One-step Immunoassay Research Articles

Development and use of an enzymatic tracer for an enzyme immunoassay of makisterone A

In an attempt to develop a convenient and reliable immunoassay for makisterone A, a biologically active form of molting hormone in several species, pure tetrameric form of acetylcholinesterase from electric eel was covalently coupled to a makisterone A-6-carboxymethoxime derivative. This conjugate was used in a classical one-step competitive enzyme immunoassay performed in 96-well microtiter plates coated with a second antibody. Using this tracer a 20-fold increase of sensitivity for detection of makisterone A was obtained compared to the original assay performed with the same antiserum and a 20-hydroxyecdysone-acetylcholinesterase conjugate as tracer. In the range of sensitivity reached (detection limit: 3 pg, 50% B/Bo: 40 pg) the assay could be applied to biological samples. Cross-reactivities relative to makisterone A (100%) for 20-hydroxyecdysone, ecdysone, and makisterone C were respectively 72, 28 and 5%. Performances of the immunoassay were exemplified by assaying crude and HPLC purified biological extracts from embryos of the cotton stainer bug, Dysdercus fasciatus.

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.

Studies of the ‘hook’ effect in the one-step sandwich immunoassay

The one-step sandwich immunoassay is increasingly replacing the traditional two-step immunoassay due to obvious advantages such as assay speed. However, the one-step sandwich immunoassay suffers from the ‘hook’ effect irrespective of the analyte characteristics. The ‘hook’ effect is dependent primarily on the analyte concentration. Three different model analytes, human growth hormone (hGH), the dimeric form of hGH (D-hGH, having a discrete number of repeating epitopes) and ferritin (multiple epitopes) having different immunological properties have been employed in studies of the one-step sandwich immunoassay. The characteristics of each of the model analytes offer new insights into general guidelines for assay procedures. These guidelines permit rapid optimization of assay conditions for an immunoassay without a priori knowledge of the immunological characteristics of the antibody or antigen. Both experimental and theoretical data show several instances where high capacity solid-phase antibodies can effectively shift the ‘hook’ to relatively higher analyte concentrations. The effect of the concentration of labeled antibody on assay response was examined theoretically.

Development and In Vitro Characterization of a New Immunoassay of Cardiac Troponin T

We describe a new one-step enzyme immunoassay of troponin T that uses two specific monoclonal antibodies and streptavidin-coated tubes as the solid phase. The monoclonal antibodies were obtained by conventional hybridoma technology, with human troponin T as antigen. The identity of the cardiac troponin T antigen was confirmed by analysis of the amino acid composition and by partial sequence analysis. The specificity of the monoclonal antibodies for cardiac troponin T was proved by immunoblot analysis and displacement curves. The capture antibody is labeled with biotin. The second antibody is conjugated to horseradish peroxidase (EC 1.11.1.7). The assay [Enzymun-TestR system (Boehringer Mannheim GmbH)] is performed in only 90 min at room temperature; the measuring range for troponin T is 0.1 to 15 micrograms/L. The assay shows excellent between-run precision (CV = 3.3-4.9%).

Enzyme immunoassay and immunochemical characterization of pancreatic stone protein in human serum.

Monoclonal antibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5 was employed as the standard in the assay. The assay's measurable range was 25-1,500 ng/ml and within run coefficient of variation was 3.7-6.4%. Analytical recovery of the assay was 101.5 +/- 5.65% (mean +/- SD). The results of experiments in which serum was fractionated by Mono S (cation exchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5. The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis.

Simultaneous use of poly- and monoclonal antibodies as enzyme tracers in a one-step enzyme immunoassay for the detection of hepatitis B surface antigen

A one-step third generation enzyme immunoassay (EIA) was developed for the detection of hepatitis B surface antigen (HBsAg) in human serum or plasma using a polyclonal (Pab-HBsAg) and two monoclonal antibodies to HBsAg (Mab 1-HBsAg and Mab 2-HBsAg). In this assay, the solid phase is coated with Mab 1-HBsAg and the specimen is incubated simultaneously with peroxidase labelled Pab-HBsAg and Mab 2-HBsAg. If HBsAg is present in a specimen it will form sandwich complexes with capture and tracer antibodies. The hook effect, observed in some HBsAg detection tests when a high concentration of HBsAg is present, was minimized in this assay by increasing the concentration of the peroxidase-labelled Mab 2-HBsAg. The sensitivity of this assay for HBsAg/ ay and HBsAg/ ad subtypes in a standard (2 h incubation) procedure was 0.6 and 0.3 ng/ml and in an overnight (16–22 h incubation) procedure 0.2 and 0.15 ng/ml, respectively. Strong elimination of the hook effect was observed with specimens containing high levels of HBsAg compared with test results using peroxidase-labelled Pab-HBsAg alone as enzyme tracer. This EIA offers a procedure, with a high specificity and wide range of sensitivity for the detection of HBsAg in human sera or plasma.

Sensitive one-step enzyme immunoassay for HIV-1 p24 antigen in human blood specimens and cell culture supernatants

An enzyme immunoassay (EIA) with a single incubation step has been developed to detect the HIV-1 major core polypeptide p24 in human blood specimens and in cell culture supernatants. In this assay, the solid phase is coated with monoclonal antibodies to p24, and the specimen is incubated simultaneously with peroxidase labelled polyclonal anti-HIV(Fab′). If HIV-1 p24 antigen is present in a specimen it will form a sandwich complex with the capture and tracer antibodies during the incubation step. The sensitivity of the assay for p24 antigen, 20 pg/ml, was equal to that of two commercial HIV antigen detection EIAs, when human blood specimens and virus isolation cell culture supernatants were analyzed. However, the assay described is simpler to perform than those previously reported, since only one incubation step is needed. Moreover this assay minimizes the handling of material containing potentially infectious HIV.

A one-step two-particle latex immunoassay for the detection of Salmonella typhi endotoxin

A simple and rapid test (LIMM, short for latex immunoassay) is described for detecting Salmonella typhi endotoxin. It involves the simultaneous binding of the antigen by two types of reagent particles contained in a micro-tube: an indicator latex particle coated with a monoclonal antibody specific for the 0–9 determinant on the endotoxin, and a magnetic bead coated with another monoclonal antibody specific for a different O-determinant. At the end of-the test, the magnetic beads are sedimented by use of a magnet, and the result is read based on the turbidity of the indicator latex suspension. Compared to a similar assay developed previously which uses only a single particle reagent (i.e., a tube agglutination system), LIMM was found to be slightly more sensitive especially when using short (< 30 min) incubation times, and was at all times easier to read. The sensitivity of LIMM, in fact, increased with increasing time of incubation. When compared to the sensitivity (25 ng/ml) of a conventional slide latex agglutination test performed using the same indicator latex reagent, this parameter was 0-, 4.9- 12.5- and 28.7-fold better after 5, 15, 30 and 60 min of incubation in the LIMM.

One-step chromatographic immunoassay for qualitative determination of choriogonadotropin in urine

One-step chromatographic immunoassay for qualitative determination of choriogonadotropin in urine

Recombinant Equine Interferon-β1: Purification and Preliminary Characterization

Equine interferon-beta 1 (EqIFN-beta 1) was purified from extracts of recombinant Escherichia coli by sequential chromatography on hydroxylapatite, anion-, and cation-exchangers. The resulting protein was greater than 98% pure as determined by sodium dodecylsulfate gel electrophoresis, gel permeation HPLC, and reverse-phase HPLC. Amino-terminal amino acid sequencing revealed that essentially all molecules contained an additional amino-terminal methionine. The specific antiviral activity of EqIFN-beta 1 determined on equine dermal fibroblasts challenged with vesicular stomatitis virus (VSV) was approximately 5 X 10(8) U/mg. Less than 0.001% of this activity was observed in antiviral assays using human (A549), murine (L-M), ovine (SCP), or bovine (MDBK and BT) cells challenged with VSV or encephalomyocarditis virus. A series of monoclonal murine IgG antibodies were developed which neutralize the antiviral activity of EqIFN-beta 1. None of these antibodies nor rabbit antiserum to EqIFN-beta 1 were able to neutralize human IFN-beta; antiserum to human IFN-beta did not neutralize EqIFN-beta 1. Two of the monoclonal antibodies were used to establish a rapid one-step solid-phase enzyme immunoassay for EqIFN-beta 1.

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 × 10 9 to 2.2 × 10 10 1/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, an 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 μg SOD/1.

A one-step enzyme immunoassay for human manganese superoxide dismutase with monoclonal antibodies

A one-step enzyme immunoassay for the determination of manganese superoxide dismutase in serum has been developed with two kinds of monoclonal antibodies. Proposed method had high sensitivity (assay range, 0.4–200 ng/ml), good recovery (recovery percentage, 102.9–106.2%) and reproducibility (intraassay, C.V. = 1.87–3.66%; interassay, C.V. = 3.03–10.4%). From these results, it is possible to apply this method to routine clinical analysis and biochemical research with various purposes.

One-step enzyme immunoassay for free thyroxin: results with dysalbuminemic sera and one serum containing autoantibodies to thyroxin.

Journal Article One-step enzyme immunoassay for free thyroxin: results with dysalbuminemic sera and one serum containing autoantibodies to thyroxin. Get access R Sapin, R Sapin Institut de Physique Biologique, Faculté de Médecine, Strasbourg, France Search for other works by this author on: Oxford Academic Google Scholar F Gasser, F Gasser Institut de Physique Biologique, Faculté de Médecine, Strasbourg, France Search for other works by this author on: Oxford Academic Google Scholar J Chambron J Chambron Institut de Physique Biologique, Faculté de Médecine, Strasbourg, France Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 35, Issue 5, 1 May 1989, Pages 888–889, https://doi.org/10.1093/clinchem/35.5.888 Published: 01 May 1989

Bispecific antibody-producing hybrid hybridoma and its use in one-step immunoassays for human lymphotoxin.

A hybrid hybridoma cell line secreting a bispecific monoclonal antibody (MAb) was constructed by fusing horseradish peroxidase (HRPO)-immunized mouse splenocytes with previously established mouse hybridomas secreting anti-human lymphotoxin (hLT). This cell line was grown in ascitic fluid in mice to obtain large quantities of hybrid MAbs and a bispecific antibody, reacting with both HRPO and hLT, was separated from the monospecific antibody or other inactive immunoglobulin populations by hydroxylapatite chromatography. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the bispecific antibody molecule contained two different types of heavy and light chains of both anti-HRPO and anti-hLT origin. The bispecific antibody was used to establish one-step enzyme immunoassays (EIAs) employing competitive and sandwich systems. The simple sandwich EIA was able to detect 1-100 U/ml of hLT and there was good correlation (r = 0.96) between hLT concentrations measured by the one-step EIA and a bioassay using L929 cell-lysis.

One-step chemiluminescent immunoassay of free thyroxin with acridinium-ester-labeled thyroxin evaluated and compared with a two-step radioimmunoassay.

This new one-step chemiluminescent immunoassay of free thyroxin (FT4) involving a thyroxin-immunoglobulin conjugate labeled with acridinium ester (Magic Lite System; Ciba Corning Diagnostics Corp., Medfield, MA) is rapid (one 1-h incubation), requires two calibrators per run, and takes 10 s per sample for the quantification step. Analytical performances were excellent: within- and between-run CVs of less than 10% in the working range, no significant effect of hemolysis, bilirubin, or lipemia, and no significant interaction between the conjugate and the thyroxin-binding proteins. Magic Lite results (y) correlated well with those obtained by the Sclavo (x) two-step radioimmunoassay (Sclavo, Siena, Italy): y = 1.35x + 1.32 (r = 0.94, n = 267, P less than 0.001, Sxy = 6.29). Clinical sensitivities (diagnostic efficiencies) for hypothyroidism and hyperthyroidism were 0.91 and 0.98 for normal interval limits of 12 and 21.5 pmol/L (95% confidence interval). Magic Lite results in situations where patient therapy, treatment, or unusual conditions can result in a lack of correlation between the clinical status and the FT4 values were qualitatively the same as those obtained by the Sclavo assay.

Clinical evaluation of Amerlite's one-step immunoassay for determining free thyroxin.

Clinical evaluation of Amerlite's one-step immunoassay for determining free thyroxin.

New one-step enzyme immunoassay for free thyroxin.

This enzyme immunoassay for free thyroxin (FT4) involves simultaneous incubation of sample and thyroxin conjugated to horseradish peroxidase (EC 1.11.1.7) in antibody-coated tubes at room temperature. The test can be performed in 90 min. It is not influenced by variations in concentrations of thyroxin-binding globulin (TBG), thyroxin-binding prealbumin, or albumin. With increased concentrations of non-esterified fatty acids, the measured FT4 equivalent concentrations increase immediately, indicating displacement of endogenous thyroxin from its binding sites. FT4 values obtained with this assay for 110 samples, including sera with low albumin and high TBG as well as sera of patients treated with heparin, agreed well with those measured by a two-step radioimmunoassay and with the T4/TBG ratio. The measurable range of FT4 with our assay is 2.5 to 65 ng/L. Intra-assay CVs range from 3.4% to 2.2%, interassay CVs from 6.6% to 2.6% at concentrations of 9 to 45 ng of FT4 equivalent per liter.

Production of monoclonal antibodies to calcitonin and development of a two-site enzyme immunoassay

A procedure is described for the production of calcitonin-specific hybridomas which involves primary and secondary immunization of Balb/c mice in the hind footpads with free, synthetic human calcitonin and cell fusion of lymphocytes from popliteal lymphonodes with a P3 × 63 myeloma line. This protocol offers the following advantages: (a) it is short and easy to perform, (b) it requires small amounts of unconjugated antigen, and (c) it gives a high yield of antigen-specific IgG-secreting hybridomas. Routine screening was carried out by ELISA on solid phase calcitonin and binding of the monoclonals to free antigen was studied with calcitonin linked to biotin through a 13 carbon atom spacer. Monoclonal couples capable of simultaneously binding calcitonin in solution were found by pairing in all possible combinations 25 purified antibodies, in their unlabelled and biotinylated form, in a checkerboard matrix experimental system. Of the over 30 positive pairs identified, four were used in a one-step enzyme immunoassay for calcitonin determination on microtiter plates in a concn range between 0.1 and 5 ng/ml. With the detecting monoclonal directly conjugated to peroxidase with a heterobifunctional crosslinker, the range of the assay with one monoclonal pair was between 25 and 1000 pg/ml with an 18 hr incubation at 4°C.

Activation of the third component of complement (C3) detected by a monoclonal anti-C3‘g’ neoantigen antibody in a one-step enzyme immunoassay

A previously produced and characterized rat monoclonal antibody recognizing a neoantigen in the human C3‘g’ fragment was used as the capture antibody in an enzyme-linked immunosorbent assay. Detection was made using a polyclonal rabbit anti-human C3d and a peroxidase-linked anti-rabbit Ig antiserum. The activity in normal human EDTA plasma was found to be 3% of that in a zymosan-activated serum pool. Fractionation experiments revealed that most of the activity in normal plasma, in vivo activated plasma and in vitro activated serum eluted in one peak with a molecular weight corresponding to iC3b. A positive correlation ( P<0.01) was found between the present assay anda previously established two-step C3d ELISA both with respect to normal plasma, individual patient samples and consecutively drawn samples following artificial in vivo activation. Complement activation assays based on specific antibodies to ‘activation antigens’ should be preferred whenever available since they enable direct, rapid and specific quantification of the actual fragment(s).

A monoclonal-based, two-site enzyme immunoassay of human insulin

A procedure is described for the efficient production of insulin-specific monoclonal antibodies, which involves primary and secondary immunization of BALB/c mice in the hind footpads with bovine or porcine insulin and fusion of lymphocytes from popliteal lymph nodes with a P3×63 murine myeloma line. With this protocol, over 200 positive hybrids were obtained from four separate fusions. Dissociation constants of 31 purified monoclonals, cross-reacting with human insulin, were determined by two different methods and ranged between 4 × 10 −10 and 2 × 10 −6 mol/1.24 monoclonals were biotinylated, paired in all possible combinations and tested by ELISA for their capacity to simultaneously bind to human insulin in a two-site assay. More than 40 monoclonal pairs were found which formed a sandwich with the hormone. The development of a simple and rapid one-step enzyme immunoassay is described, which involves a first monoclonal bound to the wells of a microtiter plate and a second monoclonal conjugated to alkaline phosphatase. With this assay, insulin can be determined in a range between 0.08 and 7.5 ng/ml in 3–4 h.