate
3. CPatient sera diluted ten times too much
4. DStop solution added before the substrate
Answer and explanation in LearnFree account
Question 17 · Medium
Compared with radioimmunoassay, chemiluminescent immunoassays using acridinium esters are preferred in modern laboratories mainly because they:
1. AWork only for large protein analytes
2. BMeasure colour change with a spectrophotometer
3. CNeed no antibody to bind the analyte
4. DGive high sensitivity without radioactive waste
Answer and explanation in LearnFree account
Question 18 · Hard
Amino acids in urine from a child are separated by TLC. The solvent front is 9.0 cm from the origin. An unknown spot is centred 3.6 cm from the origin, and a leucine standard on the same plate gives Rf 0.70. What is the Rf of the unknown?
1. A0.60
2. B2.50
3. C0.57
4. D0.40
Answer and explanation in LearnFree account
Question 19 · Easy
Serum protein electrophoresis on agarose or cellulose acetate is usually run in which buffer?
1. ABarbital buffer at pH 8.6
2. BAcetate buffer at pH 4.5
3. CCitrate buffer at pH 6.2
4. DPhosphate buffer at pH 7.4
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Question 20 · Hard
A 10-year-old tribal girl from Wayanad has had painful crises since early childhood. Hb 7.4 g/dL, MCV 86 fL, reticulocytes 12%, sickle solubility test positive. Hb electrophoresis at alkaline pH shows a band at the S position only, with HbF 8% and HbA2 2.6%; no HbA band is seen. Her parents have not been tested. What is the most likely diagnosis?
1. ASickle cell trait (HbAS)
2. BSickle cell anaemia (HbSS)
3. CSickle-HbD Punjab disease
4. DSickle-beta-zero thalassaemia
Answer and explanation in LearnFree account
Question 21 · Medium
Bridging of the beta and gamma regions on serum protein electrophoresis is a classic pattern of:
1. AIron deficiency anaemia
2. BCirrhosis of the liver
3. CAcute inflammation
4. DMultiple myeloma
Answer and explanation in LearnFree account
Question 22 · Hard
A 66-year-old woman has back pain and anaemia. Total protein 10.2 g/dL, albumin 3.1 g/dL, calcium 11.6 mg/dL, creatinine 2.1 mg/dL. Serum protein electrophoresis shows a tall, narrow band in the gamma region with low normal gamma globulins on either side. What is the next laboratory step?
1. AMeasure serum ferritin and iron
2. BQuantify alpha-1 antitrypsin
3. CRepeat electrophoresis on plasma
4. DSerum and urine immunofixation
Answer and explanation in LearnFree account
Question 23 · Hard
A 55-year-old man with diabetes has fasting glucose 220 mg/dL and fructosamine raised, but HbA1c by cation-exchange HPLC is 4.9%. The chromatogram shows an unexpected large peak after the HbA0 peak, and Hb is 13.8 g/dL with a normal reticulocyte count. What is the best action?
1. AReport the 4.9% as good glycaemic control
2. BRequest a fasting insulin level instead
3. CRetest HbA1c by a boronate affinity method
4. DRepeat on the same column after recalibration
Answer and explanation in LearnFree account
Question 24 · Medium
A narrow band appears between the beta and gamma regions on an electrophoresis run by mistake on heparinised plasma. This band is:
1. AHaptoglobin
2. BFibrinogen
3. CC-reactive protein
4. DTransferrin
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Question 25 · Hard
A 35-year-old man with a large pituitary tumour on MRI has serum prolactin 82 ng/mL by a one-step sandwich immunoassay. The clinician expects a value in the thousands. On 1 in 100 dilution, prolactin is 6,400 ng/mL. What explains the first result?
1. AMacroprolactin in the sample
2. BHeterophile antibody interference
3. CBiotin interference in the assay
4. DHigh-dose hook effect
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Electrophoresis, chromatography and immunochemical methods

A healthy 24-year-old woman from Assam has antenatal screening: Hb 11.8 g/dL, MCV 71 fL, MCH 23 pg, RBC 5.2 million/µL, serum ferritin normal. Cation-exchange HPLC: HbA 69%, HbF 0.6%, and 28% in the HbA2 window (HbA2 in beta-thalassaemia trait is usually 3.5-7%). The most likely interpretation is:

A Punjabi family's samples show a variant band in the HbS zone at alkaline pH, but the sickle solubility test is negative. Which further electrophoresis distinguishes HbD from HbS?

By which property does isoelectric focusing separate proteins?

At pH 8.0, a protein with an isoelectric point of 5.5 is applied to ion-exchange columns. It will bind to:

After the wells of an ELISA plate are coated with antigen, a protein such as bovine serum albumin is added before the patient's serum. Its purpose is to:

In capillary zone electrophoresis of serum proteins, the proteins reach the detector at the cathode end although they are negatively charged. This is due to:

On what principle do boronate columns that retain all glycated haemoglobins work?

DEAE-cellulose acts as an anion exchanger because its diethylaminoethyl groups are:

In a competitive radioimmunoassay, a higher concentration of the analyte in the patient's sample gives:

In reverse-phase HPLC with a C18 column and a water-methanol mobile phase, which compounds elute first?

Which radioisotope is most often used to label antigens in radioimmunoassay?

On serum protein electrophoresis, a broad, diffuse rise of the whole gamma region usually indicates:

Affinity chromatography separates a protein from a mixture mainly by its:

In nephrotic syndrome, serum protein electrophoresis typically shows a low albumin with a marked rise in:

In an indirect ELISA for antibodies, every well on a plate, including the blank and the negative controls, turns deep blue after TMB substrate is added. The positive control reads as expected. What is the most likely error?

Compared with radioimmunoassay, chemiluminescent immunoassays using acridinium esters are preferred in modern laboratories mainly because they:

Amino acids in urine from a child are separated by TLC. The solvent front is 9.0 cm from the origin. An unknown spot is centred 3.6 cm from the origin, and a leucine standard on the same plate gives Rf 0.70. What is the Rf of the unknown?

Serum protein electrophoresis on agarose or cellulose acetate is usually run in which buffer?

Bridging of the beta and gamma regions on serum protein electrophoresis is a classic pattern of:

A 66-year-old woman has back pain and anaemia. Total protein 10.2 g/dL, albumin 3.1 g/dL, calcium 11.6 mg/dL, creatinine 2.1 mg/dL. Serum protein electrophoresis shows a tall, narrow band in the gamma region with low normal gamma globulins on either side. What is the next laboratory step?

A 55-year-old man with diabetes has fasting glucose 220 mg/dL and fructosamine raised, but HbA1c by cation-exchange HPLC is 4.9%. The chromatogram shows an unexpected large peak after the HbA0 peak, and Hb is 13.8 g/dL with a normal reticulocyte count. What is the best action?

A narrow band appears between the beta and gamma regions on an electrophoresis run by mistake on heparinised plasma. This band is:

A 35-year-old man with a large pituitary tumour on MRI has serum prolactin 82 ng/mL by a one-step sandwich immunoassay. The clinician expects a value in the thousands. On 1 in 100 dilution, prolactin is 6,400 ng/mL. What explains the first result?