Pdf the influence of naturally occurring heterophilic. Of approximately 2600 samples screened, 81 had heterophilic antibodies. Positive heterophilic antibody interference in sandwich capture assays occurs because heterophilic antibodies bridge the capture and the tracer antibody. This is due to the highaffinity, stable interaction of streptavidin with biotin, together with the various chemical and enzymatic biotinylation methods available for use in different assay designs, in which biotinylation does not alter a molecules properties. Heterophile antibodies are of particular importance in clinical medicine for their use in detecting epsteinbarr virus the causative agent of infectious mononucleosis. Pdf interference by human antimouse antibody in twosite. Heterophile antibodies and rheumatoid factors are known to account for most of the heterophile interference in immunoassays 4, 7. Moreover, igf1 bioactivity measured by the kinase receptor activation assay was normal.
Heterophilic antibody interference has falsely elevated figure 1b results as a consequence 58,6064, although falsely low values 31,55,6567 have been reported in cases when the interfering antibody creates a complex with only one antibody from the reagent. A second antibody, which is labelled with an enzyme or chemiluminescent agent, marks the immobilised antibody by attaching to a distant site on hcg tracer antibody. When is a heterophile antibody not a heterophile antibody. Considering this, a longer sample incubation time than 90 minutes might be. Heterophilic antibodies may be a cause of falsely low total. Clinical laboratories should be alert to assay interference from antibodies. Heterophilic antibodies, defined as antibodies against animal immunoglobulins, can be a source of interference in many immunoassays. Rheumatoid factor is an autoantibody against the fc portion of igg that readily crossreacts with assay reagents. After pretreatment with specific heterophilic blocking tubes that contain blocking reagents to eliminate heterophilic antibodies, serum free thyroxine, testosterone, and igf1 levels turned out to be normal. Heterophilic antibody and hama interference can vary from 0. Abstract we verified that antibody binding substances in serum that interfere in twosite immunoassays involving murine antibodies are heterophilic antibodies.
Hypergonadotropinemianot associated to ovarian failure and induced by. Abstract heterophilic antibodies interfere in twosite immunoassays. Eliminating interference from heterophilic antibodies in a. To date, only iga and igm rheumatoid factors have been described in dogs 58, 59, but this may be related to limitations in the methods used to study them 60. Heterophilic antibodies interfering with radioimmunoassay. Sample dilution, removal of interfering antibodies, or treatment of specimen with a heterophilic blocking agent prior to assay, has been recommended to remove heterophilic antibody interference. Endogenous heterophile and human antianimal antibodies mode. Feb 12, 2009 hbt heterophilic blocking scantibodies s tube elimination of heterophilic interference f or immunochemistry assays part number.
Heterophile antibodies are well known interferences that affect many immunoassay formats. Incidence of immunoassay interferencethe prevalence of interference in modern immunoassays is low, but variable and dependent on the type of antibody interference. Positive heterophilic antibody interference in sandwich capture assays occurs because heterophilic antibodies bridge the capture and the tracer antibody 68. Interferences in quantitative immunochemical methods biochemia. Unusual results from immunoassays and the role of the clinical endocrinologist. Biotin interference, heterophilic antibodies, hook effect, immunoassay. The falsepositive pregnancy test result was due to heterophilic antibovine and antigoat antibodies in the patients serum. Patients routinely exposed to animals or to animal serum products can be prone to this interference and anomalous values may be observed. C t he heterophilic interference problem a heterophilic sample is a serum or plasma sample which contains antibodies which are able to bind to animal. This is a pdf file of an unedited manuscript that has been accepted for publication. Endogenous interfering antibodies may fall under two categories. Functional differences between resident and exudate peritoneal mouse macrophages. Sometimes, the antibody may recognize closely related molecules e. This case shows that serum heterophilic antibodies can interfere with immunoassays and result in unnecessary diagnostic procedures andor.
Our purpose was to screen for the samples containing heterophilic antibodies that react with mouse immunoglobulins and to use them to determine how to eliminate interference in various twosite immunoassays being developed in our laboratory. Heterophile antibody interference in a multiplexed fluorescent. Heterophilic antibody interference in immunometric. People with heterophilic antibodies might have unusual results in a number of different kinds of assays. Interference from endogenous antibodies in automated. Furthermore, the assay reagents introduce an error. We verified that antibodybinding substances in serum that interfere in twosite immunoassays involving murine antibodies are heterophilic antibodies. Lkpf1 100 tests, lkpfs 500 tests the sample binds to the free psaspecific monoclonal antibody coated bead. Heterophilic antibodies may be a cause of falsely low. Crossreaction is a problem in diagnostic immunoassays where endogenous molecules with a similar.
Heterophile antibodies antibodies which cross phyla in their reactivity are produced against poorly defined antigens and generally show weak avidity and are multispecies specific. Heterophilic antibodies may be a cause of falsely low total igf1 levels. Antibodies that interfere with immunoassays have been called heterophile or heterophilic antibodies. Other problems of immunoassays have been recognized for. Interference from heterophilic antibodies may also be blocked by adding any. Heterophilic antibodies ha were formally defined as broad specificity, low affinity, igm isotype antibodies that originate as a result of the maturation of the humoral branch of the immune system. Pdf interferences in immunoassays johan schiettecatte.
Hama has been shown to interfere with some twosite immunometric assays utilizing murine monoclonal antibodies, resulting in both false positive or falsenegative results. Faba 2 fragments of antibody ca were prepared by digestion with pepsin and purified by gel ifitration. Pdf we verified that antibodybinding substances in serum that interfere in two site immunoassays involving. Heterophile antibody interference in a multiplexed. The problem of crossreactivity by vitamin d 1,25sohc2d3 determin. The confirmatory test will automatically be ordered if necessary. However, until the method developer has access to authentic samples representing subject matrix and treatment condition, the ability to evaluate crossreactivity may be. These are generally weak antibodies with multispecific activities. How ever, such phantoms are not confined to heterophilic antibodies. Neutrophil attractant protein1immunoglobulin g immune. Antibody multispecificity in immunoassay interference. Labcorp test details for human antimouse antibodies. Immunoassays involving streptavidinbiotin interaction are now widely used by many reagent manufacturers. Heterophilic crossreactive antibodies sometimes present in patient populations and healthy individuals may also interfere.
There is no single procedure that can rule out all interferences. Clia igf1 immunoassays has introduced endogenous antibodies as a new source of interference. Ebv infection induces the production of several antibody classes, of which heterophile antibodies are one others include antii, rheumatoid factor and ana. Interference in immunoassay is an underestimated problem. However, all immunoassays for prl recognize macroprl to some. Endogenous interference in immunoassays in clinical chemistry. In fact interference due to heterophilic antibodies is the major problem in assays of various cancer markers. Heterophile antibodies resulted in significantly elevated cytokine values. New onset of heterophilic antibody interference in prostate. Heterophilic antibody interference in commercial immunoassays.
Thus, heterophilic antibodies are absent in the proteinfree ultrafiltrate, and. We verified that antibody binding substances in serum that interfere in twosite immunoassays involving murine antibodies are heterophilic antibodies. Jul 18, 2019 this method is also less convenient to implement in practice, because immunoassays use a variety of different antibodies, and the blocking agent is most effective when it is as similar as possible to the tracer antibody. Heterophilic antibodies are poorly defined human antibodies that cause interference by noncompetitive binding mostly to the fc region of assay antibodies. They include heterophilic antibodies, human antianimal antibodies, autoanalyte antibodies, rheumatoid factor and other proteins. However, until the method developer has access to authentic samples representing subject matrix and treatment condition, the ability to evaluate crossreactivity may be somewhat limited. Heterophilic antibodies in human serum can react with reagent immunoglobulins, interfering with in vitro immunoassays. Interferences in quantitative immunochemical methods.
Most of the interferences are typical for all immunoassays table 1 and some relate. The problem of phantom hcg was identified in the early 1970s when classic competitive immunoassays were in use. Heterophile antibodies may be present in all patients,11,12 however the potential for immunoassay interference from heterophile antibodies is less than for. With immunoassays pursuing lower detection limits, multiplexing analytes, and higher sample. Incubation of serum containing heterophilic antibodies and a murine monoclonal antibody to human choriogonadotropin hcg leads to formation of a series of soluble immune complexes. Ec50 of reagents used in blocking heterophilic interference. The patient sample is incubated with the bead during the first cycle at which time free psa in catalog number. Endogenous heterophile and human antianimal antibodies. Crossreactivity problems are well known to immunoassay kit manufacturers and the package inserts of each immunoassay include known crossreactants.
Problems with hama, heterophilic antibodies, and rheumatoid factors have clinical. Interference in immunoassays to support therapeutic antibody. Even igm lambda antibody to escherichia coli can produce false positive test results with determination of various tumor markers as well as troponin i. Some types of immunoassay interference are similar to interferences in other chemistry assays and some types are unique to immunoassays. Incubation of serum containing heterophilic antibodies and a murine monoclonal antibody to human choriogonadotropin hcg leads to formation of a ser. Heterophilic antibodies remain a problem for the immunoassay laboratory. Jun 14, 2018 varying results upon repeated testing are sometimes observed in samples with heterophilic antibodies, and may even increase the suspicion of interference. Bioanalytical interferences in immunoassays for antibody. Final patient values are reported as nanograms of precipitable. Stuart we verified that antibody binding substances in serum that. Interference of heterophilic antibodies was considered. Prevalence of interfering antibodies in dogs and cats. On the other hand, thyroid hormone autoantibodies directed against t3 and t4 are not common 8, 12and they cause interference only to the t4 and t3 measurements. The interfering substance interferent in the sample may be exogenous to the patient, for example a drug, or endogenous, for example antibodies produced by the patient.
Heterophile antibody an overview sciencedirect topics. An overview on interference in clinical immunoassays. Heterophilic and human antianimal antibodies are well known as a potential cause of interference in immunoassays. Heterophile antibodies are antibodies produced against poorly defined antigens. Issues of interferences in clinical chemistry tests including heterophilic. Heterophile antibody interference with thyroid assay. Artificial prostatespecific antigen persistence after. False diagnosis and needless therapy of presumed malignant.
There was no single assay that gave a normal concentration of prl for all. Human antianimal antibodies that develop as a result of treatments with animal immunoglobulins are antibodies with strong avidities, produced against welldefined antigens. This positive and negative interference was attributed to the presence of low affinity, polyspecific, natural heterophilic antibodies in serum and plasma samples 2, 3. Interference of heterophilic and other antibodies in.
For example, in rheumatic fever, antibodies against group a streptococcal cell walls can also react with and thus damage human heart tissues. Heterophilic iga, igg and igm antibodies have all been demonstrated previously 4 and rheumatoid factors of all these immunoglobulin classes are known to exist in people 56, 57. Serum amh antimullerian hormone levels, as detected by immunoassay kits, have been recognized as a useful diagnostic and prognostic tool in reproductive medicine because they are a reliable marker of the ovarian follicular status and a predictor of the. These antibodies exhibit a potential for interference with immunoassays. Heterophile antibodies consist of natural antibodies and. All assays used to characterize the sera were manual 3step methods. Negativeinterference in cardiac troponin i immunoassays by circulating troponin. However, for hcg testing a positive test result in serum but negative test result betahcg in urine may indicate interference in the serum hcg assay including interference due to the presence of heterophilic antibodies. Defining characteristics of heterophilic antibodies. We have developed a multiplexed fluorescent microsphere immunoassay for the. Prostate biopsy is required for the diagnosis of prostate cancer, antibody specific for free uncomplexed psa.
Heterophile antibodies are antibodies induced by external antigens heterophile antigens some crossreact with selfantigens. Interference in an immunoassay, whether enzymelinked. Almost all polyclonal antibodies used in immunoassay reactions are of the. Antistreptavidin interferences in roche thyroid immunoassays. Howe ver, crossreactivity with drugs and their metabolites is still a problem for the measurements of steroids which have an identical structure. Immunoassays are assays that use one or more antibodies as reagents. Pdf interference by human antimouse antibody in two. Interference of heterophilic and other antibodies in measurement of. As twosite immunoassays are widely used, this type of interference is the most commonly. As more attention was directed to this issue and with advances in immunodetection technology, it became apparent that igg was the predominant heterophile antibody isotype 4. Pdf heterophilic antibodies remain a problem for the. Immunoassays highly specific in vitro tests that use antigen antibody reaction to detect extremely low concentrations of a broad range of biologically important substances in blood and other body fluids antigen antibody reaction known since the end of the 19 th ct, precipitation in gel, agglutination or turbidimetry assays gradually developed. Studies with goat and rabbit antimouse antibody, as models of human antimouse antibody hama, have shown that several of the commonly used two site immunoassays e. However, in the presence of circulating interfering antibody endogenous, e.
Recent findings indicate that many endogenous antibodies exhibit multispecificity. Heterophilic antibodies can be present in the serum of patients and may interfere with the animal antibodies used in immunoassay kits. The most frequently characterized interference comes from endogenous human antimouse antibodies hama, which can react with the mouse monoclonal antibodies utilized in the assay. Pdf we verified that antibodybinding substances in serum that interfere in twosite immunoassays involving. Pdf the influence of naturally occurring heterophilic anti. Falsely elevated serum antimullerian hormone level in a. Features of heterophilic antibody interference in immunoassays include. Heterophile antibodies are a wellrecognized cause of interference in immunoassays 4, 12, 14, 19 and are present in 5 to 40% of normal blood donors 7, 8, 11. Heterophile antibodies interfere with immunoassays by a noncompetitive mechanism.
Thus, heterophilic antibodies are absent in the protein free ultrafiltrate, and monitoring free drug concentration also eliminates this interference. Among the most commonly encountered heterophile antibodies are human anti mouse antibodies hama and rheumatoid factor rf, a type of heterophilic antibody w i th nonspeci fic a nity or he. Avoiding inappropriate clinical decisions based on false. However, because the animal antibodies are used in different amounts and with other reagents in each assay system, a person with heterophilic antibodies will not always have an unusual or. The investigation of interferences in immunoassay core. Characterization of canine antimouse antibodies highlights. Reducing immunoassay interferences through innovative. This antibody is commonly a polyclonal igg of mouse, sheep, rabbit, or goat origin. These antibodies interfered with radioimmunoassays using goat antibodies. Interference is also a problem for thyroglobulin tg assays with endogenous. New onset of heterophilic antibody interference in.
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