Development of a fast solid-phase enzyme immunoassay for C-reactive protein

A fast sandwich enzyme immunoassay has been developed for C-reactive protein (CRP). This method can be used for screening CRP concentration in large numbers of samples providing a non precipitation, non agglutination and non radioactive alternative for assessment of human CRP. Advantages over previously reported CRP sandwich assays include: assay time was reduced from 4 1/2 h to 45 min, incubations were made at room temperature instead of 37 degrees C and serum dilutions required were 100-400 fold instead of 10000-20000 fold. Correlations were good with both nephelometry and phosphorylethanolamine binding assay. The 45% false positives found with the slide-latex anti C-reactive protein method were reduced to 0% by the use of the described method.     

Generation of monoclonal antibodies to prostatic acid phosphatase isoenzyme 2 and application in solid-phase enzyme immunoassay

Monoclonal antibodies specific to prostatic acid phosphatase (PAP) isoenzyme 2 were generated by using an improved hybridoma technique. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were first cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for further subculture. Out of a total of 600 colonies recovered after two cell fusion experiments, 13 were shown to exhibit affinity to PAP isoenzyme 2 by radioimmunoassay. Nine hybrid cell lines which showed high affinity and specificity were established for further evaluation. Their immunoglobulin subclass was determined to be immunoglobulin G. The association constants between PAP isoenzyme 2 and each monoclonal antibody were determined by titration curve in radioimmunoassay (RIA). Three of them (PAP 1, PAP 03, and PAP 019) were shown to be over 1 X 10(9) M-1. From the results of a matrix cross-matching procedure, a pair of antibodies (PAP 03 and PAP 1) reacting with discrete antigenic determinants were identified for preparing a solid phase sandwich enzyme immunoassay (EIA) kit. The designed EIA procedure could be performed within 40 min in a one-stage incubation protocol. The assay time was shorter than that of other commercial RIA or EIA kits, and the sensitivity was 0.4 ng/ml which was comparable to that of RIA kits. The EIA kit was shown not to cross-react with human thyroid stimulating hormone, alpha-fetoprotein, carcinoembryonic antigen, and acid phosphatases derived from tissues other than prostate. Therefore, this design was a simple and rapid method with high sensitivity and specificity for determining PAP isoenzyme 2 in human serum.     

Generation of monoclonal antibodies to alpha-fetoprotein and application in solid-phase enzyme immunoassay

By using an improved hybridoma technique, monoclonal antibodies specific to alpha-fetoprotein (AFP) were generated. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were initially cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for subculture. Out of 800 colonies recovered after two cell fusion experiments, 16 were shown to exhibit affinity to AFP by radioimmunoassay. Six hybrid cell lines which showed high affinity and specificity were selected for further evaluation. From the results of a cross-matching procedure, two pairs of antibodies (AFP 3 and AFP 05; AFP 3 and AFP 013) reacting with discrete antigenic determinants were identified for preparing solid-phase sandwich enzyme immunoassay (EIA) kits. The association constants between AFP and these three antibodies (AFP 3, AFP 05, and AFP 013) were 2.0, 3.7, and 3.8 X 10(9) M-1, respectively. The immunoglobulin subclass of them was determined to be IgG1. The EIA procedure designed could be performed within 40 min in a one-stage incubation and 70 min in a two-stage incubation. The incubation time was shown to be equal to or shorter than that of any other known commercial kits and the sensitivity was less than 1 IU/ml. In order to avoid the high-dose hook effect which occurred in the one-stage incubation procedure, a two-stage incubation protocol was advised.     

Enzyme-linked immunoassay of ursodeoxycholic acid in serum.

A sensitive and specific enzyme-linked immunoassay for the measurement of ursodeoxycholic acid in human serum was developed. Ursodeoxycholic acid conjugated to alkaline phosphatase (from calf intestine) was used as a tracer. An antiserum to ursodeoxycholic acid serum was raised in rabbits using ursodeoxycholic acid--bovine serum albumin conjugate as an antigen. The binding required 1 hr at 42 degrees C; separation of the bound tracer was achieved by addition of a second antibody, and alkaline phosphatase activity of this bound complex was measured colorimetrically. The ratio of bound to total enzyme activity decreased linearly with a logarithmic increase in ursodeoxycholic acid concentration from 20 to 900 pmol. The specificity and sensitivity of this enzyme-linked immunoassay were similar to those of a radioimmunoassay reported previously. The serum ursodeoxycholic acid levels measured by this method correlated well with those determined by gas--liquid chromatography and radioimmunoassay. Based on these findings, this enzyme-linked immunoassay of bile acid might be useful as a tool for the routine clinical analysis of serum bile acids.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.     

A solid-phase immunoassay for the binding of cartilage proteoglycan to hyaluronic acid

A solid-phase assay for detecting the binding of cartilage proteoglycan (PG) to hyaluronic acid (HA) is described. In the assay, HA is immobilized on protamine-treated microtiter wells, the wells are incubated with PG monomer and antibody to PG monomer, and then an ELISA system is used to detect binding of the PG to HA. The specificity of the assay is indicated by the failure to detect PG binding to chondroitin sulfate or albumin-coated microtiter wells, the absence of binding with tryptic fragments of PG monomer other than the HA-binding segment, the loss of binding after reduction and alkylation of PG monomer, and the inhibition of binding by preincubation of PG monomer with small amounts of HA. In contrast to the HA-PG interaction in solution, hyaluronidase digestion of HA does not affect its ability to inhibit the reaction of PG monomer with immobilized HA. The microtiter well-based assay appears to be a rapid, simple, and potentially versatile method for studying interactions with HA.     

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.     

Extending the detection limit of solid-phase electrochemical enzyme immunoassay to the attomole level

Electrochemical enzyme immunoassay methodology has been developed to take advantage of the selectivity of antibody reactions, the amplification feature of an enzyme-based assay, and the ease with which small amounts of the enzyme-generated product can be detected electrochemically. A heterogeneous sandwich enzyme immunoassay was used in this work as the model assay. In this type of assay, the antigen is sandwiched between the enzyme conjugate and a primary antibody that is adsorbed to the solid phase. Alkaline phosphatase is a suitable enzyme for electrochemical assays since it catalyzes the conversion of electroinactive phenyl phosphate to electroactive phenol. The product, phenol, is then quantitated by liquid chromatography with electrochemical detection in a thin-layer flow cell with a carbon paste electrode at 0.895 V vs Ag/AgCl. The current produced by the oxidation of phenol is directly proportional to the analyte (antigen) concentration. The problem associated with these types of solid-phase immunoassays is that the adsorption of the primary antibody is desired while the adsorption of other assay proteins is not. The detection limits are generally defined by the ability to control this nonspecific adsorption. The detection limit of a previous electrochemical assay for rabbit IgG was 100 pg/ml and was limited by a large background current observed in the absence of antigen. In the present study, each step of the assay was examined in order to determine the sources of this background current, and it was found that the major contribution was from the nonspecific adsorption of the enzyme conjugate. Using combinations of Tween 20 and bovine serum albumin as blocking agents, the level of nonspecific adsorption was reduced by 96%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maleimide derivative of hapten for coupling to enzyme: a new method in enzyme immunoassay.

Meta-maleimidobenzoyl derivative of L-thyroxine methyl ester (MBTM) was synthesized and coupled to β-galactosidase at molar ratio of over 5 to 1. More than 97% of the enzyme was found to be labeled with MBTM as examined by double antibody precipitation method in excess of anti-T4 antibody. Maleimide group of MBTM was found to be labile; about 50% was destroyed in 3 hours when prepared in a solution of 1 μg/ml phosphate buffer (pH 7.0, 0.05M). With antiserum dilution of 2,400 fold, reproducible T4 enzyme immunoassay was carried out using double antibody precipitation method. A high sensitivity in the assay was observed on the 0–10 μg/100 ml range.     

Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay.

A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.     

Development of enzyme immunoassay for the herbicide chlorsulfuron

Antibodies against the herbicide chlorsulfuron have been raised and characterized. Enzyme immunoassays (EIAs) for chlorsulfuron, involving labeled antigen or labeled antibodies, have been developed. The kinetics of antigen-antibody interactions in the EIA systems developed has been studied. Both systems exhibit equal sensitivity (1 ng/ml). The values of the coefficient of variation (CV), determined within the range of chlorsulfuron concentrations 1-100 ng/ml to be measured by the systems, are not in excess of 8%. The possibility of using glucose oxidase as a label in EIAs for chlorsulfuron has been demonstrated. Lack of cross-reactivity with a series of sulfonyl- and arylurea derivatives and triazines makes it possible to recommend the EIA systems developed for chlorsulfuron determination in the environment.     

Development of a direct enzyme immunoassay of milk progesterone and its application to pregnancy diagnosis in cows

The purposes of this study were to develop an enzyme immunoassay (EIA) for determination of progesterone in unextracted whole milk and to apply it to pregnancy diagnosis. Paper fibers covalently coupled to antibody specifically and competitively bound ³H-progesterone and 11α-hydroxyprogesterone hemisucccinate-horseradish peroxidase and were stable for 9 mo at ?20°C. The sensitivity, recovery, precision, and cross reactivity of the EIA and a radioimmunoassay (RIA) of milk progesterone were evaluated, and showed that this EIA was comparable to the RIA. Milk samples were collected once daily for one estrous cycle from ten lactating Holstein cows and the progesterone levels were determined by RIA. Milk progesterone in 70 samples measured by EIA were highly correlated (r = 0.90) with the values of RIA for the same samples. Milk samples for pregnancy diagnosis by EIA of milk progesterone were obtained daily from days 20 to 24 after 115 artificial inseminations of 85 lactating Holstein cows. Pregnancy diagnosis by EIA was confirmed by rectal palpation at 30 to 60 days after insemination or return to estrus. The accuracy based on single, two, three, four, and five consecutive samples was from 67.2 to 80.0%, 77.3 to 84.0%, 79.2 to 87.5%, 82.0 to 85.4%, and 85.4%, respectively, for pregnancy diagnosis; and from 95.0 to 98.3% for nonpregnancy.     

Development and clinical application of an enzyme immunoassay for the determination of midregional proadrenomedullin

Adrenomedullin (ADM) is a 52‐amino acid peptide with a variety of physiologic functions such as immunomodulating activity, direct bactericidal activity, maintenance of renal homeostasis, and vasodilatory activity. Midregional proADM (MR‐proADM) is derived from a larger 185‐amino acid precursor peptide, prepro‐adrenomedulin (preproADM), by posttranslational processing. It is suggested to be co‐synthesized with ADM in equimolar amounts and has the advantages over ADM in having a longer half‐life, no bioactivity, and no binding to protein. Therefore, MR‐proADM serves as a surrogate for ADM secretion. In this study, we attempted to develop an enzyme immunoassay (EIA) for quantifying MR‐proADM‐like immunoreactive substance (IS), which is applicable for monitoring plasma MR‐proADM levels. By using β‐d ‐galactosidase‐labeled preproADM(83‐94) as a marker antigen, anti‐rabbit IgG‐coated immunoplate as a bound/free separator, and 4‐methylumbelliferyl‐β‐d ‐galactopyranoside as a fluorogenic substrate, a sensitive and specific EIA was developed for the quantification of MR‐proADM‐IS in human plasma. The lower limit of quantification was 0.032 pmol/well, and the steep competitive inhibition EIA calibration curve obtained was linear between 0.16 and 10 nmol/L. By using human plasma samples containing 0.2 and 2.0 nmol/L of MR‐proADM, the interassay coefficients of variation (reproducibility) were 10.78% and 8.83%, respectively, and intraassay coefficients were 3.91% and 7.81%. Plasma MR‐proADM‐IS level was significantly higher in patients with chronic renal failure (1.39 ± 0.50 nmol/L) compared with healthy subjects (0.19 ± 0.07 nmol/L). These results suggest that our EIA may be useful to evaluate plasma MR‐proADM levels as a biomarker in various clinical settings. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk

As a result of immunization of rabbits with neomycin B (NM) conjugated to sodium periodate-oxidized (SP) transferrin, polyclonal antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive assay variant. The mean recovery rate from NM-spiked milk containing 1.5–10% fat was 111.7% and ranged from 84 to 125.2%. We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases (11/106), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the MRL was exceeded (1690 ng/ml). The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.     

Amperometric determination of alkaline phosphatase activity: application to enzyme immunoassay

An amperometric assay foralkaline phosphatase has been developed using a novel substrate, [N-ferrecenoyl]-4-aminophenyl phosphate. In the presence of alkaline phosphatase the substrate is converted to [N-ferrocenoyl]-4-aminophenol which shows an oxidation peak at + 180 mV. The change in peak current at + 180 mV was found to be related to the enzyme concentration. The assay was found to be suitable for enzyme linked immunoassay using alkaline phosphatase as the marker enzyme.     

Development of a competitive enzyme immunoassay for factor VIII-related antigen

An enzyme immunoassay system basing on a competitive method has been developed to measure factor VIII related antigen (F. VIII R:Ag). A sufficient discrimination at low F. VIII R:Ag concentrations was gained. This method appears to be sensitive to 7,8 X 10(-3) U/ml F. VIII R:Ag showing an intraassay coefficient of variation (CV) of 0,11. In comparison to the commonly used Laurell electroimmunodiffusion assay for factor VIII significant less antisera per sample for the enzyme immunoassay technique is necessary.     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D2 (PGD2) was developed in which PGD2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labeled and free PGD2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3-100 pg for PGD2. The IC50 value for PGD2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enzyme immunoassay was applied to the measurement of PGD2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD2. PGD2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD2. The in vivo level of PGD2 in rat brain was extremely low but determined to be 0.11 +/- 0.03 ng/g tissue (mean +/- S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.