An ELISA avoiding interference by heterophilic antibodies in the measurement of components of the plasminogen activation system in blood

Endogenous heterophilic antibodies in blood are known to interfere with two-site enzyme-linked immunosorbent assays (ELISAs) evoking false positive signals. In the present study, we describe an assay for the assessment of components of the plasminogen activation system (uPA, tPA and PAI-1, and their complexes) in blood which is not susceptible to interference by heterophilic antibodies. In the ELISA format, two avian (duck, chicken) antibodies are employed in the pre-analyte and two mammalian (rabbit, goat) antibodies in the post-analyte stage. The assay is compared to our earlier reported ELISA for measuring uPA, tPA and PAI-1 components in tumor tissue extracts. Applying the so-called "nonsense formats", designed against non-existent components, to the NIBSC reference preparation of rheumatoid factor (RF), no response was found with the new assay, whereas a clear RF dose-dependent interfering signal was observed with the original assay designed for tumor tissue extracts. Analysis of tumor-tissue based international reference preparations (RBG EORTC 101094 and 040297), human anti-mouse antibodies (HAMA) containing sera, and sera from patients with rheumatoid arthritis (RA), also displayed no false positive signals. In conclusion, we have developed an ELISA that permits the determination of blood levels of components in the urokinase system, free from disturbance by endogenous heterophilic antibodies.     

Monoclonal antibody two-site ELISA for human IFN-gamma. Adaptation for determinations in human serum or plasma

Mouse monoclonal antibodies (mAbs) against human interferon-gamma (IFN-gamma) were produced after immunization with recombinant IFN-gamma. Two mAbs (1-D1K and 7-B6-1) recognizing distinct epitopes on natural IFN-gamma were selected for the development of a two-site ELISA. The sensitivity was similar for IFN-gamma diluted in PBS with 1% bovine albumin, spent culture medium or fetal calf serum but reduced to approximately 50% when diluted in normal human serum. Individual normal human sera were tested and three of 14 gave false reactivities in the ELISA. One serum factor with major impact on the individual variation and the decreased sensitivity could be adsorbed to and eluted from protein A-Sepharose. Based on these observations we established a new ELISA protocol which made it possible to test for low levels of IFN-gamma in human serum and plasma samples. The modifications in this protocol are easy to apply with basic laboratory equipment.     

An ELISA method for quantification of Tamm-Horsfall protein using monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MoAbs) to Tamm-Horsfall protein (THP) were established. The isotype and reaction pattern of the MoAbs with THP from rat, rabbit, guinea pig and man were employed for the selection of clones. At least four epitopes were recognised on human THP. One of these epitopes differed from the others in its dependence on the state of aggregation of the THP. An ELISA procedure was developed for quantification of THP in urine requiring no other sample treatment than dilution in the assay buffer. In this ELISA, THP showed an increased immunoreactivity after freezing.     

Raised tryptase without anaphylaxis or mastocytosis: heterophilic antibody interference in the serum tryptase assay

Mast cell tryptase (MCT) is a key diagnostic test for mastocytosis and anaphylaxis. High serum tryptase levels are also one of the risk factors for adverse reaction in venom immunotherapy, yet occasional patients are seen with raised levels in the absence of either diagnosis. False positive results can be due to assay interference by heterophilic antibodies such as rheumatoid factor (RF) and human anti-mouse antibodies (HAMA). We therefore investigated heterophilic antibody interference by rheumatoid factor activity and HAMA as a cause of raised MCT results in the Phadia tryptase assay. Serum samples from 83 patients were assayed for MCT and rheumatoid factor before and after the use of heterophilic antibody blocking tubes (HBT). Samples with more than 17% reduction in MCT with detectable RF were then assayed for HAMA. Fourteen (17%) of the 83 samples with positive RF showed a >17% decrease in mast cell tryptase after HBT blocking. Post-HBT, eight of 14 (57%) reverted from elevated to normal range values with falls of up to 98%. RF levels were also decreased significantly (up to 75%). Only one of the 83 tested was apparently affected by HAMA in the absence of detectable IgM RF. In conclusion, any suspicious MCT result should be checked for heterophilic antibodies to evaluate possible interference. False positive MCT levels can be caused by rheumatoid factor. We suggest a strategy for identifying assay interference, and show that it is essential to incorporate this caveat into guidance for interpretation of MCT results.     

ELISA for specific anti-toxoplasma IgM antibodies: aspects related to serum interference

Two different methods were used to prepare solid-phase antigen (Ag) from soluble extracts of tachyzoites of Toxoplasma gondii: (A) physical adsorption on polystyrene beads; and (B) formaldehyde fixation of Ag previously dried in microtitration wells. In both cases a horseradish peroxidase conjugate with anti-IgM IgG was used as tracer. The assay scheme consisted of sequential incubations of diluted serum samples and tracer solution (1 or 2 h, 37 degrees C), colour development in the presence of substrate (10 min at room temperature), addition of H2SO4, and absorbance reading at 492 nm. In procedure A no cut-off value for positives could be determined owing to a large overlap between positive and negative sera. The extent of overlap directly correlated with the total IgM content of samples. With negative sera similar values were obtained with sensitized and untreated beads: thus a correction could be made by directly subtracting absorbance values determined in parallel runs with uncoated beads. Results with negative sera correlated with total IgM concentration in procedure B also, but much less variability of blank values allowed negative and positive sera to be effectively discriminated. A series of reference positive and negative sera was correctly classified by both procedures A and B. However, the latter appeared preferable, as not requiring blank correction.     

A two-site monoclonal antibody ELISA for the quantification of group V allergens in grass extracts

A two-site solid-phase enzymimmunoassay was used for the quantification of group V allergen in grass pollen extracts in mass units. The assay is based on the monoclonal antibodies (MoAbs), 1D11 and 3B2 which recognize different epitopes on the standard Phl p V. The MoAb-ELISA is very sensitive (15 100ng ml Phl p V)and highly specific for group V allergens. Six pollen extracts of different grasses demonstrated parallel binding curves. The group V content ranged between 73 and 673 μg/ml in the extracts. The International Standard of Phleum pratense (IS 82 520) contains 400 μg/nil Phl p V. A good correlation was observed between the group V content and RAST inhibition, with the exception of Poa pratensis.     

Utilization of an ELISA technic for the quantification of antipoliovirus antibodies in human sera

A double antibody enzyme linked immunosorbent assay (ELISA) was elaborated for detection of poliovirus antibodies in human sera. The IgG to be titrated were immunoabsorbed by capture on the solid phase. The antigens used were obtained from vero cell cultures (green Monkey Kidney Cells). The reaction was followed by adding rabbit antipoliovirus serum, then sheep Fab fragment prepared against rabbit IgG and labelled with horse radish peroxidase. Ortho-tolidine was used as the chromogen substrate to reveal the reaction. This enabled a first reading with the naked eye. This technique allows to keep a better track of the poliomyelitis immunization.     

Chicken antibodies: a tool to avoid interference by human anti-mouse antibodies in ELISA after in vivo treatment with murine monoclonal antibodies.

Human anti-murine antibodies (HAMA) can be found in serum of many patients who have received murine monoclonal antibodies for diagnosis or therapy. These antibodies are known to give false positive results in sandwich-type assays (e.g. ELISA or RIA). This interference problem will increase in the future as more patients are treated with murine monoclonal antibodies in vivo. HAMA can also be found in sera from patients that has not been treated with monoclonal antibodies. In this work we have studied the interference of HAMA in sandwich ELISAs containing antibodies from different species. HAMA, present in the sample, may react both with the capture antibody and the detection antibody in these assays to give a false positive reaction. HAMA did not react with chicken IgG, and if one of (or both) the capture and detection antibody was of avian origin, the interference of HAMA in sandwich assays could be avoided.     

An ELISA method for the quantification of anti-streptolysin-O antibodies

This paper describes a new assay, based on the ELISA technique, for the quantification of antibodies to streptolysin-O (ASLO). We have compared its performances with that of a standard method (inhibition of hemolysis). Using a panel of 137 sera covering the whole range of ASLO titers, the results showed a good correlation between both methods but the ELISA method was more reproducible than the standard technique, thus represents a convenient alternative for the quantification of ASLO.     

Measuring human IgG4 by a two-site immunoradiometric assay with monoclonal antibodies

By using 2 monoclonal antibodies, we developed a solid-phase 2-site immunoradiometric assay for measuring human IgG4. The measuring range (0.05-20 micrograms/ml) covered more than 2 orders of magnitude. The sensitivity level should make this assay especially useful when IgG4 concentrations are too low to be detected by conventional methods.     

Studies on HBsAg binding with polymerised human serum albumin by ELISA

A simple and sensitive ELISA was developed to characterize the interaction between polymerised human serum albumin (pHSA) and HBsAg, using pHSA-coated polyvinylmicrotitre plates as solid phase and anti-HBs-coupled HRPO as the conjugate. The interaction was found to be specific and dependent on the size of albumin polymer. pHSA-binding activity (pHSA-BA) was studied in both HBsAg-negative and HBsAg-positive sera from various liver diseases including acute viral hepatitis, fulminant hepatitis, cirrhosis of liver, chronic active hepatitis, and healthy HBsAg carriers. pHSA-BA was detected only in HBsAg-positive sera. Analysis of HBsAg-positive sera indicated pHSA-BA in high proportions of patients sera as compared to sera from healthy HBsAg carriers. pHSA-BA was detected both in the presence and absence of HBe markers, though the mean BA was relatively high in presence of HBeAg. The effect of human serum immunoglobulins (IgG, IgA, and IgM) on the BA was investigated and a correlation between pHSA-BA and HBsAg-IgM complex positivity in sera was established. Finally, the probable role of human serum IgM in facilitating the binding process was discussed.     

An improved ELISA for anti-native DNA by elimination of interference by anti-histone antibodies

We evaluated an enzyme-linked immunosorbent assay (ELISA) for antibodies to native DNA (nDNA) in which protamine was used to link DNA to polystyrene. Elevated anti-nDNA was largely restricted to patients with systemic lupus erythematosus (SLE), and within this group good correlation between ELISA and the ammonium sulfate assay was obtained. However, substantial background immunoglobulin binding to protamine coated wells was commonly observed, and it was necessary to subtract this activity from each anti-DNA determination. Many of the SLE sera also contained anti-histone antibodies, and this antibody activity showed significant correlation with the binding to protamine. In contrast, methylated bovine serum albumin (mBSA) did not bind anti-histone antibodies and provided a substrate for coupling nDNA to polystyrene. This modified ELISA allowed the quantitation of antibodies to native DNA without the simultaneous binding of anti-histone antibodies.     

Development and evaluation of a new ELISA for the detection and quantification of antierythropoietin antibodies in human sera

Assays for the analysis of antierythropoietin antibodies (anti-EPO Abs) currently suffer from a high degree of nonspecificity or are cumbersome and time consuming to perform. They are therefore not well suited for the analysis of large numbers of human sera samples, a task that has become increasingly important due to an increase in the number of patients developing anti-EPO Abs. The objective of this study was to develop and validate a sensitive and specific ELISA for the determination of anti-EPO Abs that would suit these purposes. In this new double antigen bridging ELISA, anti-EPO Abs bind via one site to recombinant human erythropoietin (rhEPO)-biotin immobilized to streptavidin-coated microtiter plates (MTPs) and by a second site to rhEPO labelled with digoxigenin (DIG). The amount of bound antibody is determined using an anti-DIG antibody coupled to peroxidase. A rabbit polyclonal anti-EPO Ab purified by immunoadsorption is used as reference antibody preparation. The dynamic range of this ELISA was 1-75 ng/ml per assay calibrated with the reference antibody preparation. The assay was specific for anti-EPO Abs and did not react with other immunoglobulins (Ig) present in human serum. The lower limit of detection (LLD) of the assay was 0.5 ng/ml, and the lower limit of quantitation (LLQ) was 1.0 ng/ml. Anti-EPO Abs could be detected in the sera of pure red cell aplasia (PRCA) patients. In contrast to previous reports, no anti-EPO Abs could be detected in the sera of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome (SS), or in the sera of dialysis patients.     

Double-antigen sandwich ELISA for detection of antibodies to SARS-associated coronavirus in human serum

The study presented here was conducted to evaluate the performance of a double-antigen sandwich ELISA to detect antibodies in human serum against the coronavirus associated with severe acute respiratory syndrome (SARS). A recombinant partial nucleocapsid protein of SARS-associated coronavirus was used as a serodiagnostic antigen in the ELISA. A total of 2892 clinical serum samples were tested with the ELISA kit, which positively identified 25 of 35 (71.4%) samples of patients with confirmed SARS infection, 286 of 407 (70%) samples of patients suspected of having SARS, 229 of 302 (75.8%) samples of convalescent SARS patients, and 0 of 544 samples obtained from healthcare workers; only 1 of 1604 clinical samples obtained from patients with other diseases demonstrated a weakly positive result. These results indicate that the double-antigen sandwich ELISA is an effective screening method for the serodiagnosis of SARS-associated coronavirus.     

Measurement of squirrel monkey serum IgG levels by a two-site sandwich radioimmunoassay with monoclonal antibodies

Monoclonal antibodies against the squirrel monkey Saimiri sciureus IgG have been produced for a more specific analysis of the antibody-related immunological aspects in experimental human or monkey malaria. Two monoclonal antibodies, 3D8/D5 and 3F11/G10, out of 64 reacted with distinct epitopes on the IgG present throughout the complete population without interfering with each other. The 2 monoclonal antibodies were used to develop a highly specific, reliable and sensitive two-site sandwich radioimmunoassay for the measurement of the serum IgG levels in 83 animals. The antibodies also allowed us to produce by a simple immunoabsorbent technique a highly purified IgG standard easy to calibrate and store. The assay permits the detection of IgG levels as low as 0.48 ng/ml. The standard curve is linear between 3.9 and 125 ng protein/ml and allows by a simple mathematical equation an accurate measurement of the serum IgG levels.     

The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs

Sandwich ELISAs have become a widely used method for the quantitative detection of serum proteins. However, they can be biased by a variety of interfering substances. As reported recently, we observed false-positive levels of interferon (IFN)-alpha and -beta in up to 27% of sera from healthy blood donors using commercial ELISAs. We now demonstrate that two different groups of naturally occurring heterophilic antibodies (IgG-type) are responsible for these titers. Group I (representing 85% of positive samples) binds to the Fab region of IgG from goat, mouse, rat, horse, and bovidae (but not rabbit). Group II (15%) recognizes an epitope in the Fc region of mouse, horse, bovine, and rabbit (but not goat or rat) immunoglobulins. The antibodies did not crossreact with human IgG subclasses but contributed to false-positive IgG rheumatoid factor levels obtained using a commercially available ELISA. To investigate the susceptibility of assays to these artifacts, various combinations of capture and detection antibodies have been tested. On this basis, we defined the relative risks that standard ELISAs might be influenced by heterophilic anti-immunoglobulin antibodies. In general, assays that use monoclonal antibodies for both capture and detection are less susceptible than others which include at least one polyclonal antiserum. However, only systems utilizing rabbit F(ab')(2) fragments have been found to be immune to this interference.     

Optimization of a competitive ELISA with polyclonal antibodies for quantification of prolamins in foods

The optimization of a sequential competitive ELISA for the quantification of prolamins in foods is described in this article. The assay was developed using polyclonal antibodies obtained by the hyper‐immunization of rabbits with commercial gliadin. The ELISA developed in this way showed a very high degree of detectability (detection limit, 1 ng ml^‐1), as well as the ability to discriminate between prolamins harmful to coeliac individuals from non‐toxic prolamins. The influence of the solvent used for extraction of the samples on the detection capability of the test was also studied. The assay proved to be useful for the evaluation of gliadins in processed foods including meat products. The assay was applied to many types of foods and was compared with a commercial kit approved by the Association of Official Analytical Chemists.     

Chicken antibodies: a tool to avoid interference by complement activation in ELISA.

MicroELISA plates coated with mammalian IgG will activate the human complement system. It has been shown that this activation of the complement system may interfere in solid-phase immunometric assays, and that there is a difference between IgG from different species and between different IgG subclasses in their ability to activate the human complement system. We have studied the ability of mammalian IgG and avian IgG to activate the human complement system. We show that chicken IgG do not activate the human complement system, and chicken IgG can thus be used in solid-phase immunometric assays to reduce interference by complement activation.     

A recombinant 70K protein ELISA. Screening for antibodies against U1snRNP proteins in human sera.

Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of U1snRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic disease were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNase ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNase but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-U1snRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-U1snRNP antibody screening may not be appropriate, because antibodies against other frequently occurring U1snRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.     

Species-dependent serum interference in a sandwich ELISA for Apo2L/TRAIL

To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different. In order to resolve the observed serum effect, studies were performed in which salts, detergents, and blocking proteins were added to the sample diluent, and optimized sample diluents that eliminated serum interference were developed for mouse, cynomolgus monkey, and chimpanzee serum. These buffers consisted of a base assay diluent (PBS/0.5% BSA/0.05% Tween-20/10 ppm ProClin 300) supplemented with: NaCl (mouse serum); NaCl, EDTA, CHAPS, bovine gamma globulin (BGG), and human IgG (cynomolgus monkey serum); and NaCl and EDTA (chimpanzee serum). Full characterization studies were performed for the "buffer" ELISA run in base assay diluent (intended for non-serum samples) as well as the assays optimized for mouse serum and cynomolgus monkey serum. Precision, accuracy, linearity, and specificity were found to be satisfactory. With the availability of a rabbit polyclonal antibody against Apo2L/TRAIL, a new pAb/mAb ELISA was developed. This assay was not only more sensitive by > or =6-fold, but it was also much less subject to serum interference.