Quality Control of ELISA
To help us to avoid the interference and variables, as the general this is the aim to reduce the errors
Definition of interference :-
*Defined as the effects of substances present in an analytical system which causes a deviation (variable value) of the measured values from the true value.
*Assay interference can be analyte dependent or analyte independent.
*May be increase or decrease the measured result.
*The increasing (positive interference) which lacks of specificity while the decreasing (negative interference) which lacks of sensitivity.
#We can put the errors and interferences as the following (must be avoid and correct these errors) :-
1. Pre analytical errors
2. Analytical errors
3. Post analytical errors
Pre analytical errors:-
As the following:-
1.Patient based:-
*Incorrect sampling time.
*Environment factors (may be changed analyte concentration and interpretation).
2.Specimen based:-
*Blood collection procedure and time of collection ( certain hormones are affected by time of collection such as cortisol).
* Must be attention to anticoagulant. some of it effects of analyte.
*The samples must be free from hemolysis, hyperbilirubinemia and lipaemia
3.Assay based:-
● Certain steps to do before the test performance as the following :-
-bring the kits to the room temperature
-check the incubator temperature
-check the date of expired of the kits
-put proper planning for assays to be performed
-attention to labelling
analytical errors:-
* It's mainly the procedural errors.
* Mostly confined to the different steps that includes addition, washing, incubation, dilution, pipetting and reading
*The errors occur due to not following the the protocol correctly or loss the understanding.
1.Washing errors:-
*High pressure washing
*Drying of well
*Splashing during washing
*Not removing of all wash solution from wells.
*Leaving the bubbles in the wells after washing
* Not tapping the wells after washing
*Use contaminated wash buffer
2.Pipetting errors:-
*Reuse of pipette tips
*Pipette tip blocked
3.Equipment errors:-
*Incubator is not maintained at right temperature
*Washer probers are blocked and contaminated
*Instrument filters are not checked
4.Procedural errors :-
*Wells are not covered during the incubation
*Not blocking when required.
*Not running all calibrators to plot a graph
*Bubbles in wells
*Use of contaminated or unclean tubes to prepare reagents
*Use kits or reagents are expired
*Using negative wells again
*Not mixing after adding stop solution
Post analytical errors:-
As the following :-
* Calculation errors
* Choosing a wrong graph scale *Comparison of result with inappropriate references interval
*Use of wrong reference values
* Use of wrong units
There are many interferences proteins which could make the errors of
measured values as the following :-
1. Albmin:-
-it may interfere as a result of its comparatively huge concentration and its ability bind as well as to release quantities of ligand.
2. Rheumatoid factors:-
-there are auto antibodies (IgM) and direct against the Fc portion of IgG.
3. Complement:-
-These proteins bind to the Fc fragment of immunoglobulin, blocking the analyte specific binding site.
4. Endogenous hormones-binding proteins:-
-There are present in varying concentration in all serum and plasma are markedly influence assays examples:-
*HBG (sex hormone binding globulin) interferes in immunoassay of testosterone and estradiol.
*TBG ( thyroxine binding globulin) and NEFA (non esterified fatty acid) interfere with the estimation of free T4
5. Heterophilic antibodies:-
-Observed in two sites sandwich reagent, in which a bridge is formed between the two antibodies forming the sandwich
Assays that affect by Heterophilic antibodies include Hcg, TSH…
6. Fibrinogen:-
-Incompletely clotted samples interfere with sampling procedures on automated immunoassay instruments.
