Immunoassay – Sources and Effects of Immunoassay Interference

What is Immunoassay?

An immunoassay is a biochemical test that measures the presence or concentration of a molecule in a solution - usually serum or urine. This is achieved through the use of an antibody or an antigen. The molecule detected (the analyte) is in many cases a protein, although it may be other kinds of molecules as long as suitable antibodies are available.

What Problems can Interference Cause in an Immunoassay Test?

Interference in immunoassay may lead to:

• the misinterpretation of a patient’s results.
• the wrong course of treatment being prescribed.
• or a serious problem missed, with potentially devastating medical and financial implications.

Examples of Sources of Interference and their Effects

Sources of pre-analytical errors:

• Haemolysis.
• Components from blood collection tubes, such as stoppers, lubricants, surfactants and separator gels, can leach into specimens or adsorb analytes.
• Special additives may also affect analyte stability.
• Delay in processing
• Incorrect storage

Below, you can find a table where each problem is detailed when an immunoassay test is compromised by incorrect storage, along with the subsequent effects.

Types of analytical errors

1. Sample to sample carry over
2. Procedural errors (e.g.  incorrect fill-volumes, incorrect additive to blood ratios, inadequate washing or clot detection)
3. The presence of substances that alter the measurable concentration of the analyte in the sample or alter antibody binding. These may arise from the sample itself, from earlier contamination as listed above or from the purified water and other reagents used.

Analytical Interferences - Biologically active species

A wide range of interferences can arise from biologically active species that are present in the sample. Species that can alter the measurable analyte concentration in the sample include hormone binding proteins and autoanalyte antibodies; while heterophile antibodies and human anti-animal antibodies can alter antibody binding. Cross-reacting species also result in over- or underestimation of sample analyte concentration if an immunoassay reagent contains antibodies directed toward molecules other than the antigen of interest. Cross-reactivity with drugs and their metabolites may be a problem for the measurement of steroids by immunoassay and hormone binding globulins can alter the measurable analyte concentration in the sample either by their removal or by blocking the analyte.

Autoantibodies can cause interference in both non-immunoassay and immunoassay methods for a number of analytes including macro-enzymes (creatine kinase, amylase), thyroid hormones in both free and total forms, thyroglobulin, insulin, prolactin and testosterone. False-positive or false-negative values may arise, depending on whether the autoantibody-analyte complex partitions into the free or the bound analyte fraction.

Heterophile antibodies interfere with immunoassays by a non-competitive mechanism. Heterophiles can bind to the conjugate, enzyme, or other parts of the detection system in reagent-limited assays, mainly of the non-RIA type, and cause interference in assays for steroid hormones, thyroid function tests, and digoxin.Other proteins that affect antibody binding and can interfere in immunoassays include complement, lysozyme and paraprotein.

Many interfering substances are unique to an individual and the concentrations can change over time. Low-affinity polyspecific antibodies can be present in high concentrations or high affinity in low concentrations.  They may interfere in a number of immunoassays for different analytes and may interfere within one or more manufacturers' immunoassay systems but not necessarily in all assays.

Cross-reaction is a problem in diagnostic immunoassays where endogenous molecules with a similar structure to the measured analyte exist or where metabolites of the analyte have common cross-reactive epitopes, and where there is administration of structurally similar medications.

Chemical and Other interferences

Other interferences can arise from the effects of reagents added to stabilise samples, to enhance other tests or to mask interferences. The effects can be multi-stage and complex. They are usually dependent on concentration but not proportionally so. Those compounds that could also be introduced as contaminants in the purified water used in the assay process are discussed in more detail below.

EDTA

EDTA is added typically at a concentration of 1 to 2 mg/mL of blood. It acts as an anticoagulant (by binding Ca needed for coagulation) and as a chelating agent.  It inhibits alkaline phosphatase, creatine kinase and leucine aminopeptidase activity.

Chelating agents, such as EDTA and citrate, do not directly inhibit serine proteases, but they do limit the activation of proteases in the coagulation system by interfering with calcium-mediated surface binding and by allowing inhibitors to dominate<

Potassium EDTA, an anticoagulant and chelating agent, interferes with calcium assays and clot generation, but it is preferred for haematology testing. EDTA binds the metallic ions europium (immunoassay reagent), zinc, and magnesium (enzyme cofactors for immunoassay reagents such as alkaline phosphatase). Insufficient sample volumes produce relatively elevated EDTA levels, which can increase the chelation of magnesium and zinc and can then affect reagent enzymes used for signal generation, such as alkaline phosphatase. Reagent antibodies recognize divalent cation complex binding sites on proteins; thus, decreased calcium and magnesium levels may induce conformational changes that decrease antibody binding.

Citrate

Citrate is also added as an anticoagulant by binding calcium ions. Trisodium citrate in a 109 mmol/L or 129 mmol/L solution is preferred for coagulation testing. It can inhibit both aspartate aminotransferase and alkaline phosphatase by the chelation of cations.

Fluoride

Sodium fluoride is added typically at a concentration of 2 mg/mL of blood as an antiglycolytic agent. It can affect many enzymatic immunoassays because of its enzyme inhibitory activity. Fluoride may also interfere with electrolyte measurements by altering cell membrane permeability and promoting haemolysis by red blood cell ATP with subsequent potassium efflux. It also affects urease for urea estimation

Sodium fluoride was found to inhibit human salivary amylase at concentrations above 50 mmol/L. This may be through fluoride-induced removal of calcium as the activity and stability of α-amylase has been shown to be dependent on the presence of chloride and calcium.

Oxalate

Potassium oxalate is added typically at a concentration of 1 to 2 mg/mL of blood. It is another calcium-chelating anticoagulant often combined with antiglycolytic agents (sodium fluoride and sodium iodoacetate) that can decrease haematocrits by as much as 10% by drawing water from cells into plasma. Oxalate can also inhibit several enzymes, such an amylase, lactate dehydrogenase, and acid and alkaline phosphatase and may precipitate calcium ions. Binding of cations present in serum, e.g., Mg²⁺ or Ca²⁺, to drugs or proteins, can change antigen conformation and the measurable analyte concentration.

Surfactants

Surfactants are commonly used to decrease non-specific adsorption, but they must be carefully selected and optimized for immunoassays since, at high concentrations, they may cause the loss of antibodies passively adsorbed onto the solid support beads used in immunoassays.

Silicone surfactants interact with ion-specific electrode membranes to increase the measured voltage during magnesium and lithium determinations. In addition, water-soluble silicone polymer coatings in separator tubes can physically mask antibodies and alter avidin-biotin binding reactions in immune-radiometric assays.

Importance of Interferences in Immunoassay.

Interference in immunoassay is one factor that contributes to the uncertainty of medical testing. Laboratories should be aware of the potential for interference in all immunoassays and how such results may cause misinterpretation of a patient’s results and a subsequent wrong diagnosis and unwarranted treatment. The recognition of such aberrant test results requires constant surveillance by both laboratory and doctor. One potential source of chemical interferences, the purified water, can be eliminated by choice of a purpose-designed system to produce high-purity water.

Dr Paul Whitehead 

After a BA in Chemistry at Oxford University, Paul focused his career on industrial applications of chemistry. He was awarded a PhD at Imperial College, London for developing a microwave-induced-plasma detector for gas chromatography. He spent the first half of his career managing the analytical support team at the Johnson Matthey Research/Technology Centre,specialising in the determination of precious metals and characterising applications such as car-exhaust catalysts and fuel cells. Subsequently, as Laboratory Manager in R&D for ELGA LabWater, he has been involved in introducing and developing the latest water purification technologies. He now acts as a consultant for ELGA.