Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA)

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.     

Increased heterogeneity of serum thyroglobulin in thyroid cancer patients as determined by monoclonal antibodies

Summary We investigated the immunological heterogeneity of plasma Tg in thyroid cancer patients using monoclonal antibodies in an immunoradiometric assay and a conventional RIA system with a polyclonal rabbit antibody. The results were compared with measurements of plasma Tg in patients with nonmalignant disease. We can demonstrate an increased immunological heterogeneity in tumor patients compared with patients with non-malignant thyroid diseases. In one case the Tg value measured by a monoclonal test system exceeded the value obtained by a polyclonal RIA system in the same sample by a factor of 25. It has to be further investigated whether this increase in heterogeneity is of diagnostic value in the follow-up of thyroid cancer patients.Abbreviations IRMA 11, IRMA 13 Immunoradiometric assay using mabTg 11 or mabTg 13 - mabTg monoclonal antithyroglobulin antibodies - Tg Thyroglobulin     

Development of an ABO-ELISA for the quantitation of human blood group anti-A and anti-B IgM and IgG antibodies

An indirect ELISA system was designed for quantitation of human blood group A and B IgM and IgG antibodies. The capturing antigens are blood group substance A or B used to sensitize polystyrol microtiter plates. Bound anti-A or anti-B antibodies are revealed either directly, by development with polyclonal anti-human immunoglobulin class-specific conjugate or with more avid mouse monoclonal anti-human isotype antibodies revealed in turn by goat anti-mouse conjugate. Reproducibly, 100 ng specific anti-A IgG provided for a significant above-background signal of 0.2 at OD405 and 15 serum samples had a mean content of 3.98 +/- 8.74 micrograms (mean +/- 2 SD) (range: 0.305-12.62) of specific anti-A IgG/g total IgG. Thus one molecule specific anti-A IgG is found per 7.9 X 10(4)-3.2 X 10(6) total IgG molecules. Statistical correlations were significant between anti-A IgG levels and agglutination titer (P less than 0.05) but non-significant when the specific anti-A IgG levels of individual serum samples were compared to their total IgG content (P greater than 0.05). Dose-response signals were similar for anti-A and anti-B IgM antibodies. Reproducibility of the assay was excellent. Specificity was ascertained by various approaches involving development of primary antibodies with heterospecific antibody conjugate and adsorption of primary antibody from serum using A and B group erythrocytes or soluble A and B substances. Separation of IgM from IgG anti-A antibodies over sizing gel resulted in fractions that were immunosorbed by mouse monoclonal anti-human IgM and IgG respectively but not vice versa.     

Detection of carcinoembryonic antigen and related antigens in sera of patients with gastrointestinal tumors using monoclonal antibodies in double-determinant radioimmunoassays

Of 14 monoclonal antibodies produced in six different laboratories, 13 bound to purified preparations of carcinoembryonic antigen (CEA). All antibodies reacted to spent medium of colorectal carcinoma cell lines. Competitive binding studies indicated that 12 different antigenic determinants representing six different groups were detected on the CEA molecule(s). Six antibodies were used in double determinant radioimmunoassays (RIA) to detect CEA and CEA-related antigens in sera of 311 patients with various gastrointestinal diseases and of normal donors. None of up to 115 sera of healthy donors had elevated antigen levels with four out of the six monoclonal antibodies tested, whereas up to 9% of sera showed elevated antigen levels when tested with two antibodies. Between 1.4% and 4.4% of sera from patients with inflammatory and benign neoplastic diseases of the gastrointestinal tract were positive. Antigen levels were elevated in 56 to 75% (depending on antibody used) of sera from patients with advanced gastrointestinal tumors. These preliminary results indicate that double-determinant immunoassays with a panel of monoclonal antibodies might improve conventional CEA assays by reducing the number of false positive sera detected by polyclonal sera in patients with benign inflammatory bowel diseases.     

Rapid biotin-avidin method for quantitation of antiviral antibody isotypes

A rapid and efficient method is described for isotype quantitation of antiviral antibodies in mice infected with Theiler's murine encephalomyelitis virus (TMEV). Serum antibodies were reacted with fluorochrome-labeled TMEV in a modified fluid-phase particle concentration fluorescence immunoassay (PCFIA). Biotin and avidin were used to attach anti-immunoglobulin isotype antibodies to polystyrene particles by the separate incubation of biotinylated goat anti-mouse isotypes (IgG1-, IgG2a-, IgG2b-, IgG3-, or IgM-specific) with avidin coupled polystyrene particles. These anti-isotype particles captured the virus-antibody complexes. Mouse myeloma proteins were used to quantitate and standardize isotype profiles of normal mouse serum using fluorescein isothiocyanate (FITC)-labeled, goat anti-mouse isotypes and polystyrene particles coated with goat anti-mouse. These assays quantitated the affinity-purified mouse serum antiviral antibodies for the standardization of antiviral isotype assays. Immunoglobulin of all serum isotypes as well as the amount of virus-specific isotypes can be quantitated rapidly and accurately.     

Production of monoclonal antibody SP-21 to colon-ovarian tumor antigen, COTA

An IgG monoclonal antibody, SP-21, directed against colon-ovarian tumor antigen, COTA, is reported. The antibody had no reactivity with CEA, normal colonic mucin, CSAp, ABO blood group antigens, or with normal human lung, liver, spleen, kidney, plasma and saliva in studies using the enzyme-linked immunoassay method (ELISA). Immunoperoxidase staining of colon, lung, kidney, and prostate cancer tissues and benign and inflammatory colon disease tissues revealed a specificity identical to that of the polyclonal (goat) anti-COTA antibodies.     

An antibody chimera technique applied to enzyme immunoassay for human alpha-1-fetoprotein with monoclonal and polyclonal antibodies

A 2-step enzyme immunoassay (EIA) for human alpha-1-fetoprotein (AFP) is proposed, which uses covalently coupled anti-AFP IgG and anti-horseradish peroxidase (HRP) IgG (antibody chimera) binding HRP as the marker enzyme immunologically. The use of polyclonal and monoclonal anti-AFP linked to anti-HRP antibodies was compared with a conventional 2-site binding EIA with HRP covalently bound to anti-AFP IgG. The sensitivity of the conventional EIA is increased by the use of an antibody chimera comprising a molar ratio of anti-AFP IgG: anti-HRP IgG of 1:8, especially if monoclonal antibodies are employed. This improved sensitivity may be achieved by a very simple coupling procedure without purification of conjugate and with very crude HRP preparations.     

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.     

Altered antigenicity of human monoclonal antibodies derived from human-mouse heterohybridomas

We have generated milligram quantities of human monoclonal antibodies (Hu-MAbs) in the ascites of pristane-primed nude mice injected with human-mouse heterohybridomas. After contaminating mouse immunoglobulins were removed by affinity chromatography, an enzyme immunosorbent assay (EIA) was used to measure the concentrations of human immunoglobulins. Ten different partially purified preparations were tested. The titration curves with all 5 IgG Hu-MAbs were unusual, reaching a plateau at a very low apparent maximum concentration of antibody. In contrast, the EIA yielded more usual titration curves and thus apparently more reliable estimates of the concentrations of 4 IgM and 1 IgA monoclonal antibodies. An analogous EIA for the quantitation of mouse IgG monoclonal antibodies also gave accurate estimates. To understand the nature of the discrepancy with human IgG, 5 Hu-MAbs of the 3 classes (2 IgG, 2 pentameric IgM and 1 IgA) were purified to homogeneity for a more detailed analysis. The inability to quantitate the human IgG monoclonal antibodies by EIA was not due to defective molecules, as shown by SDS polyacrylamide gel electrophoresis. The human IgG monoclonal antibodies were found to consist of intact heavy and light chains, as were the IgM and IgA antibodies. The possibility that the human IgG monoclonal antibodies differed antigenically from polyclonal IgG was explored by comparing the concentrations by EIA with the protein concentrations determined by absorbance at 280 nm. This analysis permitted a comparison of the detectability of antigenic determinants on Hu-MAbs with those on polyclonal Ig with goat antibodies to Ig or Ig subclass. The IgG monoclonal antibodies differed from polyclonal IgG in both their heavy and light chains. Goat antiserum monospecific for the gamma chain in fact underestimated the concentration by as much as one hundred-fold. IgM and IgA monoclonal antibodies were less antigenically distinct from their polyclonal counterparts even though their light chains were also underestimated, because goat monospecific antibodies were more efficient at recognizing their heavy chains. The molecular basis for the observed difference in antigenicity is not yet known. These findings have important implications for the analysis of the binding of IgG Hu-MAbs. A direct binding assay with the label directly conjugated to the Hu-MAb should be used in preference to an indirect assay with a labeled detecting antibody to maximize the sensitivity of the assay. The altered antigenicity of IgG Hu-MAbs may also imply decreased immunogenicity when they are given in vivo as carriers for radionuclides or cytotoxic antitumor materials.     

Characterization of a monoclonal antibody that binds to apolipoprotein E and to lipoprotein of human plasma containing apo E. Applications to ELISA quantification of plasma apo E

This study describes the development of an enzyme-linked immunosorbent assay for human apolipoprotein E (apo E). A mouse monoclonal IgG1 antibody named E01 against apolipoprotein E was selected from five antibodies secreted by hybridomas. This antibody had a high affinity for apo E [K = 1.2 x 10(7) L.M-1 for purified apo E and K = 1.05 x 10(7) L.M-1 for native apo E in very low density lipoproteins) in liquid phase and recognized every isoform of apo E but not other proteins in VLDL. Competition experiments with 125I apo E showed that its binding affinity for the E in every density class (VLDL, HDL, LDL) and in serum was the same. This antibody was used for the quantification of human apo E in serum by enzyme linked immunoassay. E01 was coated on microtiter plates and a polyclonal peroxidase-conjugate was used as second antibody. A good correlation was observed between the values obtained for apo E using both monoclonal and polyclonal antibodies.     

TNT detection using llama antibodies and a two-step competitive fluid array immunoassay

Llamas possess unique subclasses of antibodies that lack light chains, and thus are made by the pairing of two heavy chains. IgG was purified from two llamas which had been immunized with trinitrobenzene-keyhole limpet hemocyanin. Conventional IgG1 and heavy chain IgG2 and IgG3 subclasses were fractionated using affinity chromatography. The effectiveness of heavy chain antibodies for the detection of trinitrotoluene (TNT) using a competitive fluid array immunoassay was evaluated and compared to both the llama IgG1 as well as a murine monoclonal anti-TNT antibody. It was found that heavy chain antibody bound TNT with selectivity similar to conventional antibodies, yet the heavy chain antibodies possessed greater thermal stability. The titer of the heavy chain antibodies however was found to be 10-fold lower than the IgG1; thus analytical assays were best demonstrated using the llama IgG1 conventional antibody. The TNT competitive immunoassay on the Luminex fluid analyzer had a dynamic range from ∼ 100 ng/mL to 10 μg/mL. Utilizing the same two-step competitive assay format the dynamic range of the monoclonal antibody was found to have a broad range (1 ng/mL to 1 μg/mL). This method was demonstrated on TNT contaminated soil extracts using both the llama IgG1 and the mouse monoclonal validating the utility of method for analysis of field samples.     

Multiplex analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results

We hypothesized that heterophil antibodies reactive with animal proteins used in blot preparation caused nonspecific staining (NSS) on HIV Western blot (WB) studies, causing indeterminate results. We analyzed samples showing NSS on HIV WB using a multiplexed immunoassay to simultaneously measure IgG antibodies to animal IgG (bovine, goat, sheep, mouse) and bovine serum albumin. Heterophil antibodies reactive with IgG from several animal species were detected in 23 (49%) of 47 samples showing NSS on HIV WB; 15 positive samples demonstrated antibodies to all 5 antigens. Similar IgG heterophil antibodies were detected in only 2 (8%) of 24 control samples. Of the HIV WB samples with a positive HIV-1 enzyme-linked immunosorbent assay (ELISA) result at the time of WB testing (11/47), heterophil antibodies were found in 8 (73%) of 11. Preabsorption with bovine, goat, and sheep IgG removed heterophil antibodies detected by the multiplexed assay and, in some cases, eliminated reactivity in ELISA and WB testing. Heterophil antibodies are associated with indeterminate HIV immunoassay results and are an important cause of false-positive HIV ELISA results. Multiplexed immunoassays provide a powerful tool for screening patients for heterophil antibodies and resolving possible false-positive results.     

A new liquid-phase enzyme immunoassay for rabbit IgM antibodies and its application for comparing the specificity of IgM and IgG antibodies contained in the same antiserum

A new liquid-phase enzyme immunoassay (EIA) has been developed to compare the specificities of IgM and IgG antibodies when they are both present in the same serum sample and directed towards a simple hapten. The hapten viomycin (VM) was used as the model antigen and antibodies to VM were raised in rabbits. In the immunoassay VM, labelled with the enzyme galactosidase as marker (VM-GAL), was mixed with rabbit anti-VM serum. First IgM anti-VM antibodies bound to VM-GAL were precipitated with a guinea pig anti-rabbit IgM serum and galactosidase activity was measured in the precipitate. Then IgG anti-VM antibodies bound to VM-GAL were precipitated from the supernatant with a goat anti-rabbit IgG serum and enzyme activity was measured in this precipitate. The guinea pig anti-rabbit IgM and goat anti-rabbit IgG were specific for IgM and IgG respectively and did not appear to cross-react. Nine analogues of VM were used as inhibitors in this immunoassay to compare the specificities of IgM and IgG antibodies for determinants on VM. The results suggest that recognition of the fine structure of VM by IgM is less strict than recognition by IgG.     

Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies

A rapid ELISA method has been developed to quantitate myoglobin in serum. An antigen capture enzyme-linked immunoassay with IgG1 mouse monoclonal antibodies produced by cell clones MGB20-4A1.1 and MGB20-3C1.2 were used for myoglobin detection. These monoclonal antibodies are specific for different epitopes of the myoglobin molecule. Monoclonal antibodies from the hybridoma clone MGB20-4A1.1 were adsorbed to microtiter plate wells. The plates were washed with PBS containing 0.05% Tween 20 and then 20 microliter of standard serum or serum of patients and 200 microliter of peroxidase labeled monoclonal antibodies MGB20-3C1.2 were added to each well. Plates were incubated for 90 min at 37 degrees C and enzyme activity was determined using o-phenylenediamine as a substrate. The ELISA assay described is a rapid and sensitive procedure to assess the quantity of myoglobin within the range 2-1000 ng/ml serum. 120 samples can be tested in 3 h.     

Evaluation of a commercial anti-delta EIA kit for detection of antibodies to hepatitis delta virus

Anti-hepatitis delta virus (anti-HDV) antibodies were measured by solid phase IgG and IgM capture radioimmunoassays (RIA) as well as by a competitive binding enzyme immunoassay (EIA) in both acute and chronic HDV infections. EIA anti-delta test measures total delta antibody without discriminating IgM from IgG anti-delta. Low titer IgG antibodies were detected by both techniques with equal sensitivity. High titer IgG antibodies reached the end point sooner with EIA than with RIA (10(-3) versus greater than 10(-6)). When IgM anti-HDV was present without accompanying IgG anti-HDV, EIA failed to identify the antibody. Presence of high titer rheumatoid factor in the serum and lipemic samples produced false-positive results by EIA. Usage of undiluted serum samples for EIA probably exaggerates the factors contributing to false-positive reaction.     

Human interleukin 2. Quantitation by a sensitive radioimmunoassay

Using polyclonal and monoclonal antibodies to human recombinant IL-2 (rIL-2), we developed a sensitive radioimmunoassay (RIA) for quantitation of human IL-2. In this assay, microtitration plates pre-coated with an anti-rIL-2 monoclonal antibody (35H10), recognizing residues 59-72 of human IL-2, are incubated with serial dilutions of test samples. Captured IL-2 is quantitated by adding an affinity-purified rabbit anti-rIL-2 antibody followed by an 125I-labeled goat anti-rabbit IgG. Antibodies to chemically synthesized IL-2 peptides could replace the polyspecific rabbit anti-rIL-2 antibody as the second specific reagent in the assay. This configuration was more sensitive than others tested, approaching the level of detection of the conventional IL-2 bioassay, thus allowing detection of as little as 100-200 pg IL-2. Serum or plasma fluids, however, inhibited the assay, reducing its sensitivity by approximately 5-fold. This RIA correlated well with the conventional bioassay in measuring IL-2 levels in sera from IL-2-treated patients. This, and similar, RIAs may serve as a useful adjunct or alternative to the conventional IL-2 bioassay in detecting and quantitating human IL-2 in culture supernatants and clinical samples.     

Serum prostatic acid phosphatase (PAP): monoclonal enzyme-linked immunoassay compared to polyclonal radioimmunoassay

A new enzyme linked immunosorbent assay (ELISA) kit which utilizes a monoclonal antibody against prostatic acid phosphatase (PAP) was compared with current radioimmunoassay (RIA) methodology which uses a polyclonal antibody. Both assays are double antibody immunoassays with the major difference being the method of antibody preparation. A study of intrarun precision using control material showed an average within run coefficient of variation (CV) percent as 7.0 percent and 6.9 percent for ELISA and RIA, respectively, while between run CV percent averaged 11.1 percent and 11.5 percent, respectively. Thus, precision results compare similarly between the two assays. The specificity showed significantly different results. Patterns of correlation between the two methods indicate differing specificities of the primary antibodies. The values for ELISA were greater than RIA for control sera and patient samples when values fell outside the reference range; however, RIA values exceeded ELISA values with patient samples which fell within the reference ranges as provided by each manufacturer. Therefore, there exists a question of specificity of antibody employed in each of the two assays. The PAP antigen is prepared from two different sources for each kit. The ELISA manufacturer prepares antigen from seminal fluid and RIA manufacturer prepares antigen from normal human prostate. The question of specificity may be influenced by: (1) source of antigen used in immunizing animals and (2) monoclonal versus polyclonal means of producing antibody.     

Interference in double-determinant monoclonal antibody-based assay for carcinoembryonic antigen (CEA)

We investigated the false positive phenomena in immunoassays of carcinoembryonic antigen (CEA) using mouse monoclonal antibodies, and studied the properties of interfering substances and the method to eliminate non-specific interference. Serum samples from 2,250 patients, which indicated more than 10 ng/ml of CEA with EIA test kit (Boehringer Mannheim Yamanouchi; BMY) in about 25,000 samples of CEA tests, were subjected to extraction by heating at 70 degrees C followed by CEA measurement of the supernatant. The overall correlation was good between CEA values of the original and extraction methods, but CEA values in 55 cases were found to be remarkably higher in the non-extracted method than the extraction method. CEA titers of 20 samples of the discrepant cases were measured by various commercial kits, and the result indicated that non-specific interference was unavoidable in any kit using a monoclonal antibody-based double-determinant immunoassay, in spite of difference in degree and frequency of interference. The interfering activity of two patients serum with remarkably discrepant values eluted at the void volume of a large molecular size of more than 1,000 kDa in gel chromatography of Sephacryl S-300. The non-specific interference was reduced by addition of mouse gamma-globulin to the buffer of BMY kit and abolished by pretreatment of the serum with heat-killed cells of Staphylococcus aureus, suggesting that interference might be caused by some species of immunoglobulin being able to bind mouse gamma-globulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of methods to assess airborne rat and mouse allergen levels. I. Analysis of air samples

Airborne laboratory-animal allergens can be measured by several methods, but little is known about the effects of important differences in methodology. Therefore, methods used in research projects in The Netherlands, the UK, and Sweden were compared. Seventy-four sets of three parallel inhalable dust samples were taken by a single operator in animal facilities in the three countries, and analyzed in parallel by the three institutes for rat and mouse urinary allergen. Rat-allergen levels measured by RAST inhibition (UK) were 3000 and 1700 times higher than levels measured by enzyme immunoassay (EIA)-sandwich methods with polyclonal rabbit (The Netherlands) or monoclonal mouse (Sweden) antibodies, while the difference between the two EIA-sandwich methods was much smaller: a factor of 2.2. For mouse allergen, an inhibition radioimmunoassay (RIA) with rabbit antimouse antibodies (UK) gave 4.6 and 5.9 times higher concentrations than sandwich EIAs with rabbit polyclonal antibodies (Sweden and The Netherlands), while the difference between the two sandwich EIAs was, on average, 1.6-fold. Thus, although levels of rat and mouse aeroallergens are significantly correlated, the assay type gives large differences in absolute concentrations, and interlaboratory technical differences affect even the same assay type. Conversion factors can aid comparison between studies, and, in the long term, assay standardization is desirable.     

Interference of complement with the binding of carcinoembryonic antigen to solid-phase monoclonal antibodies

Six mouse monoclonal antibodies against carcinoembryonic antigen were evaluated for use in a solid-phase immunometric assay. Three IgG2 antibodies, when attached to polymer particles or microtiter wells, were found to be severely inhibited by fresh serum. The remaining three antibodies, which were of the IgG1 subclass, were inhibited only slightly or not at all when used in the same way. With the aid of labelled antibodies against C1q and C3, it was shown that antibody inhibition was accompanied by the binding of large amounts of these complement factors. Experiments involving heat inactivation or dilutions of serum, or the addition of EDTA, consistently revealed the same correlation between binding of complement factors and inhibition of CEA binding to antibody. It is suggested that the significant inhibition of the solid-phase IgG2 antibodies was caused by classical pathway activation of complement by the solid-phase antibody even in the absence of antigen. The slight inhibition of solid-phase IgG1 antibodies observed at high serum concentrations was probably due to complement binding by the alternative pathway. Preliminary evidence suggests that complement interference is not restricted to CEA assays, but is a potential problem in all types of serum immunoassays using solid-phase antibodies.