helena BioSciences SAS-3 Immunofix Instrucciones De Uso página 8

2. Non-Specific Precipitation in All Immunoglobulin Lanes
Occasionally a completed IFE plate exhibits a precipitate band in the same position in every pattern
across the plate. This may result from:
a) IgM monoclonal immunoglobulins
IgM monoclonal proteins can adhere to the gel matrix. A band will appear in all 5 antiserum lanes
of the gel. However, where the band reacts with a specific antiserum for the heavy chain and light
chain, there will be an increase in size and staining intensity of the band, allowing the
immunoglobulin type to be identified.
discrimination between the IgM-antibody reaction and the non-specific staining of precipitated IgM
protein in other lanes, simplifying the diagnosis.
b) High Titres of RF or Immune Complexes
Samples with high titres of Rheumatoid Factor or other immune complexes may show a prepitate
band at the sample application point. Reducing the sample with DTT or β-2-mercaptoethanol can
eliminate this non-specific reaction (Mix 190µL of diluted serum to 10µL of 1% (w/v) DTT in
0.85% saline solution or mix 100µL of serum with 10µL of a 1:10 dilution of β-2-mercaptoethanol
in water. Perform the IFE as usual.
β-2-mercaptoethanol).
c) Fibrinogen
Fibrinogen, if present in the sample, will show as a discrete band in all lanes of the immunofixation
pattern. Fibrinogen is present in plasma, and sometimes in the serum of patients on anticoagulant
therapy.
3. Reaction With Kappa or Lambda Light Chain Antisera but No Reaction with IgG, IgA or
IgM Heavy Chain Antisera
Samples showing this pattern may either have a free light chain monoclonal gammopathy or they
may have an IgD or IgE monoclonal protein. In this situation, the IFE should be repeated,
substituting IgD and IgE antisera for two of the other heavy chain antisera. Failure to obtain a
reaction with IgD or IgE antisera would be indicative of free light chain disease.
4. Band In Gamma Region Showing No Reactivity With IFE Antisera
C Reactive Protein (CRP) may be detected in patients with acute inflammatory response
CRP appears as a narrow band at the cathodic end of the serum protein pattern. Elevated Alpha1-
Antitrypsin and Haptoglobin are supportive evidence for CRP. Patients with a CRP band will
probably have an elevated level when assayed for CRP. A narrow band on the point of sample
application can sometimes be seen which can be caused by chylomicrons in the serum or
precipitated protein in samples which have been stored frozen.
5. Non-Reactivity With Kappa and Lambda Antisera
Occasionally a sample will have a reaction with a heavy chain antiserum but no light chain reaction
is obvious. In this situation, the following need to be ruled out - a) Heavy chain disease, b) Very
high concentrations of light chains, leading to antigen excess, c) Low concentrations of light chains,
d) Atypical light chain molecule that does not react with the antiserum, e) Light Chains with
'hidden' light chain determinants (as sometimes seen with IgA and IgD). To obtain definitive
results, testing may include: a) A higher or lower dilution of the sample to optimise the antibody /
antigen equivalence, b) Antisera from more than one manufacturer to aid in the identification of
atypical immunoglobulins, and c) Treat the sample with β-2-mercaptoethanol to 'reveal' the light
chains.
Additional dilution of the sample will improve the
NOTE: Always work in a fume hood when using
6
12-13
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