Evaluation of an automated commercial immunoluminometric assay for alpha-foetoprotein.

We evaluated a two-site immunoluminometric assay (AFP LIA-mat Byk Sangtec) for the determination of alpha-foetoprotein (AFP). The assay, involving two monoclonal antibodies which recognize two different alpha-foetoprotein epitopes, is rapid (4 h) with a wide working range (0-600 x 10(3) IU/l), a good lower limit of detection and good reproducibility (CV less than 10%). The regression equation for the AFP LIA-mat (y) and the immunoradiometric assay AFP Bridge Serono (x) was y = 1.175x - 2.27 (n = 95, r = 0.996) for serum and y = 1.16x + 479.2 for amniotic fluid. It did not display a hook effect, and when we assessed the linearity by assaying a high concentration of alpha-foetoprotein, it gave a linear response down to 3.1 x 10(3) IU/l. We also evaluated the clinical response of AFP LIA-mat in 278 patients with different diseases. Eighteen of the 19 patients with hepatocellular carcinoma had alpha-foetoprotein levels greater than 100 x 10(3) IU/l. In contrast, only 5 of the 47 patients with cirrhosis showed values above 50 x 10(3) IU/l, demonstrating that this assay discriminates fairly well between hepatocellular carcinoma and cirrhosis. Data in agreement with the literature were obtained for testicular tumours; all of seven seminomatous tumours presented values below 5 x 10(3) IU/l, whereas 50% of the non-seminomatous tumours (n = 8) presented values above 20 x 10(3) IU/l.     

Serum thyroglobulin concentrations in the first weeks of life as measured with an immunoluminometric assay

In addition to the determination of thyrotropin (TSH) on the 5th day of life as a screening parameter for congenital hypothroidism, serum thyroglobulin concentrations were first measured with a commercially available immunoradiometric assay in those cases presenting with elevated thyrotropin levels. As this thyroglobulin assay required at least 400 microliter serum for a duplicate determination, it was decided to employ a sensitive immunoluminometric assay (detection limit 50 amol/tube) instead, the amount of serum needed being reduced to 100 microliter, the sensitivity to under 3 micrograms/l. We describe the serum thyroglobulin concentrations determined by the immunoluminometric assay in both cord and venous blood and in both full-term and pre-term babies divided into 4 main groups with respect to thyroid function. The criteria for the groups with thyroid dysfunction were determined as a result of a) isolated thyrotropin elevation measured in the dry blood spot test (thyrotropin above 4 mU/l), b) lowered thyroxine:thyroxine binding globulin ratio together with elevated thyrotropin both prior to and under substitution with L-thyroxine. In full-term babies thyroglobulin levels in serum fell steadily over the first months of life. The same effect was seen in pre-term babies. The effect of L-thyroxine on the suppression of serum thyroglobulin levels appeared to be dose-dependent, as newborns from experimental criterium group a) (defined above) showed no suppression of serum thyroglobulin levels when under partial substitution with L-thyroxine (median 84 micrograms/l), whereas those in group b), i.e. with full L-thyroxine substitution showed a noticeable suppression of serum thyroglobulin (median 27 micrograms/l).     

Measurement of plasma hydroperoxide concentration by FOX-1 assay in conjunction with triphenylphosphine

INTRODUCTION: Hydroperoxides are well-recognized reactive oxygen species which are associated with oxidative stress, a phenomenon of current clinical interest as oxidative stress is associated with a number of disease condition. Ferrous ion oxidation xylenol orange (FOX) methods of hydroperoxide estimation has outdated other methods available for hydroperoxide estimation. Two versions FOX assays are described in the literature, FOX-1 and FOX-2, in which FOX-1 is more sensitive. METHODS: we increased the sensitivity of FOX-1 assay by stabilizing the reagent pH 1.7-1.8. Analogous to FOX-2 assay, we have modified FOX-1 assay by using it in conjunction with triphenylphosphine and butylated hydroxytoluene, thus increasing the specificity of FOX-1 assay for hydroperoxide. By modified FOX-1 method, we estimated plasma hydroperoxide concentration of normal human subjects and of diabetic patients and compared with FOX-2 method. RESULTS: The FOX-1 method showed a significant high value of plasma hydroperoxide concentration compared to FOX-2 method both in normal subjects and diabetic patients with a significant correlation. By modified FOX-1 method, the recovery percentage of cumene hydroperoxide was better in biological samples when compared to FOX-2 method. CONCLUSION: The modified FOX-1 method is equally specific for hydroperoxide determination when compared to FOX-2 but is more sensitive.     

Technical evaluation of a new immunoradiometric and a new immunoluminometric assay for thyroglobulin

BACKGROUND: After removal of differentiated thyroid carcinoma (DTC), serum thyroglobulin (Tg) can indicate persistent or recurrent disease. We describe two novel two-step assays designed to measure low Tg concentrations. METHODS: We evaluated prototypes of the new IRMA, DYNOtest Tg-pluS, and the new immunoluminometric assay (ILMA), LUMItest) Tg-pluS. In the first step, a high-salt incubation buffer leads to dissociation of Tg-Tg antibody complexes in serum and is intended to reduce nonspecific interference and interference of potential Tg autoantibodies in the system. We studied recovery of human Tg (from thyroid glands) added to horse serum. We also studied 58 patients with DTC in whom Tg values under thyroid-stimulating hormone (TSH) suppression and TSH stimulation (without thyroxine) were available. RESULTS: The detection limits were 0.04 microg/L Tg for the IRMA and 0.02 microg/L for the ILMA. Intraassay imprecision (CV) was <10% over the range of the calibration curve in both assays. The day-to-day CV was <20% at 0.2 microg/L for the IRMA and at 0.06 microg/L for the ILMA. No high-dose hook effect was seen with up to 200 000 microg/L added Tg or in dilutions of 12 patient sera with Tg values of 307-38 880 microg/L. Mean recovery of 50 microg Tg/L was 96% in those patients. Among 77 samples with Tg antibody values of 65.2-8150 kilounits/L, recovery by the IRMA was disturbed in 7 cases (9%) and by the ILMA in 9 cases (12%). Tg increased as measured in both assays in 50 of 58 patients after thyroxine withdrawal. CONCLUSIONS: The new assays have improved precision for Tg <1 microg/L, and even low measured Tg concentrations respond physiologically to thyroxine withdrawal. The assays are free of a high-dose hook effect up to a Tg concentration of at least 38 000 microg/L and may further reduce Tg antibody interference.     

Measurement of corticotropin in unextracted plasma: comparison of a time-resolved immunofluorometric assay and an immunoradiometric assay, with use of the same monoclonal antibodies

We describe a time-resolved immunofluorometric assay (IFMA) for corticotropin in unextracted human plasma, based on the use of two monoclonal antibodies: europium-labeled antibody 1A12 and antibody 2A3 coated onto microtiter wells. We compared the results of this assay with those of an immunoradiometric assay (IRMA) performed with the same antibodies working ranges (CV less than 10%) were 25 to 1000 ng/L and 22 to 1000 ng/L for the IFMA and IRMA, respectively, and both assays had comparable detection limits (IFMA 4.0 +/- 1 ng/L, IRMA 3.5 +/- 0.8 ng/L). Results by both assays for 130 patients' samples containing corticotropin within the range 3-100 ng/L and greater than ng/L correlated well (r = 0.88 and 0.92, respectively), and samples with corticotropin in the range 80-624 ng/L gave results that paralleled those for the standard curve. Corticotropin concentrations in apparently healthy subjects were consistent with those reported previously. The IFMA is a simple, precise, and robust assay that can be completed within one day. Its nonisotopic label is stable for at least 50 weeks.     

Measurement of beta-2-microglobulin in serum and plasma by an enzyme-linked immunosorbent assay (ELISA)

A simple technique for the measurement of beta-2-microglobulin (beta 2M) in serum was developed. The method was designed as a sandwich technique using rabbit anti-human antibodies, employing commercially available reagents in an enzyme linked immunosorbent assay (ELISA). The assay was of high specificity, sensitivity, accuracy and reproducibility. beta 2M in serum was strongly correlated with age (p less than 0.005), but independent of sex. Values in heparin and citrate plasma were significantly lower than in serum (p less than 0.001), whereas values in serum and EDTA plasma were similar. Release of beta 2M from normal blood cells was not observed in vitro before the test procedure. An excellent correlation between the results obtained in the ELISA and a RIA was demonstrated (rS = 0.99, p less than 0.0001).     

Measurement of brain natriuretic peptide in plasma samples and cardiac tissue extracts by means of an immunoradiometric assay method

Background: The mechanisms of oxygen‐induced effects on blood vessels (vasoconstriction in hyperoxaemia and vasodilatation during hypoxaemia) are uncertain. Many investigators have suggested that the vasoconstriction seen during hyperoxia/hyperoxaemia is mediated through the endothelium as a result of either increased release or activity of vasoconstrictors (oxygen radicals, endothelin, norepinephrine, angiotensin II, or serotonin (5‐HT)), or reduced activity of vasodilators (prostaglandin E₂ and nitric oxide). Serotonin has been assumed to have a central role. Methods: Eight healthy volunteers were exposed to F_iO₂ of 1.0 for 20?min and serum concentrations of serotonin and activated platelets were measured (indicated by concentrations of β‐thromboglobulin (β‐TG)). Results. During hyperoxaemia in humans, serum concentrations of serotonin and β‐TG remained unchanged. Conclusion: If serotonin is involved in oxygen‐induced vasoconstriction, the mechanism is more likely to be either a potentiating effect of serotonin on other vasoconstrictors or increased activity of serotonin on its receptor.     

Measurement of brain natriuretic peptide in plasma samples and cardiac tissue extracts by means of an immunoradiometric assay method

We evaluated the analytical characteristics and clinical usefulness of a commercial immunoradiometric assay (IRMA) kit for brain natriuretic peptide (BNP). Mean (+/-SD) plasma BNP concentrations measured in 129 normal subjects were 2.9+/-2.7 pmol/l (median 2.2 pmol/l; range 0.1-12.4 pmol/l). The mean (+/- SD) value observed in healthy men (2.1 +/- 2.0 pmol/l, n = 49) was significantly (p=0.0009) different to that found in women (3.4 +/- 2.9 pmol/l, n=80). A positive relationship (R=0.214, p=0.0174) was found between BNP values and age. In 65 patients with cardiac diseases, BNP levels increased with the progression of clinical severity of disease; patients with more severe disease [NYHA functional class III-IV, mean (+/- SD) BNP +/- 254 +/- 408 pmol/l, n=22] showed significantly (p<0.0001) increased values compared to patients with mild symptoms of disease (NYHA functional class I-II, mean (+/- SD) BNP=19.6 +/- 17.2 pmol/l, n=43). Furthermore, in 32 patients with chronic renal failure, greatly increased (p<0.0001) BNP values were found both before (mean +/- SD=88. 1+/- 111.1 pmol/l) and after haemodialysis (mean +/- SD=65.6 +/- 76.7 pmol/l), with a significant reduction after haemodialysis (p=0.0004) compared to pre-haemodialysis. The mean (+/- SD) BNP value found in atrial extracts collected during aorto-coronary bypass operations in 15 patients was 14.5 +/- 51.9 pmol/g of cardiac tissue. Moreover, the mean (+/- SD) tissue levels of BNP in 7 heart transplant recipients were 128.4 +/- 117.2 pmol/g of cardiac tissue in atrium, 68.4 +/- 76.7 pmol/g in ventricle, and 10.9 +/- 8.5 pmol/g in interventricular septum. Finally, BNP values found in cardiac tissues of two subjects collected at autopsy were considerably lower (on average 1/1000) than those observed in cardiac tissues of patients with cardiac diseases. The IRMA method for BNP determination evaluated in this study showed a good degree of sensitivity, precision and practicability. Therefore, this method should be a reliable tool for the measurement of plasma BNP levels for both experimental studies and routine assay.     

Measurement of the alpha-mannosidase activities in human plasma by a differential assay.

A differential assay based on their difference in thermal stability has been used to measure the acidic and true intermediate alpha-mannosidases in the plasma of cntrols and individuals homozygous or heterozygous for mannosidosis. The intermediate activity was found to be independent of age, sex or mannosidosis genotype. The acidic alpha-mannosidase did not vary significantly with age or between sexes for groups of the same age. The concentrations of acidic and intermediate alpha-mannosidase showed a positive correlation for adults but not for children. The ratio of acidic to true intermediate alpha-mannosidase might therefore be a useful secondary test for the detection of adult heterozygotes for mannosidosis.     

Evaluation of a sensitive immunoluminometric assay for the determination of C-reactive protein (CRP) in serum and plasma and the establishment of reference ranges for different groups of subjects

A sensitive immunoluminometric assay originally designed to measure C-reactive protein (CRP) in neonates and minimal serum volumes was adapted to measure this protein in a routine method without prior sample dilution. The concentration range covered without prior dilution was 10 micrograms/l to 20 mg/l using a sample volume of 5 microliters serum and a total assay time of less than 2 h. Serum samples were assayed from participants in a community medicine programme (SHIP--Study of Health in Pomerania) of the University of Greifswald, Germany (n = 414), as well as from mother-child pairs at birth (n = 30) and women attending the infertility clinic (n = 36). The validation of the assay was compared with a commercial latex-enhanced turbidimetric immunoassay (Roche Diagnostics--Integra 700) using routine serum samples (n = 60) from hospital patients. Comparison was made with the routine assay used in the SHIP study (Roche Diagnostics--Hitachi 717/Tina Quant). From 414 SHIP samples measured in the immunoluminometric assay, 289 were below the detection level in the turbidimetric (Tina Quant) assay. A significant positive correlation (p < 0.01) between log C-reactive protein concentration with age was found, both in the non-screened (all CRP values) (n = 414, r = 0.222) and selected (CRP < 5.00 mg/l = 90th percentile) (n = 370, r = 0.242) SHIP participants. Women were found to have significantly higher CRP levels than men (women: median age 47 a, median CRP 1.29 mg/l; men: median age 55 a, median CRP 1.00 mg/l--p = 0.016) in the non-selected SHIP participants. The situation was different in the selected group, (median age: men 54 a, women 48 a) where no significant difference in median CRP values between the sexes was seen (men: 0.874 mg/l, women 0.951 mg/l, p = 0.206). The distribution of CRP values in a "Normal Healthy Population" is skewed (mean/median--SHIP: all--2.08; selected--1.49). From the 414 SHIP samples measured in the immunoluminometric assay, 289 were below the detection level (2.5 mg/l) in the turbidimetric (Tina Quant) assay. From the 125 remaining samples the correlation between both methods was acceptable (r = 0.813), the regression line y = a + bx being: CRP (ILMA) = 1.83 + 0.842*CRP (Tina Quant). The Tina Quant assay gave values significantly higher than the ILMA in the range 2.5-25 mg/l CRP (p < 0.001). The total information loss in 289/414 subjects with a CRP < 2.5 mg/l with the Tina Quant assay makes it no longer suitable for epidemiological studies in which CRP is to be studied as a risk factor for cardiovascular events. The comparison between the immunoluminometric assay and the latex-enhanced immunoturbidimetric assay (Roche Integra) was much better. The latter measured down to less than 0.3 mg/l, thus being more suitable for epidemiological studies than the Tina Quant assay from the same producer. The correlation and regression data between the ILMA (x) and the Roche Integra assay (y) were: r = 0.971; CRP (Roche Integra) = 0.635 + 0.984*CRP (ILMA); n = 50.10 sera with CRP levels between 25 and 460 mg/l showed no high-dose hook effect in either assay. The remaining 50 sera were measurable in both assays. The turbidimetric assay gave rise to marginally but significantly higher values than the immunoluminometric assay (p = 0.004). The mothers at birth had a median CRP of 3.64 mg/l (range 1.49-12.6 mg/l), the neonates a median CRP of 34 micrograms/l (range 4-288 micrograms/l). All births were without complications, with gestational periods between 38 and 42 weeks. There was no correlation between maternal and neonatal CRP at birth. Mothers at birth had significantly higher CRP levels than healthy non-pregnant women (p < 0.001). Women attending the infertility clinic had CRP-values similar to age-matched healthy non-pregnant women (median 0.698 mg/l, range 0.05-9.97 mg/l). Interassay coefficients of variation at CRP concentrations of 0.85 and 7.9 mg/l were 8.99 and 7.93%, respectively, for the immunoluminometric     

An immunoluminometric assay for N-terminal pro-brain natriuretic peptide: development of a test for left ventricular dysfunction

Measurement of plasma levels of brain natriuretic peptide (BNP) has been used to assess left ventricular dysfunction and prognosis. Levels of the N-terminus of the precursor of BNP (NT-proBNP) have been reported to be elevated to a greater extent than BNP in left ventricular dysfunction. We have devised a non-radioactive sensitive and specific assay for NT-proBNP based on a competitive ligand binding principle. The chemiluminescent label 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium 9-carboxylate fluorosulphonate was used to label peptides representing domains in the middle and C-terminal sections of NT-proBNP. Assay of the C-terminal section of NT-proBNP (amino acids 65-76) in patients with proven left ventricular dysfunction [left ventricular wall motion index median 0.9 (range 0.3-1.4)] revealed elevated values [median 639 (386-911) fmol/ml] compared with normal controls [left ventricular wall motion index of 2 in all, NT-proBNP median 159 (120-245) fmol/ml, P<0.001]. Measurement of the middle section of NT-proBNP (amino acids 37-49) was not a discriminating test. It is thus possible to derivatize small peptides with a methyl acridinium label and preserve immunodetection with specific antibodies. Such methodology may allow non-radioactive immunoluminometric assays to be devised.     

Measurement of antigen concentration by an ultrasound-enhanced latex immunoagglutination assay

A novel ultrasonic technique that increases the rate and sensitivity of latex agglutination tests (LATs) has recently been described. The technique is based on the fact that suspended latex particles become concentrated in an ultrasonic standing wavefield, thereby increasing the rate of particle-particle collisions compared to the standard LAT procedure. The present work extends earlier qualitative assessments of agglutination and seeks to establish whether quantitative measurement of agglutinate size may be used as an indicator of antigen concentration. The agglutination of latex microparticles coated with antibody to C-reactive protein (CRP) is studied here as a model system to determine the dependence of agglutinate size on analyte (CRP) concentration. Agglutinate size is characterised by image analysis techniques. The results show that agglutinate size decreases with decreasing CRP concentration. A near linear relationship is shown between analyte concentration and the size of agglutinate formed over a 100-fold dilution range. The threshold concentration of 230 pg/mL for detection of CRP in the ultrasonic test is 2560 times lower than that required for a conventional test-card CRP latex agglutination assay.     

Measurement of ferritin in serum by an indirect competitive enzyme-linked immunosorbant assay

This indirect competitive enzyme-linked immunosorbant assay (ELISA) for ferritin, unlike other currently available ELISAS, does not require use of an anti-ferritin antibody-enzyme conjugate. Designed for use on microtiter plates, the method has a precision and sensitivity similar to those of other immunoassays. The detection limit is 20 pg of ferritin per test (corresponding to 2.0 micrograms/L in serum samples). Comparison of results obtained on serum from 57 patients by this method with those from a conventional radioimmunoassay gave a correlation coefficient of 0.92.     

Development of an enzyme-linked immunosorbent assay of apolipoprotein E-AII complex in plasma

Measurement of apolipoprotein (apo) E-AII complex in human plasma is important in determining the role of apoE in lipoprotein metabolism. In this paper, we demonstrate a new and simple method to determine apoE-All complex by using an enzyme-linked immunosorbent assay. Anti-apoE IgG (goat) was used as a capture antibody, and captured apoE-All complexes were detected by an anti-apoAll (rabbit) horseradish peroxidase-conjugated anti-rabbit IgG (goat) system. With this method, apoE-All complex was specifically determined without the interference of apoAll and was not affected by apoE monomer less than 250 mg/L. The content of the complex in reference serum, a normolipidemic serum pooled from five subjects with phenotype E3/E3, was arbitrarily defined as 100%. The coefficients of variation were 3.5%-6.3% within assay and 8.8%-11.6% between assays.     

Quantification of proteolytically active pregnancy-associated plasma protein-A with an assay based on quenched fluorescence

BACKGROUND: Maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) are used to predict the occurrence of Down syndrome. In pregnancy, PAPP-A primarily circulates as a covalent 2:2 complex with the proform of eosinophil major basic protein (proMBP), which inhibits the proteolytic activity of PAPP-A. At term, however, approximately 1% of PAPP-A exists as an active, uncomplexed dimer with proteolytic activity directed specifically toward insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5. No assays have been developed that allow quantification of PAPP-A proteolytic activity. METHODS: We developed a sensitive and specific immunocapture assay for PAPP-A activity based on intramolecular quenched fluorescence. We used a 26-residue synthetic peptide derived from IGFBP-4 in which specific positions on each side of the PAPP-A cleavage site were substituted with 3-nitrotyrosine and o-aminobenzoic acid. RESULTS: The assay detected the activity of recombinant PAPP-A as well as PAPP-A in serum samples from pregnant women. The limit of detection (mean signal of blank plus 3 SD) of the active PAPP-A subunit was 13 pmol/L, and the intra- and interassay CVs were <10% and <15%, respectively. Interestingly, the fraction of active PAPP-A decreased gradually from week 7 to week 19 of pregnancy. CONCLUSIONS: This method allows the measurement of PAPP-A enzymatic activity and because of its specificity it is relevant to the study of the biological function of PAPP-A. The method may also be useful in the diagnosis of pregnancy disorders.     

Measurement of intact human parathyrin by an extracting two-site immunoradiometric assay

This is an immunoradiometric assay of intact human parathyrin, hPTH(1-84). One antibody, directed against the N-terminal part of the hormone, was produced in goats and conjugated covalently to cellulose particles. hPTH(1-84) and the N-terminal fragments were extracted from EDTA-treated plasma by these particles and thus concentrated. Another antibody, against synthetic hPTH(53-84), was raised in rabbits; this bound to the C-terminal part of the hormone. The final step was labeling the second free binding site of this antibody with 125I-labeled Tyr52-hPTH(53-84) and measuring the bound radioactivity. This assay can detect intact PTH in concentrations as low as 0.6 pmol/L (1.2 X 10(-16) mol per tube). The assay did not cross react with hPTH(1-34), hPTH(1-44), hPTH(28-48), hPTH(39-84), hPTH(44-68), or hPTH(53-84) in concentrations up to 6400 pmol/L. In 60 normal subjects, hPTH(1-84) concentrations ranged from 1.9 to 6.8 pmol/L; in 32 patients with primary hyperparathyroidism, from 7.0 to 80 pmol/L. The hormone was not detected in four patients with hypoparathyroidism.