An enzyme-linked immunoassay for the collagen cross-link pyridinoline.

An inhibition immunoassay method for the determination of pyridinoline was developed with the use of microtitre plates coated with a pyridinoline--gelatin conjugate and rabbit antisera directed against pyridinoline linked to bovine serum albumin. The sensitivity of the assay is about 2pmol of pyridinoline, and the presence of related pyridinium and lysine-derived compounds does not significantly interfere with the procedure. Its application to tissue and human urine samples is described.     

Radioimmunoassay of detomidine, a new benzylimidazole drug with analgesic sedation properties

A sensitive and specific radioimmunoassay was developed for detomidine, 4(5)-(2,3-dimethylbenzyl)imidazole. The antibodies were raised in rabbits against a conjugate of detomidine and bovine thyroglobulin prepared by diazo reaction. Detomidine was iodinated with chloramine-T and immunoreactive tracer was purified in cation exchange chromatography. The sensitivity of the RIA was 1.6 fmol/tube allowing direct detomidine measurements from minute serum and urine samples (0.1-0.2 microliter) as well as tissue homogenates (10 microliters). For concentrations below 16 pmol/ml chloroform extraction was used to extend the measurement range to 0.3 pmol/ml. Detomidine (80 micrograms/kg iv and im) was given to one horse and two calves and blood samples were taken and urine collected for 24 h whereafter the horse was slaughtered and tissue samples taken for RIA analyses. Serially diluted serum, urine and tissue samples produced a linear displacement curve parallel to synthetic detomidine in RIA. HPLC studies showed that serum and tissue immunoreactivity was unchanged detomidine whereas most immunoreactivity in the urine was due to an unknown metabolite.     

Radioimmunoassay for neopterin in body fluids and tissues

Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioimmunoassay of epitestosterone: methodology, thermodynamic aspects and applications

A radioimmunoassay system for 17 alpha-hydroxy-4-androstene-3-one (epitestosterone) was developed and evaluated using rabbit antisera against 17 alpha-hydroxy-4-androstene-3-one-3-(O-carboxymethyl)-oxime-bovine serum albumin conjugate, and radioiodinated homologous histaminyl derivative of epitestosterone as a tracer. Two antisera with different specificity were used for radioimmunological determination of epitestosterone in hydrolyzed urine and plasma, respectively. Apparent intrinsic association constants and derived thermodynamic variables for ligand-antibody interaction were measured under various conditions in order to set up an optimal assay protocol. The mean plasma epitestosterone levels for healthy subjects were 0.44 +/- 0.15 nmol.l-1 (means +/- SD) in males and 0.092 +/- 0.056 nmol.l-1 in females. The corresponding concentrations in urine were 320 +/- 70 nmol.l-1 (males) and 273 +/- 100 nmol.l-1 (females).     

Development of a competitive enzyme immunoassay for 17α-19-nortestosterone

Because 17β-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17α-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17α-19-nortestosterone-3-carboxy-methyloxime—bovine serum albumin (17α-19-NT-3-CMO-BSA), the competitive incubation of 17α-19-NT and the 17α-19-nortestosterone-3-CMO—horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17α-19-nortestosterone was used to produce an antibody with selective affinity for the 17α-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B₀ 50% of ± 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.     

Development of an enzyme-linked immunosorbent assay of thromboxane B2 using a monoclonal antibody

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody against thromboxane B2 (TXB2). As a specific antigen, the bovine serum albumin conjugate of TXB2 was adsorbed onto polystyrene microtiter plates. The sensitivity of the monoclonal antibody was compared by means of three different enzyme conjugates, all commercially available. The detection limit with immunoglobulin conjugates of alkaline phosphatase and horseradish peroxidase was 0.04 ng of TXB2 per sample. The use of horseradish peroxidase coupled with an avidin-biotin complex allowed a tenfold increase in sensitivity to 0.0045 ng of TXB2 per sample. The suitability of the assay was checked with TXB2-containing human serum and urine samples, which yielded unchanged standard curves. Recovery experiments had an accuracy of r = 0.960 and r = 0.987. Validity was confirmed by a good correlation between radioimmunoassay and ELISA (r = 0.949). Results of an inhibition experiment with platelet-rich plasma in the presence and absence of ibuprofen demonstrated the practical applicability of this method.     

Quantitative detection of C-reactive protein using phosphocholine-labelled enzyme or microspheres

C-reactive protein (CRP) is a positive, acute-phase protein. Plasma levels rise dramatically in response to tissue injury or inflammation and fall rapidly after recovery or treatment. Antibody-based human CRP test systems do not readily detect CRP from other animals due to the species specificity of antibodies directed against human CRP. Thus, generic systems for CRP detection, based solely on the interaction between CRP and phosphocholine (PC), have been developed. PC-bovine serum albumin (PC-BSA) conjugates were produced and either labeled with horseradish peroxidase to facilitate CRP detection in a CRP enzyme-linked sorbent assay (ELSA) or coupled to carboxy-modified microspheres to facilitate the nonenzymatic, turbidimetric detection of CRP. The CRP-ELSA is a competitive assay, where the total assay time is 45 min, the assay sensitivity is 1.06 mg/L CRP, and the dynamic range of the assay is 0-500 mg/L. When PC-BSA conjugate is covalently coupled to carboxylated microspheres, agglutination occurs in the presence of CRP, the extent of which depends on the quantity of CRP present in the sample. Total assay time is 5 min with a dynamic range of 25-500 mg/L. Both assay formats are capable of accurately detecting human CRP and the CRP-ELSA can detect canine CRP as a disease state indicator.     

Urinary desmosine as a biomarker in acute lung injury.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

A cyclic undecamer peptide mimics a turn in folded Alzheimer amyloid β and elicits antibodies against oligomeric and fibrillar amyloid and plaques

The 39- to 42-residue amyloid β (Aβ) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aβ immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aβ, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aβ-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aβ(22-28)-YNGK' elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound--although more weakly--to Aβ(1-42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aβ deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice.     

Production of monoclonal antibodies and ELISA for thebaine and codeine

The ratio of hapten and bovine serum albumin in antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. Monoclonal antibodies against thebaine and codeine were produced by hybridoma fused with the sprenocytes immunized with thebaine- and codeine-bovine serum albumin conjugate and HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. No cross-reaction of anti-thebaine antibody against morphine was observed. Very small cross-reaction appeared in codeine (0.004%). The cross-reaction of anti-codeine antibody against morphine and thebaine was 2.97 and 5.98%, respectively. The full measuring range of the assay extends from 60 pg mL to 1 ng mL for thebaine and 1 ng mL to 100 ng mL for codeine.     

Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese

A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorption-glutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and sensitive sandwich enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxin B in cheese.

A rapid and sensitive screening sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of staphylococcal enterotoxin B (SEB) in cheese by using a highly avid anti-SEB antibody (Ab) as the capture Ab (CAb) and as the biotinylated Ab conjugate. The glutaraldehyde fixation method for the immobilization of CAb on polystyrene dipsticks was superior to the adsorption fixation and the adsorption-glutaraldehyde fixation methods. The glutaraldehyde fixation method resulted in a higher surface-saturating CAb concentration as evaluated by the peroxidase saturation technique and by the ability of the CAb-coated dipstick to discriminate between positive and negative controls (index of discrimination). Of nine blocking agents used alone or in pairs, lysine-human serum albumin, bovine serum albumin, human serum albumin, and gelatin effectively saturated available sites on the CAb-coated dipsticks without causing interference with the antigen-Ab reactions. The addition of 1% polyethylene glycol to the diluent of the biotinylated anti-SEB Ab conjugate improved the detection of SEB. A concentration of 4% polyethylene glycol allowed a 5-min reaction time for the streptavidin-biotin-horseradish peroxidase conjugate. Cheddar cheese homogenate reduced the sensitivity of the SEB assay; however, the sensitivity was restored when 1.6% (wt/vol) of either a nonionic detergent (Mega-9) or two zwitterionic detergents (Zwittergent 3-10 and 3-12 detergent) was added to the diluent. By using the rapid sandwich ELISA, a minimum of 0.5 to 1.0 ng of SEB per ml was detected within 45 min. The whole procedure for the analysis of the cheddar cheese samples was completed within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of antibody against aflatoxin Q1.

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

Production and characterization of antibody against aflatoxin Q1

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.     

Higher urine desmosine levels are associated with mortality in patients with acute lung injury

Desmosine is a stable breakdown product of elastin that can be reliably measured in urine samples. We tested the hypothesis that higher baseline urine desmosine would be associated with higher mortality in 579 of 861 patients included in the recent Acute Respiratory Distress Syndrome Network trial of lower tidal volume ventilation (1). We also correlated urine desmosine levels with indexes of disease severity. Finally, we assessed whether urine desmosine was lower in patients who received lower tidal volumes. Desmosine was measured by radioimmunoassay in urine samples from days 0, 1, and 3 of the study. The data were expressed as a ratio of urine desmosine to urine creatinine to control for renal dilution. The results show that higher baseline (day 0) urine desmosine-to-creatinine concentration was associated with a higher risk of death on adjusted analysis (odds ratio 1.36, 95% confidence interval 1.02-1.82, P=0.03). Urine desmosine increased in both ventilator groups from day 0 to day 3, but the average rise was higher in the 12-ml/kg predicted body weight group compared with the 6-ml/kg predicted body weight group (P=0.053, repeated-measures model). In conclusion, patients with acute lung injury ventilated with lower tidal volumes have lower urine desmosine levels, a finding that may reflect reduced extracellular matrix breakdown. These results illustrate the value of evaluating urinary biological markers that may have prognostic and pathogenetic significance in acute lung injury.     

Urinary desmosine as a biomarker in acute lung injury

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/α₁-antiprotease complex concentration or P_aO₂/F_iO₂ ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

Electrophoresis '82 : Advanced methods. Biochemical & clinical applications. Proceedings of the International Conference on Electrophoresis,Athens, April 1982 Edited by D. Stathakos Walter de Gruyter; Berlin, 1983 xvi + 867 pages. DM 260.00

A competitive solid-phase immunoassay for the determination of testosterone in serum samples using time-resolved fluorescence is described. The solid phase is a testosterone-3-(O-carboxymethyl)-oxime-ovalbumin conjugate coated to polystyrene microtiter strips. Europium-labelled polyclonal and monoclonal antibodies against testosterone-3-(O-carboxymethyl)-oxime-bovine serum albumin were compared. Their behavior was quite similar although the polyclonal antibody was more sensitive, giving a detection limit of 15 fmol testosterone per assay. Correlation with RIA was very good (r = 0.982 and y = ?0.150 + 0.969x).     

Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibody against ginsenoside Rb1 was produced by fusing splenocytes immunized with a ginsenoside Rb1- bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very small cross-reaction appeared with ginsenoside Rc and ginsenoside Rd. The full measuring range of the assay extends from 20 ngml-1 to 400 ngml-1 of ginsenoside Rb1. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 &#119 l each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.