Optimisation of a peptide-based indirect ELISA for the detection of antibody in the serum of HIV-1 seropositive patients

A modified peptide-based indirect ELISA technique for the detection of HIV-1 specific antibodies in the sera of HIV-1 seropositive individuals is described. We found that the reduction of non-specific binding of HIV-1 seropositive sera to the ELISA plate was essential for the reliable detection of serum antibodies in the peptide based indirect ELISA. Optimal results were obtained using Immulon microtitre plates, different concentrations of denatured, purified grade of casein in the blocking (1%) and washing (0.25%) solutions and by diluting HIV-1 seropositive sera 1 in 1600. These conditions reduced non-specific binding and improved assay sensitivity. We show that the inclusion of a control peptide is essential to reducing the incidence of false positive and false negative results. Taken together, the modifications described in this report improve reliability of the peptide-based indirect ELISA without compromising its sensitivity and have particular relevance for those wishing to apply the peptide-based indirect ELISA technique to serum samples which exhibit high levels of non-specific binding. To illustrate this, levels of antibody in the sera of HIV-1 seropositive and seronegative donors that are specific for peptides derived from a conserved region of HIV-1 gp120 sharing homology with the FAS apoptosis antigen were analysed using this technique.     

Use of monoclonal antibodies to quantify subclasses of human IgG. II. Enzyme immunoassay to define antigen specific (anti-filarial) IgG subclass antibodies

Because of their single epitope specificity, monoclonal antibodies (Mcabs) may perform with different levels of efficiency in immunoassays depending on the accessibility of the particular epitope recognized. In order to develop assays capable of detecting specific antibodies of each of the four human IgG subclasses, we have evaluated by ELISA the performance characteristics of a panel of Mcabs raised to the subclass proteins. At least one Mcab to each of the four subclasses was identified that was specific in its ability to capture its own relevant IgG subclass without any associated light chain, allotype or isoallotype activity and that was able to function effectively as a probe in an optimized, quantitative ELISA. When IgG subclass antibodies were measured in sera from patients with filariasis using specific filarial antigen, the sensitivities of each subclass antibody assay varied; for IgG1 and IgG4 antibodies the sensitivity of detection was 50 ng/ml and for IgG2 and IgG3, 10 ng/ml. The potency of the Mcab, determined by its titration for use as a probe, did not correlate with the sensitivity of the assay. These Mcabs were also capable of defining IgG subclass antibody responses qualitatively in immunoblot analyses with little or no non-specific binding. The availability of such highly characterized Mcabs for use in quantitative and qualitative definition of specific IgG subclass antibody responses should greatly improve our detection and subsequent understanding of the role of these IgG subclasses in various disease states.     

Use of monoclonal antibody in diagnosis of candidiasis caused by Candida albicans: detection of circulating aspartyl proteinase antigen.

To develop a serological diagnosis of invasive candidiasis based on detection of circulating secreted aspartyl proteinase (SAP) antigen of Candida albicans, three different enzyme-linked immunosorbent assays (ELISAs) were compared. The first was a standard ELISA to detect anti-SAP antibodies, and the others were an antigen capture ELISA and an inhibition ELISA to detect circulating SAP antigen with monoclonal antibody (MAb) CAP1, which is highly specific for SAP. These tests were applied to 33 serum samples retrospectively selected from 33 patients with mycologically and/or serologically proven invasive candidiasis caused by C. albicans. Serum samples from 12 patients with aspergillosis and serum samples from 13 healthy individuals were also included. The sensitivities and specificities were 69.7 and 76.0% for the standard ELISA and 93.9 and 92.0% for the antigen capture ELISA, respectively. However, these values reached 93.9 and 96.0%, respectively, for the inhibition ELISA. Serum samples from 31 of 33 patients had detectable SAP antigen, with concentrations ranging from 6.3 to 19.0 ng/ml. These results indicate that the inhibition ELISA with MAb CAP1 is effective in detection of circulating SAP antigen and that this assay may be useful for diagnosis and treatment monitoring of invasive candidiasis.     

Improved sensitivity and specificity of sandwich, competitive and capture enzyme-linked immunosorbent assays for allergen-specific antibodies

Indirect ELISA is widely used to detect specific antibodies but can suffer from high non-specific binding-particularly of IgG. The use of affinity-purified rabbit antibody-coated microtitre plates to bind antigen greatly increases sensitivity without a significant increase in non-specific binding of IgG. Capture, competitive and sandwich assay procedures gave comparable results for IgG antibodies; but only the sandwich assay was suitable for detection of IgE antibodies.     

Noise control in solid-phase immunoassays by use of a matrix coat

This paper describes the matrix coat noise control technique as applied in an ELISA to detect IgG antibody to the semen protein p30 (also known as prostate-specific antigen). Background noise due to non-specific binding of IgG in solid-phase immunoassays has recently been shown to be highly charge dependent. In this technique, the surface antigen (here, p30) is co-coated on the test surface with an anionic macromolecule, the noise reduction matrix component, to reduce non-specific IgG binding. A second matrix component, the noise balancing component, is added if necessary to balance non-specific IgG binding between a detecting well which contains matrix plus antigen and a control well which contains matrix alone. Sample noise can then be measured in the control well and subtracted to yield a noise-corrected signal. This approach both minimizes background noise and then precisely quantitates residual noise. In this study, human alpha 1-acid glycoprotein (AGP) served as the noise reduction component and bovine serum albumin (BSA) as the noise balancing component. Optimal coating concentrations of AGP and BSA for the matrix were determined by a new technique, the tetrad method of signal and noise analysis. A signal probe (immune serum), a noise probe (non-immune serum) and a set of four test wells are employed to analyze the noise control properties of a given combination of matrix components. For each such tetrad of wells, three ratios are calculated: the sensitivity, signal-to-noise and noise balance ratios, along with a composite index, the matrix index. These can be used to select the combination and concentrations of matrix components which optimize assay noise control and minimize losses of assay sensitivity. When an ELISA using matrix coat noise control (MCNC-ELISA) was compared with ELISAs using standard blocking techniques, the MCNC-ELISA was superior in its ability to control for false positive results due to non-specific IgG binding and binding of the polycation poly-L-lysine. Two subjects (14%) out of a clinic population of sexually active women were found to have significant serum levels of anti-p30 IgG (P less than or equal to 0.001) by MCNC-ELISA.     

Use of Monoclonal Antibody in Diagnosis of Candidiasis Caused by Candida albicans: Detection of Circulating Aspartyl Proteinase Antigen

To develop a serological diagnosis of invasive candidiasis based on detection of circulating secreted aspartyl proteinase (SAP) antigen of Candida albicans, three different enzyme-linked immunosorbent assays (ELISAs) were compared. The first was a standard ELISA to detect anti-SAP antibodies, and the others were an antigen capture ELISA and an inhibition ELISA to detect circulating SAP antigen with monoclonal antibody (MAb) CAP1, which is highly specific for SAP. These tests were applied to 33 serum samples retrospectively selected from 33 patients with mycologically and/or serologically proven invasive candidiasis caused by C. albicans. Serum samples from 12 patients with aspergillosis and serum samples from 13 healthy individuals were also included. The sensitivities and specificities were 69.7 and 76.0% for the standard ELISA and 93.9 and 92.0% for the antigen capture ELISA, respectively. However, these values reached 93.9 and 96.0%, respectively, for the inhibition ELISA. Serum samples from 31 of 33 patients had detectable SAP antigen, with concentrations ranging from 6.3 to 19.0 ng/ml. These results indicate that the inhibition ELISA with MAb CAP1 is effective in detection of circulating SAP antigen and that this assay may be useful for diagnosis and treatment monitoring of invasive candidiasis.     

Specific detection of antibodies in cancer patients following immunotherapy with anti-idiotype

Assays were compared for specificity and sensitivity in detecting in cancer patients' sera antibodies (Ab) raised during the course of immunotherapy with goat anti-idiotypic antibodies (Ab2) bearing the internal image of a colon carcinoma-associated antigen defined by monoclonal antibody (MAb) CO17-1A (Ab1). The human Ab were tested for binding to tumor cells, isolated tumor antigen (Ag), and Ab2, and for the capacity to inhibit binding of Ab2 to Ab1. Chimeric (human/mouse) MAb CO17-1A was used as a positive control in all assays. Of the four different cell binding assays used, the mixed hemadsorption assay (MHA) showed the highest specificity and sensitivity. For detection of Ag-binding human Ab, the enzyme-linked immunosorbent assay (ELISA) with Ag as target and peroxidase (PO)-labeled anti-human IgG antibodies as tracer for detection of human Ab binding to the target, showed higher specificity and sensitivity as compared to radioimmunoassay (RIA). For detection of human Ab binding specifically to Ab2, three different ELISAs and three RIAs were used. Best results were obtained in the ELISA with anti-human IgG antibodies as target and biotinylated Ab2 as tracer for detection of human Ab binding to the target. Of four different inhibition assays used, the ELISA which measures inhibition of binding of biotinylated Ab2 to Ab1 by human Ab or chimeric antibody at 37 degrees C was the most sensitive and specific. These assays have general applicability for the characterization of human Ab responses in Ab2 vaccination approaches to various tumors and pathogens and therefore provide the basis for the establishment of a correlation between Ab responses and clinical outcome of the disease.     

Reverse ELISA for IgG and IgM antibodies to detect Lassa virus infections in Africa.

BACKGROUND: Anti-Lassa antibodies are detected by indirect immunofluorescence assay (IFA) or by enzyme-immunoassay (ELISA). Both methods have problems to detect low amounts of specific antibodies. OBJECTIVES: We report here highly sensitive and specific reverse ELISAs to detect Lassa virus IgG and IgM antibodies. Due to the reverse techniques, serum samples could be applied at dilutions of 1:10 without increasing non-specific background reactions. STUDY DESIGN: For IgM antibody detection microtiter plates were coated with anti-IgM antibodies and for IgG antibody detection with rheumatoid factor (RF) (Sachers M, Emmerich P, Mohr H, Schmitz H. Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA). J Virol Methods 1985;10:99-110). In both assays a tissue culture antigen was used in combination with a labeled anti-Lassa monoclonal antibody (Hufert FT, Ludke W, Schmitz H. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Arch Virol 1989;106(3-4):201-12). RESULTS: The reverse ELISA turned out to detect virus-specific IgG and IgM antibody in all 20 samples of West African patients collected 2-8 weeks after onset of Lassa fever. Moreover, both IFA and reverse ELISA found IgG antibodies in 53 out of 643 samples of healthy West Africans (sensitivity of 100%). Six of the 643 samples were positive by reverse IgG ELISA only. Thus, the specificity compared to IIF was 99.0%, but it may be even higher, because compared to IFA the IgG ELISA was clearly more sensitive in detecting low antibody titers. CONCLUSIONS: In Ghana 3% seropositives were found by IFA, but 4% by the reverse ELISA. The reverse ELISAs can be performed with high sensitivity and specificity under field conditions in Africa.     

Use of avidin-biotin complex in an ELISA system: a quantitative comparison with two other immunoperoxidase detection systems using keratin antisera

Three systems--the indirect, peroxidase-antiperoxidase (PAP), and avidin-biotin peroxidase complex (ABC) detection system--were compared in quantitative ELISA titration and inhibition assays and tissue labeling with keratin antisera. The ABC system was found to be the most sensitive of the three in ELISA titration assays, being approximately 4-fold more sensitive than the indirect method and 2-fold more sensitive than the PAP method. In addition, the ABC method demonstrated increased sensitivity when compared on tissue sections. Furthermore, the secondary and tertiary components of the PAP and ABC systems were titrated and optimal concentration ranges determined for use in ELISA inhibition assays. In ELISA inhibition assays the broadest usable inhibitor range and maximal sensitivity were obtained using the ABC system as compared with indirect and PAP detection systems. Finally, the effects of varying microtiter plate-coating concentrations on range and sensitivity were examined, and possible explanations are discussed including the possibility of surface-bound antigen being a competitor for the antibody of the solution phase antibody-antigen complex.     

Binding properties of antibodies to prothrombin and beta2-glycoprotein I (beta2-GPI) assayed by ELISA and dot blot

Most anti-phospholipid antibodies (aPL) associated with the anti-phospholipid syndrome are autoantibodies with specificity towards beta2-GPI (anti-beta2-GPI) or prothrombin (anti-II). They are mainly screened by ELISA using polyoxygenated plates. However, some authors have claimed that immunoblotting can also be used. Exposure of cryptic epitopes or increase of antigen density on its binding to either phospholipids or suitable plastic surfaces are the two hypotheses proposed for the interaction of beta2-GPI or prothrombin with their antibodies. Forty-five patients with aPL were studied: 25 with lupus anti-coagulant (LA) and anti-cardiolipin antibodies (aCL), 10 with LA alone and 10 with aCL but negative LA. All patients with LA and aCL were positive for anti-beta2-GPI by ELISA and dot blot, while 15/25 had anti-IIELISA and 14 of them also had anti-II by dot blot assay. No patient with LA alone tested positive for anti-beta2-GPI by ELISA or dot blot, whereas 6/10 had anti-IIELISA (five of them were also positive by dot blot). Four out of 10 aCL-positive patients had anti-beta2-GPI by ELISA and dot blot, while none of this group had anti-II by ELISA or dot blot. Antibody binding to beta2-GPI or prothrombin in both ELISA and dot blot was significantly reduced by phospholipid liposomes mixed together with beta2-GPI or prothrombin, whereas liposomal eluants retained it in both assays. Parallel fluid-phase inhibition experiments using increasing concentrations (up to 200 microg/ml) of beta2-GPI or prothrombin demonstrated that antibody binding reduction was more evident on dot blot than on ELISA. It was almost completely abolished on dot blot, while on ELISA a moderate inhibition was achieved even at the highest protein concentration. However, antibody binding on ELISA was virtually abolished when diluted sera were incubated with high protein concentrations applied to nitrocellulose membranes. We could infer that ELISA and dot blot detect antibodies with some differences in avidity but directed against native epitopes on beta2-GPI and prothrombin.     

Detection of fish antigens aerosolized during fish processing using newly developed immunoassays

BACKGROUND: Aerosolization of fish proteins during seafood processing has been identified as a potential route for allergic sensitization and occupational asthma among workers involved in high-risk activities. The aim of this study was to develop immunological assays for the quantification of aerosolized fish antigens in a fish-processing factory. METHODS: Polyclonal antibodies to the main fish species processed in the factory (anchovy and pilchard) were generated in rabbits and compared by ELISA inhibition assay and immunoblotting. These antisera were utilized to develop ELISA assays for the detection of fish antigens. The ELISA inhibition assays were evaluated by analyzing environmental air samples collected from three areas in a fish-processing factory: pilchard canning, fish meal production and lobster processing. RESULTS: By immunoblotting, the rabbit polyclonal antibodies demonstrated IgG antibody binding patterns comparable with IgE antibodies of fish-sensitized patients, particularly in regard to the major fish allergens parvalbumins. The sensitivity of the fish-specific ELISA assays developed was 0.5 microg/ml. The ELISA inhibition assays were able to differentiate between the two different fish species of interest but did not recognize a crustacean species. Notable differences in exposure levels to canned pilchard and anchovy antigens were demonstrated in the three different working areas of the factory, with assays having a detection limit as low as 105 ng/m(3). CONCLUSION: These ELISA-based assays are sensitive and specific to quantify differential exposure levels to fish antigens produced during fish processing, making it possible to investigate exposure-disease response relationships among workers in this industry.     

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.     

Real-time biospecific interaction analysis of antibody reactivity to peptides from the envelope glycoprotein, gp160, of HIV-1.

A new assay designed to quantitate antibody reactivity to specific peptides using biospecific interaction analysis (BIAcore) has been developed. Peptides of various lengths (15-40 amino acids) and isoelectric points (pI = 4.5-13) were covalently linked (immobilized) to a biosensor and interacted with polyclonal human sera. The immobilization procedure was highly reproducible, with bound peptides retaining high antibody reactivity. The assay was rapid, requiring only 25-30 min to immobilize the peptide and 2-8 min for each subsequent peptide/serum binding interaction. The same peptide surface has been used for up to 90 cycles of serum binding and regeneration with only a 0.3% decay in reactivity over cycle number. The quantitative BIAcore signal, measuring peptide/antibody binding interactions, was directly related to the antigen/antibody concentrations within the biosensor. The assay allowed interactants to be studied in their native form and without the need of additional secondary detection antibodies. Correlation between conventional peptide ELISA and BIAcore was obtained. The BIAcore linear range persisted over a series of eight two-fold dilutions. This extended linear dynamic response range is an improvement over conventional ELISA measurements. The sensitivity for monoclonal antibody detection is similar to conventional ELISAs and 4.9 ng/ml was readily detected.     

A highly sensitive enzyme-linked immunosorbent assay for idiotype-bearing antibodies

A sensitive and specific immunoenzyme assay (ELISA) for quantitation of total and cross-reactive idiotype-bearing (CRI) anti-ABA antibodies is described. Total anti-ABA antibodies are directly assessed in ABA-BGG coated polyvinyl wells with enzyme-labelled rabbit anti-mouse immunoglobulins. By interpolation on a standard curve absorbance values give the concentration of anti-ABA antibodies with a sensitivity of 30 ng/ml. CRI+ antibodies are quantitated by inhibition of enzyme-labelled monoclonal CRI+ antibody binding to solid-phase coated rabbit anti-CRI immunoglobulins. The concentration of CRI+ antibodies, evaluated by interpolation on a standard inhibition curve, can be measured at the level of 10 ng/ml. This highly sensitive, rapid, specific and reproducible assay is easily used, with minor modifications, to detect specific antibodies in any idiotype system.     

ELISA for the detection of anticardiolipin antibodies. High specificity based on the use of adult bovine serum as buffer and systematic subtraction of non-specific binding

We have used an ELISA procedure to compare the reactivity of samples with or without IgG anticardiolipin antibodies (ACA) on cardiolipin-coated wells (target wells) and cardiolipin-free wells (control wells), using 10% fetal calf serum (10% FCS), 10% adult bovine serum (10% ABS) or 1% bovine serum albumin (1% BSA) as buffer. With 1% BSA, ACA reactivity was very low which suggests that this buffer would be inappropriate for use in ELISA procedures for ACA. 10% FCS induced non-specific binding of normal IgG only on target wells, particularly in the case of IgG hypergammaglobulinemia. With 10% ABS, this non-specific binding occurred on the solid phase with and without cardiolipin and could be accurately evaluated by subtracting the absorbance of control wells. Results of assays on 35 systemic lupus erythematosus sera using 10% ABS as buffer showed that highly specific results-could be obtained only if non-specific binding was systematically subtracted.     

A novel method to investigate the heterocliticity of antibodies

Monoclonal and anti-dinitrophenyl and anti-trinitrophenyl IgE antibodies were used to measure heterocliticity using competitive inhibition assays with homologous and heterologous haptens. The antibodies or antibody-containing ascites fluids were diluted to give 50% of the maximum binding to wells of antigen-coated microtiter plates. The % inhibition of binding of the antibody to the antigen by various concentrations of homologous and heterologous haptens at a standard dilution of antibody can then be compared. The advantages of this method of determination of heterocliticity are that it is fast, simple, quantitative and does not need radiolabeled reagents.     

Solid phase enzyme immunoassay of cyclic adenosine 3',5'-monophosphate. Effect of coating strategy upon assay performance in comparison with radioimmunoassay.

We have evaluated two novel enzyme-linked immunosorbent assays (ELISAs) used to quantitate cyclic AMP. In one assay ELISA plates are coated with antigen consisting of a cyclic AMP-polylysine conjugate. Cyclic AMP samples added to plates are quantified by their ability to decrease the binding and anti-cyclic AMP antibodies to the coated antigen. A second ELISA utilizes a plated anti-immunoglobin technique in which plates are coated first with anti-goat IgG and then with goat anti-cyclic AMP antiserum. Cyclic AMP samples are quantified by their ability to compete with cyclic AMP-peroxidase conjugates for binding to the plated anti-cyclic AMP antibodies. The plated anti-immunoglobin ELISA proved to be somewhat more sensitive than the plated antigen ELISA and was comparable in sensitivity to an automated RIA for measuring cyclic AMP in standards and urine samples. Our data fit with the generalization that optimal ELISA sensitivity is obtained through the use of plated anti-immunoglobins rather than plated antigens. Further they demonstrate the practicality of utilizing small ligand-enzyme conjugates for ELISAs.     

Enzyme-linked immunosorbent assay for titration of Haemophilus influenzae capsular and O antigen antibodies.

The enzyme-linked immunosorbent assay (ELISA) was elaborated for the detection of immunoglobulin M (IgM) and IgG antibodies against capsular and O antigens of Haemophilus influenzae. Purified capsular polysaccharide and lipopolysaccharide were used as antigens, with optimal coating concentrations being about 50 and 100 micrograms/ml, respectively. The antibody content was expressed as the highest serum dilution (-log10) showing an absorbance of 0.2 above the background level. The titers of hyperimmune sera (reference sera) ranged between 5 and 7 -log10. The sensitivity of the method was about 80 ng/ml with regard to anticapsular antibodies and 3 to 5 ng/ml with regard to anti-lipopolysaccharide antibodies. For detection of antibodies against capsular polysaccharide in sera obtained after primary immunization, ELISA was about 100-fold more sensitive than the indirect hemagglutination assay, whereas in hyperimmune sera, ELISA was about 10-fold more sensitive than the indirect hemagglutination assay. The sensitivity of ELISA for detecting anticapsular antibodies after primary and booster immunizations was 50-fold higher than that of the bactericidal assay using capsulated bacteria, whereas the sensitivity of the two methods was the same when hyperimmune sera were tested. ELISA performed with lipopolysaccharide as the antigen was about 50- and 150-fold more sensitive than the complement fixation and bactericidal assays tested with noncapsulated variants after primary injection and hyperimmunization, respectively.     

Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays were used to study (i) binding of rabbit antibodies (raised against litter mate liver plasma membrane fraction) to the immunizing membrane fraction, and (ii) binding of human antibodies to liver membrane fractions and to liver-specific lipoprotein (a liver membrane-derived antigen complex). When assays were conducted using the non-ionic detergent Tween 20 as blocking agent, high non-specific binding was encountered. With the low titre rabbit antisera high binding of non-immune test antibody and of second antibody (anti-rabbit IgG) to the immunogen, and also directly to the solid phase, was found. This was abolished by replacement of Tween 20 in the antibody diluent buffers by a non-reactive protein, casein proving to be a more effective blocking agent than either bovine serum albumin or gelatin. With human sera, high binding of human IgG to the solid phase was noted. This too was blocked by casein, but only when the anti-microbial agent Thimerosal was included in the casein buffer, and when Tween 20 in the wash buffer was replaced by casein-Thimerosal so that the solid phase was exposed to casein before incubation with the test serum. The casein buffers described may prove of general value in solid-phase assays where high non-specific binding is encountered.     

Enzyme-linked immunosorbent assays for determination of plasma aldosterone using highly specific polyclonal antibodies

Two enzyme-linked immunosorbent assays were established and compared for the estimation of plasma aldosterone. In the first method immobilized aldosterone-protein complexes on the ELISA plates compete with aldosterone to be determined for the binding of certain amount of anti-aldosterone antibodies. The sensitivity of this method depends on the protein carrier used to conjugate with aldosterone. In the second method, anti-aldosterone antibodies adsorbed on ELISA plates compete for binding of known amount of the enzyme-labeled aldosterone and aldosterone to be determined. The highly specific rabbit anti-aldosterone antibodies were obtained by injection of aldosterone-oxime thyroglobulin. The detection limit of aldosterone in both methods ranged between 2-20 pg. The proposed assays are suitable for the determination of aldosterone in biological fluids compared with other reported ELISA assays, as well as with RIA.