Various washing cycles can be programmed. Careful
maintenance is essential, since they are prone to machine
errors, such as having a particular nozzle being blocked.
¾ Follow procedure for preparation of wash buffer
¾ Check washer alignment daily as part of routine
instrument start-up procedures
¾ Ensure that the plate is levelled
¾ Make certain well is completely filled, when washing,
to ensure residual conjugate is removed
¾ Examine that the plate is levelled
¾ Make certain well is completely filled, when washing,
to ensure residual conjugate is removed
¾ Examine the fill volume (a slight dome should be
observed at the top of the well)
¾ When washing does not allow wells to overflow
¾ Reduce pressure in wash system
¾ Check washers before use to determine they are
working properly. Perform routine maintenance
¾ Allow approximately 20 seconds between the addition
of wash solution and subsequent aspiration
¾ Examine the wells for complete aspiration of contents
¾ Upon completion of wash cycle, blot to remove residual
¾ Calibrate pipettes regularly according to manufacturer’s
¾ Avoid touching sidewall of well with tips
¾ Avoid splashing of sample and reagents
¾ Avoid blowing out tip contents
¾ Use a new tip for each sample/control/reagent addition
¾ New tips should be used on the multichannel pipettes
¾ Reverse pipette when using the multichannel pipette to
add conjugate and substrate solution
¾ Forward pipette when using the multichannel pipettes
¾ Check pipette tips are long enough to provide air space
between top of tip and pipette barrel
¾ Check pipette barrel for residual fluid or dried material,
¾ Ensure pipettes tips are fitted tightly
¾ Service pipettes periodically by the manufacturers or
¾ Do not open the pipette without proper tools.
¾ Bring microplate pouches to room temperature before
¾ Level microwells evenly in microplate frame as the
individual breakaway wells have very flexible plate
frames leading to bowing of wells and yield poor washes
¾ Place plates in dark immediately after addition of
substrate solution, provided the substrate is sensitive
¾ Grasp holder on grip marks when tapping to avoid
¾ Rotate strips 180oC and reinsert or use correct holder if
¾ Seal unused wells in pouches along with the desiccant
¾ Date the pouches when first opened
¾ Clean bottom surface of plates with wash buffer to
¾ Make sure microwells are at level during washing,
reagent addition and plate/strip reading
¾ Wipe the bottom of the plate with a lint-free cloth/towel
¾ Do not allow microwells to become dry once the assay
¾ Use freshly prepared substrate A and substrate B
¾ Do not hold substrate solution longer than 1 hour
¾ Follow procedure of working substrate solution
¾ The temperature of solution is important because it
affects rate of color reaction
¾ Do not add fresh substrate to reagent bottle containing
¾ Clean old substrate solution bottle with H2SO4 and
thoroughly rinse with distilled water.
¾ Store at recommended temperature
¾ Never store exclusively diluted conjugate for use at
¾ Always make up the working dilution of conjugate just
¾ Neverleave conjugates on the bench for excessive time.
¾ Ensure all necessary items are chosen before starting
¾ Maintain a logbook on calibration and results data
¾ While performing the assay, do not divert attention.
A fundamental problem with the analysis of components in
biological materials is the effect of the extremely complex
and variable mixture of proteins, carbohydrates, lipids,
and small molecules and salts constituting the sample.
The effect of these compounds on analytical techniques is
It can be defined as “the sum of the effects of all the
components, qualitative or quantitative, in a system with
the exception of the analyte to be measured.”
Assay buffers: The ionic strength and pH of buffers
are vitally important, particularly in the case of
monoclonal antibodies with pH values of 5–9. The use of
binding displacers (blockers) may change the binding
characteristics of antibodies, particularly those of low
Labels have a profound effect on assays. The structure of
most molecules, especially haptens, may be dramatically
changed by labeling, e.g. by attachment of a radioactive
iodine atom to a steroid. Labeling antibodies with enzymes
is less of a problem because of their large size.
Separation of the Antibody-bound and
The proportion of free analyte in the bound fraction
and vice versa is known as the “misclassification error”.
Antibody bound fraction may be efficiently separated
from the free analyte using solid-phase systems in which
the antibody is covalently linked to an inert support, e.g.
the reaction tube, a polystyrene bead, a cellulose or nylon.
Interfering proteins of general relevance include the
It may interfere as a result of its comparatively huge
concentration and its ability to bind as well as to release
These are autoantibodies usually IgM class, and directed
including systemic lupus erythematosus, scleroderma and
These proteins bind to the Fc fragment of immunoglobulins,
blocking the analyte specific binding sites.
Strongly associates with proteins having low isoelectric
points (pI). Immunoglobulins have a pI of around 5 and
lysozyme may form a bridge between the solid-phase IgG
Endogeneous Hormone-binding Proteins
These are present in varying concentrations in all serum
and plasma samples and may markedly influence assay
performance. For example, 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
Abnormal forms of Endogeneous binding Proteins
These are present in the plasma of some patients. They are
present in familial dysalbuminemic hyperthyroxinemia
(FDH) in which albumin molecules bind to thyroxine (T4).
Individuals with FDH can be diagnosed as thyrotoxic, in
They may arise as a consequence of intimate contact,
either intentional or unintentional, with animals. The most
formed between the two antibodies forming the sandwich.
Assays that are affected by heterophilic antibodies include
CEA, CA 125, hCG, TSH, T3, T4, free T4, prolactin, HBsAg
Fibrinogen from incompletely clotted samples interferes
with sampling procedures on automated immunoassay
instruments and may produce spurious results.
Paraproteinemia causes interferences in many assays
by increasing the viscosity of the sample. They may also
nonspecifically bind either analytes or reagents that may
Nonspecific interference may arise from excessive
concentrations of other constituents of plasma. Free fatty
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