Thursday, November 7, 2019

Quality Control of ELISA

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.

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