Multiple epitope interactions in the two-step sandwich immunoassay.

The 'hook' effect as related to the two-step sandwich immunoassay has been investigated experimentally and theoretically. The multiple epitope interactions between the analyte and the labeled antibody cause a 'hook' in the two-step sandwich immunoassay. Three different analytes and monoclonal antibodies were chosen to carefully demonstrate the effect of the analyte characteristics on this immunoassay. Two monoclonal antibodies against two different epitopes of biosynthetic human growth hormone (hGH) was the simplest model for this study. The sandwich immunoassay for hGH shows no 'hook' effect. The non-covalent dimeric form of hGH (D-hGH) possesses two repeating epitopes which is the simplest model for an analyte having a discrete number of repeating epitopes. The D-hGH assay demonstrated a 'hook' effect in the two-step sandwich immunoassay if the labeled antibody was allowed to interact with more than one epitope. In a third system multiple epitope interactions with the labeled antibody were observed using ferritin. The effect of the analyte concentration and the liquid-phase antibody have been examined to elucidate the nature of these various interactions. The cause of the 'hook' effect in the two-step sandwich immunoassay is attributed to the desorption of the bound analyte due to a conformational change after the labeled antibody interacts with several epitopes of the adsorbed analyte.     

Studies of the low dose 'hook' effect in a competitive homogeneous immunoassay.

The interactions of two monoclonal antibodies with human growth hormone (hGH) have been investigated. The individual antibodies showed normal behavior in a competitive binding assay, but mixtures of the antibodies demonstrated a 'hook' attributable to cooperative interactions. Cooperativity was observed in titrations which preceded the competitive binding assay. Size exclusion chromatographic data suggest that the cooperativity is explained by the formation of higher molecular weight complexes (up to 700 kDa). The major complex is probably linear, consisting of three antibody molecules. Circular and linear complexes with four antibody molecules (octameric complexes) are also possible. Theoretical models also support the formation of cyclic complexes in a competitive binding assay.     

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA).

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.2 X 10(10) l/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, and additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 micrograms SOD/l.     

Quadroma-secreted bi(interferon alpha 2--peroxidase) specific antibody suitable for one-step immunoassay.

A mouse hybridoma (quadroma) was prepared by fusing hybridomas producing monoclonal antibody of G1-isotype to human interferon-alpha 2 with hybridomas producing monoclonal antibody of G2a-isotype against horseradish peroxidase. The established quadroma line secreted immunoglobulins of both G1/G2a-isotypes which manifested parental and bispecific binding characteristics. Culture supernatant containing the bifunctional antibody cross-linking interferon and peroxidase was used for a one-step immunoassay. The developed sandwich ELISA was able to detect the human interferon-alpha 2 at a concentration of 10 units/ml (0, 1 ng/ml) within 2-3 hours.     

Modeling and Optimization of High-Sensitivity,Low-Volume Microfluidic-Based Surface Immunoassays

Microfluidics are emerging as a promising technology for miniaturizing biological assays for applications in diagnostics and research in life sciences because they enable the parallel analysis of multiple analytes with economy of samples and in short time. We have previously developed microfluidic networks for surface immunoassays where antibodies that are immobilized on one wall of a microchannel capture analytes flowing in the microchannel. This technology is capable of detecting analytes with picomolar sensitivity and from sub-microliter volume of sample within 45 min. This paper presents the theoretical modeling of these immunoassays where a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We then derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance, which we think will allow further development of microfluidic networks for immunoassay applications.     

Solid-phase sandwich enzyme immunoassays of human chorionic gonadotropin using monoclonal antibodies

A two-site sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) employing monoclonal antibodies directed against beta- and alpha-subunits is described. Monoclonal anti-beta-hCG antibody was used for coating microtitration plates and monoclonal anti-alpha-hCG antibody labelled with 1 of the 3 enzymes namely horseradish peroxidase, alkaline phosphatase or beta-galactosidase was used as tracer. The assay is able to detect up to 1 ng hCG/ml. No significant difference was observed with respect to sensitivity and range of assay with the 3 enzymes. The assay can be performed as a 'two-step' assay or reduced to a 'one-step' procedure with a linear relationship between absorbance and hormone concentration up to 31.25 ng hCG/ml. Beyond these concentrations an inflection of the dose curve was observed. This can, however, be avoided by increasing the concentration of antibody-enzyme conjugate. A higher sensitivity enabling detection up to 0.25 ng hCG/ml was attained in the sandwich enzyme immunoassay with the use of biotin-avidin interface. The hCG values obtained on 47 human urine samples either by the 'one-step' or 'two-step' procedure were similar with a correlation coefficient of 0.996. Results obtained by 'two-step' sandwich enzyme immunoassay on 22 human urine samples correlated well (r = 0.968) with the values obtained by radioimmunoassay.     

Sandwich type ELISA and a fluorescent cytometric microbead assay for quantitative determination of hepatitis B virus X antigen level in human sera

The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.     

Comparison of three multiplex cytokine analysis systems: Luminex, SearchLight and FAST Quant

Multiplex cytokine analysis technologies have become readily available in the last five years. Two main formats exist: multiplex sandwich ELISA and bead based assays. While these have each been compared to individual ELISAs, there has been no direct comparison between the two formats. We report here the comparison of two multiplex sandwich ELISA procedures (FAST Quant and SearchLight) and a bead based assay (UpState Luminex). All three kits differed from each other for different analytes and there was no clear pattern of one system giving systematically different results than another for any analyte studied. We suggest that each system has merits and several factors including range of analytes available, prospect of development of new analytes, dynamic range of the assay, sensitivity of the assay, cost of equipment, cost of consumables, ease of use and ease of data analysis need to be considered when choosing a system for use. We also suggest that results obtained from different systems cannot be combined.     

Improved fluoroimmunoassays using the dye Alexa Fluor 647 with the RAPTOR,a fiber optic biosensor

The performance of the fluorescent dye Alexa Fluor 647 (AF647) was explored as an alternative to Cy5 for immunoassays on the RAPTOR, a fiber optic biosensor. The RAPTOR performs sandwich fluoroimmunoassays on the surface of small polystyrene optical waveguides for analyte detection. Fluorescence and immunoassay data were examined at various dye-to-protein (D/P) ratios for both Cy5 and Alexa Fluor 647. Primarily, due to the self-quenching characteristics of Cy5, Alexa Fluor 647 is substantially more effective in fluoroimmunoassays, yielding over twice the signal for any given analyte concentration. Alexa Fluor 647 can be attached to antibodies at higher ratios, D/P=6, before self-quenching begins to limit the dye's effectiveness. Furthermore, while Alexa Fluor 647 becomes quenched at high dye-to-protein ratios, D/P=9, the net fluorescence yield reaches a maximum, as opposed to Cy5-labeled proteins, which become nearly nonfluorescent at high labeling ratios, D/P> or =6. The limitations of Cy5 were elucidated with an immunoassay for ricin, while the advantages of Alexa Fluor 647 were demonstrated in both direct binding assays as well as in a sandwich immunoassay for staphylococcal enterotoxin B.     

Quantitative microarray pesticide analysis

To replace a pesticide immunoassay based on microtiter plates, we have developed a quantitative, competitive microarray immunoassay, which permits rapid and highly sensitive quantification of the dichlobenil degradation product 2,6-dichlorobenzamide (BAM), and the prominently used herbicide atrazine. The pesticide analysis is based on the competitive binding of fluorescence conjugated monoclonal antibodies (mAb) to their respective analytes. Lowest detection limits were calculated to 1 ng/l (5 pM) for BAM and 3 ng/l (10 pM) for atrazine. Corresponding IC(50) values were, 10 ng/l (50 pM) for BAM and 34 ng/l (160 pM) for atrazine, respectively. In comparison to the existing microtiter plate immunoassay, the microarray was found to be up to 20-fold more sensitive. Compared to the gas chromatography with mass spectroscopy (GCMS) analysis performed on more than 1000-fold concentrated samples, the microarray-based immunoassay was even 10-fold more sensitive using non-concentrated samples. Measuring both analytes simultaneously did not affect assay sensitivity compared to single analyte quantification. Besides a gain in sensitivity and the possibility of multiplex quantification, assay times and assay complexity were reduced drastically with the microarray platform compared to the microtiter plate immunoassay and GCMS, suggesting that the microarray based immunoassay is a viable method for measuring picomolar amounts of analytes, e.g. clinically relevant analytes.     

Monoclonal antibodies to human growth hormone can distinguish between pituitary and genetically engineered forms

Monoclonal antibodies (MABs) prepared against human pituitary growth hormone (hGH) have been compared for their binding to pituitary-derived and genetically engineered methionyl growth hormone (met-hGH). The antibodies bind to four non-overlapping epitopes of which two are completely shared with human choronic somatomammotropin (hCS). The determinant defined by MAB NA27 was expressed on met-hGH to a lesser degree than on hGH of pituitary origin. However, another antibody, QA68, which binds to a determinant closely related to NA27, failed to discriminate between hGH and met-hGH. A further two MABs (EB1 and NA71) were similarly ineffective in distinguishing between the two forms of the hormone. The determinant recognized by antibody EB2 was equally represented on hGH and met-hGH when assessed by a liquid-phase radioimmunoassay: however, measurement of the binding in a solid-phase assay resulted in a two-four-fold lower binding to met-hGH. Bioactivity assessed by both an in vitro cell proliferation assay and an in vivo cartilage sulphation bioassay failed to distinguish between the two hormones. It is therefore concluded that the NH2-terminal methionine on bacterially derived growth hormone results in altered antigenicity of the hormone without any measurable effect on bioactivity.     

Advances in the development of a reliable assay for the measurement of stool decay-accelerating factor in the detection of colorectal cancer

We have previously shown that stool concentrations of decay-accelerating factor (DAF; CD55), a membrane-bound complement-regulatory protein, are significantly elevated in patients with colorectal cancer and that the measurement of stool DAF may be a valuable test for the detection of colorectal cancer. Accordingly, we are working to develop a clinically useful immunoassay for fecal DAF. A requirement for such assay is a plentiful and reliable supply of anti-DAF antibodies. We developed a sandwich enzyme-linked immunosorbent assay (ELISA) for DAF in stool specimens, using two monoclonal anti-DAF antibodies recognizing different epitopes on the DAF molecule. When we first used a biotin-labeled antibody and enzyme-linked streptavidin method, we often observed stool interference, probably due to the presence of a substance(s) with biotin activity which non-specifically bound to the Fc portion of IgG of the first anti-DAF antibody on the ELISA wells. By the use of inorganic salts in the sample-dilution buffer and HRP-labeled anti-DAF as second antibody, we circumvented the stool interference and established that the new ELISA system could reliably measure DAF at low concentrations in stool specimens. Because the new assay system uses only monoclonal antibodies, we can now consistently supply ample amounts of antibodies for routine measurement of stool DAF.     

A sensitive two-site sandwich enzyme immunoassay for human angiotensin converting enzyme utilizing monoclonal antibodies.

A sensitive enzyme immunoassay was developed for human angiotensin converting enzyme. Monoclonal antibodies specific for two unique converting enzyme epitopes were utilized to develop a two-site sandwich enzyme immunoassay. Alkaline phosphatase conjugated to the detecting antibody hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) to NAD. Subsequently, NAD is cycled between its reduced and oxidized forms by an alcohol dehydrogenase/diaphorase catalyzed redox cycle. Each cycle converts iodonitrotetrazolium violet to a highly colored formazan which is quantitated. With this assay, as little as 94 pg/ml of native converting enzyme is detectable without interference from either therapeutic or endogenous converting enzyme inhibitors.     

应用单克隆抗体测定纤维连结蛋白ELISA方法的建立

本文介绍用McAb作为包被和检测抗体进行双抗体夹心ELISA来测定人血浆纤维连结蛋白。通过实验找出最佳条件,即两种McAb混合使用,在McAb和酶结合物稀释液内加入4％PEG及使用TMBI底物的最适浓度等,该法敏感性可达80ng/ml,可作为临床常规方法使用。     

Multianalyte microspot immunoassay. The microanalytical 'compact disk' of the future.

Throughout the 1970s, controversy centered both on immunoassay 'sensitivity' per se and on the relative sensitivities of labelled antibody and labelled analyte methods. Our own theoretical studies in this period revealed that radioimmunoassay (RIA) sensitivities could be surpassed only by the use of very high specific activity non-isotopic labels in 'non-competitive' designs, preferably based on the use of monoclonal antibodies. The time-resolved fluorescence methodology known as Delfia - developed in collaboration with the instrument manufacturer LKB/Wallac - represented the first commercial 'ultra-sensitive' non-isotopic technique based on these theoretical insights, the same concepts being subsequently adopted in comparable methodologies relying on the use of chemiluminescent and enzyme labels. However, a second advantage of high specific activity labels is that they permit the development of 'multi-analyte' immunoassay systems combining ultra-sensitivity with the simultaneous measurement of tens, hundreds or thousands of analytes in a small biological sample. This possibility relies on simple, albeit hitherto unexploited, physicochemical concepts. The first is that all immunoassays rely on measurement of Ab occupancy by analyte. The second is that, provided the Ab concentration used is 'vanishingly small', fractional Ab occupancy is independent of both Ab concentration and sample volume. This leads to the notion of 'ratiometric' immunoassay, involving measurement of the ratio of signals (eg fluorescent signals) emitted by two labelled Ab's, the first ('sensor' Ab) deposited as a microspot on a solid support, the second a 'developing' Ab directed against either occupied or unoccupied sensor Ab binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The antigenic topography of human growth hormone

Murine monoclonal antibodies (MAb) have been used as tools to probe the antigenic topography of human growth hormone (hGH). Mapping experiments were carried out by testing the ability of paired MAb to bind simultaneously or separately to 125I-hGH. A putative three-dimensional model of the relative distribution of 20 hGH epitopes indicated that they covered the entire molecular surface, showing the following essential characteristics. A domain of unique hGH specificity representing approximately 20% of the whole area was detected, as well as the presence of a discontinuous band of immunological identity between hGH and human placental lactogen (hPL) occupying 30% of the molecular surface. The rest of the surface (about 50%) displayed only partial cross-reactivity with hPL. Three restricted antigenic areas were also recognized. One of them appeared to correlate with a conformational change induced by the adsorption of the protein to plastic surfaces and the other two showed cross-reactivity with human prolactin and heterologous GH, respectively.     

Monoclonal antibodies to human growth hormone induce an allosteric conformational change in the antigen.

We re-investigated the properties of a monoclonal antibody (mAb), 4D11, to human growth hormone (hGH) that showed a very weak affinity, recognizing hGH only when the hormone was solubilized on a solid surface. MAb4D11 did not significantly bind 125I-hGH. It was found that three mAb directed to different hGH epitopes (mAb 3C11, 10C1 and NA71) were able to induce the binding of the soluble antigen to mAb 4D11. The co-operative effect could be demonstrated by the formation of binary complexes (Ag:Ab, 1:2) detected by high-performance liquid chromatography (HPLC) and by the increase of radioactivity found when the synergistic mAb were added to 125I-hGH incubated with mAb 4D11 immobilized on polyvinyl microplates. Other possible explanations, such as the formation of cyclic complexes or the generation of a new epitope in the Fc fragment of the first antibody (Ab), were dismissed because the Fab fragment of one of the enhancing mAb (3C11) gave the same effect as the intact Ab. The data suggest that the hGH molecule undergoes a localized conformational change after binding to mAb 3C11, NA71 or 10C1 and that mAb 4D11 binds with high affinity to the modified region of the hormone. The formation or not of ternary complexes (Ag:Ab, 1:3) was used to localize the 4D11 epitope on the surface of the Ag. It is suggested that mAb 4D11 recognizes a conformational change produced in the region defined by the AE5/AC8 epitopes, which is close to the hGH antigenic domain only expressed when the protein is immobilized on plastic surfaces.     

Multiple copies of identical epitopes on the adenovirus hexon

A double monoclonal antibody (MAb) sandwich enzyme-linked immunosorbent assay (double MAb ELISA), which uses the same MAb as solid-phase immunosorbent (capture MAb) and as detector MAb (peroxidase-labeled), was developed to quantify the specific epitopes of adenovirus hexon. Four MAbs directed against crystallized adenovirus type 1 (Ad h 1) hexon were tested by this assay with homologous and different heterologous hexons. The lowest reacting concn with the homologous and heterologous hexon types both in direct and double MAb ELISA was determined and compared. At least two copies of four different epitopes were identified by the MAbs. Evidence is presented that more than one copy of identical or closely related epitopes exist on the homologous as well as on the heterologous hexon molecules. However, their presence could be detected only in higher concn of hexon preparations of subgenera A, B and D.     

Rapid detection of group B streptococcal antigen by monoclonal antibody sandwich enzyme assay.

Group B Streptococcus (GBS) is the most common cause of neonatal sepsis and meningitis. Infants at greatest risk to develop invasive disease are delivered to women colonized with GBS in their birth canals and lacking immunity to the colonizing serotype. We have investigated the sensitivity and specificity of a recently developed monoclonal antibody sandwich enzyme immunoassay for detection of GBS antigen. The sandwich enzyme immunoassay detected types II and III GBS at a concentration of 5 X 10(4) CFU/ml and types Ia and Ib GBS at 5 X 10(5) CFU/ml. No cross-reactions were noted when each of the GBS serotypes was reacted with antibodies of differing serotypes specificities. Type III GBS native antigen was detected at a concentration of 1 ng/ml. The sandwich enzyme assay is more sensitive than other methods currently in use for rapid detection of GBS and is serotype specific. This assay system should prove useful for the detection of GBS colonization during labor and for identification of neonates with invasive disease.     

Relative distribution of various antigenic determinants on the human growth hormone surface

The relative distribution of 12 antigenic determinants on the surface of the human growth hormone (hGH) molecule has been established. The necessary information was obtained by testing the ability of paired monoclonal antibodies (MAb) to bind simultaneously or not, to 125I-hGH which leads to the formation of 1:2 or 1:1, Ag-Ab complexes, respectively. The results obtained indicate that the epitopes occupy a large percentage of the total hGH molecular surface and revealed the existence of; an antigenic region specific for hGH; at least two independent domains of immunological identity between hGH and human placental lactogen (hPL), one of them also shared by heterologous GH; and other independent areas of partial cross reactivity with hPL. MAb competition experiments in a solid-phase RIA showed the unreliability of this technique for mapping purposes. The distribution of the hGH epitopes suggested in this work is in accord with present views on protein antigenicity and also explains data existing in the literature concerning the behavior of some of the MAb tested here.