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This report is about the various interferences in immunoassays. Often, the accuracy of immunoassays is compromised as a result of certain interfering contaminants in the assay and these contaminants can create a positive or negative bias. Although interferences exist, they can be minimized by using statistical tools and data to identify the bias results. In this report, we will be talking about the various sources of interference in immunoassays as well as the methods to prevent, or minimize, these interferences from occurring in immunoassays.
Firstly is the blood Collection. The plasma protein concentration will increase if prolonged usage of tourniquet is applied. This is called venous stasis and would elevate plasma protein concentration comparatively by 5%. The ligand bound to the serum or plasma concentration will also be elevated thus giving fasle results. This does not interfere directly to the assay but the interpretation of the results may be affected.
Secondly is the nature of the sample. The collection of sample is important as different collection tubes (of different components (e.g. EDTA, Lithium Heparin) may result in the interference in immunoassay. Serum is the matrix of choice for most immunoassays. However, samples anticoagulated with anticoagulant such as EDTA can be useful for reducing interference from calcium modulated complement activation. Sodium fluoride tube may be improper for some enzymatic immunoassay methods. Sodium azide preservation may also interfere with the results. The usage of gel serum separator has been proven to interfere with some immunoassays.
Thirdly, Lipaemia which is a condition whereby increased amounts of lipids are present in the blood Lipaemia may cause fat soluble compounds such as steroids to interfere with some assay. Lipaemia will also interfere immunoassay that turbidimetric end-points.
Interferences In Immunoassays
3.1 Matrix Effects
First factor is pertaining to assay buffers. Monoclonal antibodies usually have pI value of 5-9 thus, ionic strength and pH of buffers are crucial. The use of binding displacers (blockers) may alter the binding characteristics of antibodies, especially those of low affinity. Excessive concentrations of detergents may denature proteins and deadsorb analytical antibodies from the solid phases. Detergents may contain peroxidase which prevents antigen-antibody reaction.
Second factor is on polyclonal antibodies which are antibodies that come from different B cell line. Polyclonal antibodies may not bind to specific antigen molecules only but also to metabolites and fragments having the right epitopes. Antibody mixtures lacking the Fc fragment results in lesser binding of non-specific binding agents.
Thirdly is on monoclonal antibodies which comprises the IgA, Ig G or IgM subclass and this may create problems in immunoassays. This usually happens when the capture antibodies namely, anti-IgA, anti-IgG, anti-IgM are used to bind the monoclonal antibodies to the solid support or as labels in polyclonal-monoclonal systems. Moreover, IgA and IgM can be polymeric with four and ten binding sites per immunoglobulin molecule.
Lysozyme interferes with immunoassays by directly creating a bridge between the solid-phase Immunoglobulin G (IgG) and the signal antibody. This happens due to the fact that lysozymes are attracted to proteins of low isoelectric points (pI) and that immunoglobulins have a low pI of 5. The interference of lysozymes can be reduced by adding Cu2+ ions as well as by adding ovalbumin38 in buffers.
Endogenous Hormone-Binding Proteins
This group of proteins can directly interfere with immunoassays substantially. Unless ligand binding sites are inhibited, assay for total hormone measurement can be directly affected, causing the method to be inaccurate. To obtain total ligand measurement, all the ligands that are attached to the proteins must be unbound and binding of signal-ligand complex must also be prevented. Changes in more specific binding proteins also have more direct influence over the results of immunoassays, reducing the accuracy of the assay.
Abnormal Forms Of Endogenous Binding Proteins
An example would be familial dysalbuminaemic hyperthyroxinaemia (FDH) in which half of the albumin molecules is 50 times more likely to bind to thyroxine molecules than normal. Patients with FDH are found to have more thyroxine in their body and are more likely to be diagnosed as thyrotoxic. Furthermore, in these patients, the binding of labelled hormone is greater than that of normal serum.
Autoantibodies To Thyroid Hormones
Antoantibodies to thyroid hormones are quite common in our population and may affect the accuracy of some thyroid disease test. In order to minimise interference to testing, proteins must be separated from the hormones to ensure that the antibodies are not present to affect the results of the testing. Thyroglobulin autoantibody interference, however, creates the most problem among the testing and as of yet, none of the methods are not affected by the interference.
Heterophilic antibodies may be caused as a result of close contact with animals or from the introduction of monoclonal antibodies to the human body. These antibodies can interfere with several different types of assay. In reagent-limited assays, heterophilic antibodies actively reduce the amount of binding sites on primary antibodies, leading to inaccuracy. In reagent-excess assays however, the formation of a bridge between two heterophilic antibodies may also lead to inaccuracy on its part.
Non-specific interference may be caused by the components of blood plasma. Fatty acids from plasma may assays for T4 by unbinding it from endogenous binding proteins. Drug assays may also be affected by the plasma itself while plasmas from different people also produce different results for norgestrol acetate assays. Chinese medicines, on the other hand, are also known to cause digoxin-like immunoreacitivty in certain assays.
Minimisation Of Interference
Cross reactivity within immunoassays is one of the interferences that occur. Interferences by cross-reactivity serve as a major obstacle in getting accurate results. While in most cases this phenomenon cannot be avoided, it is possible to reduce the degree of interference to a minimal level.
Thus, in order to obtain accurate results from immunoassays, it is essential to eliminate or at least minimize cross reactivity interferences. There are many various ways to reduce cross reactivity in immunoassays, namely by using more specific antibodies, separating cross reactants from the analytes, applying physical and/or chemical methods to reduce or block cross-reactivity, the usage of two-site assays and pre-treatment techniques.
Cross-reactivity in immunoassays can be minimized by the utilizing monoclonal antibodies (MAb). MAb have high specificity as they only recognize one type of antigen. When choosing the appropriate MAb that is specific for the analyte, pools of several different MAbs with different specificities are screened to select the most specific clone(s). Currently, research is being conducted to produce antibodies specific to steroid hormones. However, cross-reactivity may occur to a certain degree even though MAbs are used in immunoassays. Thus, other methods can also be used in conjunction like positioning the attachment of haptens on carrier proteins or onto the support column material in the affinity purification process will also increase antibody-binding specificity and thus minimizing the interferences caused by cross-reactivity.
Chemical and physical methods can also be used to reduce interferences caused by cross-reactivity in immunoassays. In cases where the analyte and the cross-reactant differs in their susceptibility for a certain chemical, the cross-reactant can be converted to a less active form without compromising the activity of the analyte via a chemical reaction. For example, we can eliminate the extraction of testosterone (cross-reactant) that comes along with dihydrotestosterone (analyte) by oxidation as oxidation destroys testosterone and does not affect dihydrotestosterone (DHT). Other methods include the addition of chemicals to split the disulfide bonds, thus inactivating interfering antibodies.
Physical methods such as adjusting incubation time and temperature also reduce interferences caused by cross-reactivity. Cross-reactivity decreases with increasing incubation time and is at the minimum when equilibrium of the antigen-antibody reaction is reached. Generally, it takes hours to days for most CB immunoassays to reach equilibrium and thus it is essential to find a balance between increased productivity and decreased specificity when performing immunoassays. Cross-reactivity also changes with temperature as higher temperature causes equilibrium of the reaction to be attained in a shorter period of time. However, the equilibrium constant also shifts with temperature, thus making the overall effect of temperature on cross-reactivity unpredictable.
The principle of two-site assay revolves around the usage of two antibodies with specificity for two distinct epitopes on the same analyte of interest. With appropriate selection of antibodies, specificity of the immunoassay can be increased greatly as these two antibodies are required to bind to the analyte. However, there is a drawback as only analytes large enough to bind the two antibodies at the same time can be utilized for two-site immunoassays.
For the 'block' approach to solving interference problems, particularly for antigen assays, one may attempt to use non-immune immunoglobulin from the species used to raise the reagent antibodies. This technique blocks the interfering antibody and has been the most common immunological approach to reduce interference. Other pre-treatment techniques include precipitation of all unwanted, endogenous immunoglobulins and heating to denature. For reducing interference in specific antibody assays, one could also use highly purified capture antigens to reduce nonspecific binding sites, which may result in interference. This is made possible now through DNA recombinant technology.
Interferences will always be present to some degree in immunoassays, due to the subtle nature of the reactions. It is thus necessary for a laboratory technician to accept a compromise. However, when possible, it is best to reduce interferences to a minimal level in immunoassays, by employing certain scientific strategies.
In a case where interferences get in the way of the immunoassay, then it is best to assay the sample by an alternative method. After all, it is the duty of the clinician to be aware of assay interference problems, and doing their best to bring it to the minimal level.
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National Centre for Biotechnology Information, 2004. Interferences in Immunoassay [online]. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904417/ [Accessed 12 January 2010].
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