Microparticle-enhanced nephelometric immunoassay of human plasminogen

A microparticle-enhanced nephelometric immunoassay was developed for plasminogen quantitation in human plasma. It is based on the nephelometric measurement of the light scattered by microparticle clusters formed during a sandwich reaction between plasminogen, microparticle—anti-plasminogen conjugate, and the free antibodies of anti-plasminogen rabbit antiserum. This immunoassay was sensitive (detection limit in reaction mixture, 34 μg/L) and could be performed in 500-fold diluted human plasma, excluding any interference or sample pretreatment. It allowed the quantitation of plasminogen on a large range of concentrations (17–550 mg/L), with a security in antigen excess reaching 1,100 mg/L, with accuracy (linear recovery in dilution-overloading assay and correlation with conventional immunonephelometry), and precision (within- and between-run coefficients of variation lower than 8%). A normal reference range from 54 to 148 mg/L (mean ± 2 SD) was calculated from plasminogen concentration in plasma from 130 adults. Easy to perform (no washing or phase separation) and rapid (two steps of 30 minutes then 1 hour of incubation at room temperature), this microparticle-enhanced nephelometric immunoassay could be an interesting alternative method for human plasminogen quantitation. © 1996 Wiley-Liss, Inc.     

Measurement of eleven serum proteins by microparticle-enhanced nephelometric immunoassay.

Eleven proteins (immunoglobulins IgG, IgA, IgM, orosomucoid, alpha 1-antiproteinase, haptoglobin, ceruloplasmin, C-reactive protein, transferrin, prealbumin and alpha 2-macroglobulin) in human serum were quantitated by a new microparticle-enhanced nephelometric immunoassay. This is a one step competitive assay, based on the nephelometric measurement of light scattered by clusters of protein-coated microparticles specially synthesized for that use. Statistical evaluation (precision, recovery and method comparison) shows that the determination of serum proteins is reliable and accurate for wide ranges of concentration and that the method is quite adequate for strongly increased concentrations. This microparticle-enhanced nephelometric immunoassay appears to offer an alternative method for routine measurement of a great variety of serum proteins at high and intermediate concentrations, which are usually quantified by radial immunodiffusion or conventional immunonephelometry. On account of its sensitivity, it can also be used for the determination of relatively low concentrations of analytes.     

Microparticle-enhanced nephelometric immunoassay of lactoferrin in human milk

A microparticle-enhanced nephelometric immunoassay was developed for lactoferrin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immunoagglutination of a microparticle-lactoferrin conjugate with an antilactoferrin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 0.2 mg/L) and can be performed in diluted milk (1/3,000 in reaction mixture), excluding any interference or sample pretreatment. It allowed the quantification of lactoferrin on a large range of concentrations (0.675–21.6 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 3% to 6%). Changes in the lactoferrin concentration of human milk during lactation were determined in 190 samples. The concentration and ratio of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.9 g/l, 29%) than in transitional milk (2.9 g/L, 22%) or mature milk (2.5 g/L, 24%). J. Clin. Lab. Anal. 11:239–243, 1997. © 1997 Wiley-Liss, Inc.     

Detection of antithyroglobulin autoantibodies with defined epitopic specificity by a microparticle-enhanced nephelometric immunoassay.

To hydrophilic, polyfunctional spherical microparticles of predetermined diameter, produced by copolymerization of acrylic monomers, we covalently bound human thyroglobulin. The thyroglobulin-microsphere conjugate was agglutinated, in the presence of antimouse immunoglobulins antiserum, by four monoclonal antibodies, each recognizing a different antigenic domain on the thyroglobulin molecule. These agglutinations were quantified by measuring with a specially designed nephelometer the light scattered by clusters of the conjugates. Agglutination with the monoclonal antibody recognizing antigenic domain II of the thyroglobulin molecule was specifically inhibited by some human sera that contained antithyroglobulin autoantibodies. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for these autoantibodies with defined epitopic specificity. Using this assay, we detected and quantified antithyroglobulin autoantibodies in serum samples from all eight patients examined with Hashimoto disease and from most (75%) patients with untreated Graves disease.     

Microparticle-enhanced nephelometric immunoassay for human C-reactive protein.

Polyfunctional hydrophilic microspheres of 125-nm diameter can be produced by copolymerization of acrylic monomers. Purified c-reactive protein (CRP) was covalently bound to these new micropheres, and the conjugate obtained was used as reagent in a microparticle-enhanced nephelometric immunoassay for human CRP. This assay was based on the measure, with a specially designed nephelometer, of the light scattered by aggregates formed during the immunoagglutination of the conjugate with anti-CRP antiserum. Sensitive inhibition of this agglutination by free CRP (6 ng/ml) allowed CRP quantitation in highly diluted serum samples (1/500-1/2,000), excluding any interference or sample pretreatment. The CRP assay was easy to perform (no washing or phase separation), reliable (coefficients of variation ranged from 1.3% to 9.3% for within-run and between-run determinations), and accurate (mean percentage of recovery: 104%; correlation coefficients with accepted analytical methods greater than or equal to 0.97) over a large range of concentrations. The inhibition mode excluded errors in the antigen excess zone and provided total security at high concentrations.     

Quantitative automated latex nephelometric immunoassay for determination of myoglobin in human serum

We have evaluated a new latex nephelometric test for the quantitation of myoglobin in human serum. The assay consists of incubating the diluted serum sample (20-fold) for 12 min at room temperature with latex particles covalently coated with anti-myoglobin antibodies and then quantifying the change of light-scatter produced. The assay is fully automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. There is no interference from bilirubin (up to 340 mumol/l), haemoglobin (up to 7,000 mg/l), or rheumatoid factor (up to 1,100 int. units/ml). Myoglobin standard curve extends from 20 to 380 micrograms/l. Assay detection limit lies around 6 micrograms/l. Coefficient of variation ranged from 2.7 to 7.6%. Correlation coefficient between latex immunoassay and an RIA method was 0.987, calculated from the assay of 37 samples. A statistically significant difference was found between the distribution for females and males. The serum level of myoglobin showed an age-dependent variation. Concentrations up to 60 micrograms/l are considered to be normal.     

Changes in the kappa-casein and beta-casein concentrations in human milk during lactation.

A microparticle-enhanced nephelometric immunoassay was developed for kappa-casein quantification in human milk. Together with a previously reported beta-casein comparable immunoassay, it was applied to 862 samples milk, collected from 82 mothers, to investigate the changes in casein concentrations in human milk during the first twelve weeks of lactation. kappa-casein immunoassay is sensitive (detection limit in the reaction mixture, 0.02 mg/L) and can be performed in diluted milk, excluding any interference or sample pretreatment. It allowed the quantitation of kappa-casein over a large range of concentrations (0.14-4.56 g/L) with accuracy and precision (coefficients of variation from 3 to 10%). beta- and kappa-casein concentrations and percentages among milk total proteins increase between colostrum (2.6 g/L, 14.3% and 1.2 g/L, 6. 5%, respectively) and transitional milk (4.4 g/L, 33.2% and 1.3 g/L, 9.5%), decrease at different rates from the third to the eighth week, then remain stable at least up to the end of the third month of lactation (2.7 g/L, 25.3% and 0.9 g/L, 8.5%). The beta-casein/kappa-casein ratio is higher in colostrum (0.61) than in transitional and mature milk (0.30) and could be related to a better digestibility of colostrum casein micelles by the neonate during the first days of life.     

Quantitative nephelometric assay for determining myoglobin evaluated

A recently introduced automated nephelometric immunoassay involving shell/core particles for determination of myoglobin (Behringwerke) was evaluated with the BNA Nephelometer. Method precision was good: the intra-assay CV varied between 1.5% and 6.1%; with daily calibration, the interassay CV ranged between 1.5% and 7.5%. For usual sample dilutions, the assay response varied linearly with myoglobin concentrations up to 23.1 nmol/L. After automatic dilution by the instrument, concentrations up to 2310 nmol/L could be measured without high-dose "hook" effect. Further manual dilution allowed measurement of myoglobin concentrations up to 26,000 nmol/L. Calibration was stable for at least seven days. We detected no significant interferences from hemoglobin, haptoglobin, bilirubin, iodine-containing contrast media, and rheumatoid factors. Treating lipemic samples with Lipoclean (Behringwerke) decreased test results. Simultaneously drawn serum and plasma samples from the same subject showed no consistent differences in myoglobin concentrations. The mean reference myoglobin concentration was 1.380 (SD 0.82) nmol/L for men and 0.878 (SD 0.45) nmol/L for women. In patients with renal insufficiency, serum creatinine values were moderately related to serum myoglobin values (r = 0.465). Although a commercial radioimmunoassay (Byk-Sangtec) and the nephelometric assay intercorrelated well (r = 0.929), values obtained by nephelometry were significantly lower (P less than 0.05). By both assays, results for heart and skeletal muscle tissue extracts showed no correlation, a finding that suggests the existence of multiple forms of myoglobin in human tissues. We conclude that immunonephelometry is a rapid, practical, and reliable method for measuring myoglobin in serum.     

Development of a microparticle-enhanced nephelometric immunoassay for quantitation of human lysozyme in pleural effusion and plasma

A microparticle-enhanced nephelometric immunoassay, based on polystyrene beads coated with antihuman lysozyme antibody, has been developed for lysozyme quantification in sera and pleural effusions. The standard curve extends from 0.58 mg/l to 18.75 mg/l and no antigen effect was observed. The results showed a good serial precision. The intra-assay precision (n = 20) expressed as CV was between 2.2 and 4.2 in three different concentrations. The inter-assay precision, with different calibration curves (n = 12) was between 6.4 and 7.1. The analytical assay showed a sufficient linearity (r > 0.999). There were no interferences either with haemoglobin (up to 4 g/l), lipids (up to 0.5%, expressed as 1% Lipofundina content), or bilirubin (up to 5 mg/dl). The analytical sensitivity was lower than 0.6 mg/l. The correlation with a Micrococcus lysodeikticus turbidimetric assay showed a correlation coefficient of 0.915. We have studied 92 patients with pleural effusion. In each case, pleural fluid adenosine deaminase activity and pleural fluid to plasma lysozyme ratio were determined. The lysozyme ratio showed similar clinical sensitivity and specificity as to adenosine deaminase.     

Automated latex nephelometric immunoassay of theophylline in human serum.

A latex test was adapted for the nephelometric quantitation of theophylline in human serum. The assay is fully automated on the Behring Nephelometer Analyser with a sampling rate of 150 samples per hour. There is no interference from bilirubin (up to 340 mumol/l), haemoglobin (up to 7000 mg/l), Intralipid (up to 5 g/l), or rheumatoid factor (up to 1100 x 10(3) IU/l). The theophylline standard curve extends from 1.25 to 40 mg/l. The coefficient of variation ranged from 2.4 to 7.4%. The correlation coefficient between the latex immunoassay and the TDx theophylline procedure was 0.988, calculated from the assay of 74 samples.     

Automated quantitative nephelometric latex immunoassay for determining ferritin in human serum.

We describe a rapid and sensitive latex nephelometric immunoassay for quantifying ferritin in human serum. This latex immunoassay procedure uses commercially available ready-for-use reagents [Tina-Quant (a) Ferritin, Boehringer Mannheim] that have a long shelf life. The assay consists of incubating the diluted serum sample (5-fold) for 12 min at room temperature with latex particles covalently coated with anti-ferritin antibodies, and then quantifying the change of light-scatter produced. The assay is fully automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. The method has an analytical range of 3 to 260 micrograms/l. Maximal intra- and inter-assay CVs were 4.0 and 6.2%, respectively. Analytical recoveries ranged from 91.3 to 103.6%. Assay detection limit was less than 3 micrograms/l. Linearity of the test is given throughout the measuring range. There was no interference from bilirubin (up to 340 mumol/l), haemoglobin (up to 7 g/l), or rheumatoid factor (up to 1,100 IU/ml). Turbid and lipemic samples interfere. This interference may be avoided by pretreating these samples prior to assay. Results correlated well with those obtained by an automated ELISA test (r = 0.995) and with those of two commercial RIA methods (r greater than 0.97). This latex nephelometric procedure is a convenient method and represents an interesting alternative to other immunoassays for measuring ferritin in human serum.     

Measurement of serum myoglobin by a turbidimetric latex agglutination method.

We evaluated an immunoturbidimetric quantitation for serum myoglobin by the latex agglutination method using an automated biochemical analyzer. This method is rapid, specific, accurate, precise, and has wide dynamic range. The total assay time is 10 min and is performed at 37 degrees C with continuous monitoring at 570 nm. The assay results were compared with radioisotopic immunoassay results and showed a good correlation coefficient, r = 0.99; Y = 0.98 x + 9.3; N = 79. Sera from healthy adults has a myoglobin concentration in the range of 15-80 ng/ml(N = 362). Sex- and age-related differences were observed. The serum myoglobin levels in males and elderly people showed higher concentration than in females and younger people. The peak elevation of serum myoglobin compared with other cardiac markers was observed within 6 hours after onset of chest pain as well as the CK-isoform ratio (MM3/MM1). All of the serum from 21 patients with definite acute myocardial infarction showed increased serum myoglobin levels (100-1200 ng/ml) upon admission and within 6 hours. The results suggest that assays for serum myoglobin levels are helpful in the early diagnosis of acute myocardial necrosis.     

Examination of a competitive enzyme-linked immunoassay (CELIA) technique and a laser nephelometric immunoassay technique for the measurement of apolipoprotein B

A competitive enzyme-linked immunoassay (CELIA) technique for quantitative measurement of apolipoprotein B (Apo B) was developed. The method is a non-isotopic immunoassay that utilizes a soluble enzyme/antibody complex as a universal labeling reagent. The method was characterized according to precision, sensitivity, recovery and parallelism. The CELIA Apo B method was compared to a commercially available laser nephelometric immunoassay. We found that the nephelometric results were highly correlated with triglyceride levels and the nephelometric assay was susceptible to interference from lipemia or turbidity. The range of values obtained on 56 apparently healthy, fasting young adults was 0.35-1.25 g/l by the CELIA method and 0.40-1.00 g/l by the nephelometric immunoassay. The nephelometric method was more precise (coefficient of variation 5%) than the CELIA technique (CV 10%); however, the CELIA method seems to be less sensitive to interferences.     

Immunoassay of human muscle enolase subunit in serum: a novel marker antigen for muscle diseases

A sandwich enzyme immunoassay method for measurement of beta subunit of muscle enolase in human serum was developed by use of purified antibodies to enolase beta subunit and beta-D-galactosidase from Escherichia coli as label. The assay was specific to the beta subunit with no cross-reaction with the alpha and gamma subunits of human enolase. The measurable range was from 10 pg to 10 ng per assay tube or 1 to 1000 ng/ml serum. Coefficients of variation within-run and between-run for the assay of serum beta subunit were less than 14%. Normal adult sera contained about 6 ng/ml of the beta subunit, and the levels were significantly elevated in sera of patients with muscular dystrophy and those with myocardial infarction. Serum levels of the beta subunit correlated well with those of creatine phosphokinase, but poorly with those of myoglobin in the same samples. The specific distribution of beta subunit in skeletal muscle and heart was confirmed by measuring the levels in various tissue extracts.     

Automated latex nephelometric immunoassay for the measurement of serum lipoprotein (a)

A sensitive immunoassay based on latex particle agglutination has been developed for measuring lipoprotein Lp(a) concentrations in serum or plasma. Carboxylated latex particles (diameter 240 nm) covalently coated with F(ab')₂ fragments of anti-lipoprotein Lp(a) antibodies are incubated with diluted sample (400-fold) for 12 min at room temperature, with the resulting agglutination quantified by measuring the change of light-scatter produced. The assay has been automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. This assay generates a standard curve in the range of 27 to 1750 mg/L, showing inter-assay precision of less than 8%. There were no interferences from plasminogen, bilirubin, Intralipid?, haemoglobin, rheumatoid factor, and apolipoprotein B. No significant differences were observed when fresh and frozen samples were compared. Sample pretreatment with “Lipoclean” clearing agent and sample lyophillization decreased the agglutinating reaction. In two separate studies using 77 and 112 patient sera the Lp(a) values, determined by the latex nephelometric method, the Terumo Macra? Lp(a) ELISA test, and the Pharmacia Apo(a) radioimmunoassay method, gave correlation coefficients of 0.948 and 0.974, respectively. Physiological lipoprotein (a) values were determined in a blood donor group, with the distribution of serum Lp(a) highly skewed, with a mean (SD) and median values of 213(236) mg/L and 116 mg/L, respectively. Concentrations of Lp(a) were found to be age-and sex-independent. This latex nephelometric procedure is a convenient method and an interesting alternative to other immunoassays for routine measurement of human lipoprotein (a). © 1993 Wiley-Liss, Inc.     

A sensitive sandwich enzyme immunoassay for human myoglobin using Fab'-horseradish peroxidase conjugate: methods and results in normal subjects and patients with various diseases

A sensitive sandwich enzyme immunoassay (EIA) for human myoglobin (Mb) was developed. Serum Mb was assayed by incubation with an anti-Mb rabbit IgG-coated polystyrene ball and then with affinity-purified anti-Mb Fab'-horseradish peroxidase (HRP) conjugate. The HRP activity bound to the polystyrene ball was assayed fluorimetrically. The sensitivity was 3.1 pg/tube and serum Mb levels of 0.4-1000 micrograms/l could be determined. The recoveries of Mb added to human sera at 3 concentrations were 92.8-99.8%. The within- and between-assay coefficients of variations (CV values) were both below 10%. The regression equation of values determined by EIA and radioimmunoassay (RIA) was y(EIA) = 0.964x (RIA)--2.30 and the correlation coefficient was 0.984. The mean normal serum concentration of Mb was 21.5 +/- 6.0 micrograms/l (mean +/- SD) in males and 16.9 +/- 5.8 micrograms/l in females. The significance of serum Mb determination by the EIA in various diseases was confirmed. The present EIA for Mb is more sensitive and economical than RIA and should be useful for determining Mb in biological materials.     

Immunoquantification of lipoprotein(a): Comparison of nephelometry with electroimmunodiffusion

A new fully automated nephelometric immunoassay for Iipoprotein(a) quantification in human serum was evaluated using the Behring Nephelometer Analyzer. The assay exhibited a good linearity in the concentration range of 110-1,770 mg/l; at higher concentrations, samples were automatically diluted by a factor of 4. The method is simple, robust, and shows an excellent stability of the calibration curve over several weeks. Intraassay and day-to-day coefficients of variation were 2% and 4.5%, respectively. The method correlated well with electroimmunodiffusion (r = 0.977; n = 123; P = 0.0001). Unspecific turbidity as expressed by an elevated blank value occurred in 3% of all freshly measured samples (n = 392). Storage of the samples for 1 week at 4°C had no significant influence on the results. Frozen sera, on the other hand, cannot be assayed by this method. We believe that this assay is well suited for use in clinical routine work. © 1993 Wiley-Liss, Inc.     

Monoclonal antibody–based immunoassays for serum myoglobin quantification in acute myocardial infarction

We have developed a monoclonal antibody–based enzyme immunoassay and a solid-phase radioimmunoassay for human myoglobin. Both assays are based on competition for the monoclonal antibody between the free myoglobin present in the standards or serum samples and the myoglobin coated to the wells of microtiter plates. Consequently, the absorbance at 630 nm and the radioactivity are inversely related to the concentrations of free myoglobin. The sensitivity of both assays was 10 μg/L with linearity up to 1,000 μg/L. There was no interference with other serum proteins, as judged from analysis of specimens with high concentrations of lactate dehydrogenase, creatine kinase, or hemoglobin. The average serum myoglobin concentration in 30 normal individuals was 67 μg/L. Five patients with cardiac arrhythmias had normal values (average, 63 μg/L) while four patients with myocardial infarction had abnormally high concentrations of myoglobin (300–1,000+ μg/L). In a typical case of myocardial infarction, serum myoglobin rose 21 hr earlier and peaked 12 hr earlier than creatine kinase and its cardiac isoenzyme. These rapid imrnunoassays appear to be useful for the early detection of increased serum myoglobin indicative of myocardial infarction.     

Myoglobin and creatine kinase isoenzyme MB mass assays: intermethod behaviour of patient sera and commercially available control materials

The low biological variation of myoglobin and creatine kinase isoenzyme MB mass (CK-MBm) requires accurate measurements. In the standardization process, in order to effectively measure and correct intermethod variability, the intermethod behaviour of control materials must be the same of patient sera, i.e. they must be commutable. In this work we checked the commutability of some commercially available control materials in pairs of methods for myoglobin and CK-MBm measurements; we assessed the impact of commutable and non-commutable control materials when used for equalizing patient sera results by two different methods and discussed the problems related to external quality assessment schemes. Myoglobin and CK-MBm were measured in sets of 49 and 56 patient sera and in 13 commercially available control materials with two automatic analytical systems. The non-commutability rate was 8.3% for myoglobin and 23.1% for CK-MBm. Recalculation of serum samples results with a control material as calibrator lowered or increased the bias originally present according to whether the material itself was commutable or not. We conclude that also for myoglobin and CK-MBm assays it is necessary to check the commutability of materials to be used in external quality assessment schemes, or to normalize patient results by different methods.     

Rapid and sensitive radioimmunoassays for human myoglobin

A radioimmunoassay for quantitation of serum myoglobin in healthy individuals and patients with different diseases is described. Purified myoglobin was labelled by an 125I-labelled ester (N-succinimidyl 3-(-4 hydroxy, 5-[125I]iodophenyl) propionate), a commercially available antiserum was used, and the antigen-antibody complex was precipitated with polyethylene glycol 6000. The rapid assay can be performed within 1 h at 37 degrees C with a detection limit of 45 micrograms/l. Prolonged incubation at 4 degrees C for 18 or 72 h gives a detection limit of 6 and 2 micrograms/l, respectively. The mean coefficient of variation of the routine assay was 11%. In healthy human subjects a significant difference in mean serum myoglobin concentration was found between 43 women (34 +/- 17 micrograms/l) and 51 mean 47 +/- 15 micrograms/l). In twenty patients admitted to hospital with the clinical diagnosis acute myocardial infarction, the serum myoglobin concentration profiles were in close agreement with the final diagnosis. In three patients with myocardial infarction serum samples were taken every 2 h after the acute episode, and serum myoglobin levels were compared with the levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase and creatine kinase isoenzyme-MB.