Lab Study 2 Flashcards
What does the biuret reagent contain?
The Biuret reagent contains:
- Copper sulfate pentahydrate - source of cupric ions (Cu2+)
- Sodium hydroxide - creates an alkaline environment
- Sodium potassium tartrate - forms complex with cupric ions to prevent their precipitation within the alkaline environment
- Potassium iodide - antioxidant
What colour will the proteins change to with the biuret reagent? Why?
Light pink (small peptide chains) or violet (presence of many peptide bonds).
In an alkaline environment, the copper atoms will form a complex with the nitrogen of peptide bonds in polypeptides producing a color change from blue to a light pink (presence of small peptide chains) or violet (presence of many peptide bonds) as the chelate forms. No color change indicates the absence of peptide bonds, and therefore, is negative for proteins.
What is the reference range for total protein? What does total protein consist of?
The reference range for total protein is 60-80 g/L. Total protein consists of albumin and globulins. Each protein fraction can be quantified using other methods.
For pre-analytical considerations, what problem samples should be avoided? What do you do if you can’t avoid these issues?
- a) Lipemic, icteric, and hemolyzed samples should be avoided as it interferes with the test
b) Free hemoglobin causes elevated results - If unavoidable can correct with a serum blank.
For pre-analytical consideration, how are ambulatory patients results affected? Why?
- Protein values are approximately 5 g/L higher for ambulatory patients (slight hemoconcentration) than patients that are supine or recumbent
- Reduction in serum albumin and fluid shift toward the extracellular compartments
How does pregnancy affect total protein counts?
Lower total protein results are seen in pregnancy.
What is the preferred sample type? (i.e. plasma or serum).
Serum is the preferred sample type. Plasma can be used but will give a slightly higher value due to the fibrinogen content.
How much sample is used in Serum Protein Electrophoresis?
2 - 10 uL applied to a support medium.
What support medium materials are used in serum protein electrophoresis?
The support medium, a neutral polymer such as agarose, cellulose acetate, or polyacrylamide provides structural support for the sample to travel through.
After ~5 mins of allowing the sample to diffuse into the support medium, the support medium is put into an electrophoresis chamber containing buffer, what does the buffer do? What pH is typically used?
The buffer maintains the pH and ionic strength within the electrophoresis environment. Generally, a buffer with a pH of 8.6 is used for separation of serum proteins. It is important that the buffer maintain contact with the support medium to maintain consistent and constant electrical flow.
Where are the serum samples placed? Where do the serum proteins migrate to?
There are two electrodes, the cathode (negative electrode) and the anode (positive electrode). Serum samples are applied close to the cathode end of the support medium and will migrate towards the anode as many serum proteins carry a net negative charge in a buffer at pH 8.6. So, the current travels between the electrodes through the buffer and medium separating the proteins from the sample.
What is the next two steps after electrophoresis for serum proteins?
- Once the appropriate amount of time has passed, the support medium is removed and placed in a fixative (usually an acidic solution) or dried to prevent further migration and prevent loss of sample from the medium.
- The support medium is then stained in order to visualize the banding pattern. Excess stain is washed away to ensure it does not interfere with interpretation.
What is the order of the bands in terms of type of proteins?
The dark band closest to the anode is albumin (dark because it is the most abundant serum protein), then α1, then α2, then β (actually comprised of a β1 and β2 band), and finally γ (more faint and wider in normal serum) located closest to the cathode.
How do you know which protein is in the greatest quantity in your sample without using a densitometer?
The amount of dye taken up by the band is proportional to the amount of sample present in that area.
What can be used to estimate the quantities of each protein in the sample?
The stained support medium can be read using a densitometer, an instrument that accurately quantifies the amount of protein fraction present using optical density (remember Beer’s Law!). The wider and darker the band, the wider and higher the peak produced in the densitometric pattern. The area under each peak represents the proportion of that protein fraction in the sample.
How do you interpret the banding or densitometric pattern?
We must first understand what the expected normal values are in regards to the serum fractions as part of total protein values.
See table in lab. Protein Frac. Ref Range(SI) %of total Pro. Albumin 35-50 g/L 53-65% α1 1-3 g/L 2.5-5% α2 6-10 g/L 7-13% β 7-11 g/L 8-14% γ 8-16 g/L 12-22%
What do increased or decreased values potentially mean for each protein fraction, albumin, alpha1, alpha2, beta, and gamma?
Protein Fraction / Increased Values Seen In / Decreased Values Seen In:
Albumin: I: Dehydration
D: Malnutrition,
Inflammation, Liver and
Kidney Disease
α1 I: Infection and inflammation
D: Liver Disease
α2 I: Inflammation, infection and
kidney disease
D: Liver Disease
β I: Iron deficiency anemia
D: Poor nutrition
γ I: Infection, inflammation, liver
disease, “M” spike with Multiple
Myeloma
D: Hypogamma-globulinemia
What occurs with alpha2 and beta fractions when samples are hemolyzed?
It will sometimes just appear as an increased beta band.
What do you see in the cases of cirrhosis?
A beta-gamma bridge.
What do you see if plasma is used instead of serum?
A narrow band will appear between beta and gamma fractions.
What variables can influence the success of your electrophoresis run?
Human competence, pH, buffer strength, gel integrity, voltage, time, etc.
What affect can too much heat cause with electrophoresis of proteins?
Wick flow:
Heat causes evaporation of buffer, drawing the support medium in from the edges causing an uneven pattern
What is ‘Electroendosmosis’ and how can it affect the results?
With basic buffers, proteins take on a negative charge and migrate towards the positive electrode (anode). The buffer contains positive ions that form a positive ion cloud which migrates to the cathode. This movement in opposite directions slows migration, or causes the large or less charged proteins to migrate in the opposite direction
What are some simple problems that can cause no migration?
No power,
No buffer, and
No sample.
What are some reasons that could cause indistinct bands?
- voltage too high,
- incorrect migration time (long patterns = time too long;
- bands not separated = too short), or
- low ionic strength buffer
What could cause holes in the pattern?
Air bubbles in support medium or in sample.
What are immunoassays?
Immunoassays consist of a wide range of methods used to identify and quantify analytes by manipulating the known characteristics of antigen and antibody interactions.
What are antibodies?
Antibodies are glycoprotein molecules that bind “lock and key” to antigens.
In body –> Function to remove “non-self” antigens (such as those on bacterial surfaces) through the processes of neutralization and/or opsonization.
What is the specific and unique area of the antigen that the antibody binds to called?
Epitope.
How can be use antigen or antibodies for analysis?
If the antigen or antibody used in the method is known, then we can determine the presence, absence, or quantity of the other.
Known antigen investigate unknown antibody and vice versa.
What type of substances can be investigated for using antibody/antigen reactions?
Presence of pathogens Hormones, Drugs Current infection Long term immunity
What are the benefits of antigen-antibody reactions?
Quick
Accurate
Precise laboratory results.
What is the difference between performing a heterogeneous and homogeneous immunoassay?
Heterogenous immunoassays require a physical SEPARATION step of the Ag:Ab complex from other sample components.
Homogeneous immunoassays DO NOT.
What is a “competitive” immunoassay?
Competitive Immunoassay:
A analyte in the patient sample competes with a labelled analyte in the reagent.
How are the amounts of labelled antigen related to the patient analyte?
The amount of signal produced from the labelled Ag:Ab complex is inversely proportional to the amount of analyte present in the patient sample.
What is a noncompetitive immunoassay?
Noncompetitive Immunoassay:
No competition between the analyte and reagent.
What are different types of noncompetitive immunoassays?
- Direct (using one labelled analyte in reagent).
- Indirect. E.g. Two antibodies can be used, one unlabeled and fixed to the well and the other labeled that attaches to the antigen present in the patient sample, forming a sandwich.
Note: Confirm understanding re #2.
How is the amount of signal produced from the labelled Ag:Ab complex related to the amount of analyte present in the patient’s sample in noncompetitive immunoassays?
Directly proportional.
What are heterophile antibodies and how can it interfere with the results?
Heterophile antibodies are antibodies in patient serum that can react with reagent antibodies causing both false positive and false negative results.
