A two-particle turbidometric latex immunoassay for the detection of specific IgM antibodies

A simple two stage assay using latex particles as a reaction indicator has been developed for the detection of IgM antibodies to Trichinella spiralis. In the first stage, magnetic polystyrene beads (Dynabeads) coated with T. spiralis antigen were incubated for 30 min with the test serum. After washing, in the second stage, the assay was developed for 1 h using anti-mu-coated latex particles. After sedimentation of the Dynabeads the turbidity of the resultant latex suspension was measured spectrophotometrically at a wavelength of 400 nm. A decrease in turbidity of more than 20% from that of the control, unreacted, suspension was considered positive. Using an IgM phosphorylcholine-binding monoclonal antibody which was reactive with T. spiralis, the sensitivity of the assay was determined to be 110 ng/ml of antibody. This was 20-fold less than the sensitivity achieved in an indirect enzyme-linked immunosorbent assay (ELISA). When the assay was applied to sera obtained from CBA/N or BALB/c mice, which were either normal or immunized against T. spiralis, the expected results were obtained with titers up to 1/640 observed, and confirmed (r = 0.93, P less than 0.001) in the ELISA.     

Rapid detection of spontaneous bacterial peritonitis by granulocyte elastase latex immunoassay and reagent strip

Spontaneous bacterial peritonitis (SBP) is a serious complication in patients with liver cirrhosis that requires rapid recognition for effective antibiotic therapy. Elevated levels of granulocyte elastase (GE), an enzyme that is released from degranulated polymorphonuclear neutrophils(PMN), have been reported in ascitic fluid of SBP patients. The aim of this study was to assess the utility of GE measurement by a latex immunoassay (LIA) and by reagent strips for rapid diagnosis of SBP. In 26 ascitic samples which had differing GE concentrations, the results of this LIA method closely correlated with those of a GE/alpha1-PI complex ELISA and an EIA using monoclonal antibodies against GE. The evaluation parameters of linearity (r > 0.99), analytical recovery (96-107%) and within-assay variation[coefficient of variation(CV): 0.97-2.35%] were found to be satisfactory. In 58 ascitic samples from patients with liver cirrhosis, GE levels confirmed by LIA in SBP ascites (n=14) at the time of diagnosis were higher (1436.9 +/- 715.1 ng/ml) than those in non SBP ascites (n=44)(13.1 +/- 3.9 ng/ml). The receiver operating characteristic (ROC) curve showed that ascitic GE by LIA enabled discrimination between SBP and non-SBP, and a cut-off value of 49.5 ng/ml had a sensitivity of 85.7% and specificity of 97.7%. In addition, the usefulness of reagent strips designed for testing cervical mucus for rapid bedside detection of SBP was assessed for GE. The sensitivity, specificity, and positive and negative predictive values of the reagent strips for diagnosis of SBP were 92.9%, 90.9%, 76.5%, and 97.6%, respectively. These results indicate that GE-LIA and GE reagent strips are rapid and sensitive and can aid diagnosis of SBP.     

Use of microtiter plates for latex agglutination immunoassay of serum alpha 2-macroglobulin

A simple, sensitive turbidimetric immunoassay is described for analysis of alpha 2-macroglobulin (AMG) in serum, using microtiter plates and a commercial kit that features a sensitive latex reagent. Using microwells as reaction vessels and the latex reagent made it possible to minimize the sample size and the amounts of reagents. The procedure involves two pipetting steps and a brief incubation (10 min) at room temperature; the assay measures AMG concentrations ranging from 0.12 to 5 g/L and is unaffected by icterus, hemolysis, lipemia, or rheumatoid factor, even at high concentrations. Recovery of AMG added to three pooled serum samples averaged 95-105%. CVs for replicate analyses ranged from 1.9 to 4.8% (within-run) and 6.1 to 9.3% (between-run). Based on paired analyses of 70 serum specimens, AMG concentrations obtained by this turbidimetric microtiter immunoassay were in close agreement (correlation coefficient = 0.99) with results obtained by an enzyme-linked immunosorbent assay (ELISA). Serum AMG concentrations in 48 healthy adults (mean +/- 2SD) were 1.87 +/- 1.00 g/L.     

Determination of plasma noradrenaline by automatic high pressure liquid chromatography with electrochemical detection

A method for the plasma noradrenaline (NA) analysis by reversed-phase liquid chromatography with electrochemical detection is described. The plasma sample preparation includes two-steps: ion-exchange and alumina adsorption. The use of a data system and an automatic sampler enables the injection of samples in series. Using this technique, determination of plasma NA concentration in 34 healthy subjects (27.4 +/- 7.2 years) gave the following results: 0.28 +/- 0.10 micrograms/l after 30 min of resting in the supine position and 0.52 +/- 0.17 micrograms/l after 5 min of upright position.     

Serum FDP assay with latex photometric immunoassay and its analysis by immunoblotting

Sixty patients' sera with FDP-E value ranged from 200 to 2,500 ng/ml by LPIA system were assayed for FDP-Total (FDP-T) by the same system and FDP-DD by ELISA. Correlations of values between FDP-E, FDP-T and FDP-DD were excellent and ratio of FDP-T/FDP-E (T/E) and FDP-DD/FDP-E (DD/E) from same 60 sera were 32.4 + 7.4 and 1.55 + 0.50, respectively. Ratio of T/E from sera of FDP-E value demonstrating more than 500 ng/ml were classified into three groups, namely, 25.0-35.0, less than 18.0, and more than 50.0. Ten sera in each group were analyzed for FDP fragments of D, DD, Y, DY or X (DY/X) and high molecular weight (HMW) with SDS-PAGE and immunoblot method. It was found that relative concentration and proportion of D and DD.E fragments showed its characteristic pattern in each group.     

Homogeneous latex immunoassay for thyroid hormone testing. Determination of thyroxine and triiodothyronine

We describe here two latex immunoassays for total thyroxine (T4) and total triiodothyronine (T3). These homogeneous 60 min assays are quantified by optically counting the monomeric particles remaining after agglutination. When precision is assessed, both methods display coefficients of variation of 3-7% for within-run assays and 4-10% for between-run assays. The accuracy of the methods, as tested by dilution and spike recovery experiments, was found to be satisfactory. Two correlation studies were carried out to compare the present method with leading commercial methods. The coefficients obtained were: r = 0.92 and r = 0.93 with 150 sera for T3, and r = 0.95 (150 sera) and r = 0.93 (108 sera) for T4.     

Ultrasound enhanced detection of individual meningococcal serogroups by latex immunoassay

AIMS: To examine A, C, Y, and W135 Neisseria meningitidis serogroup characterisation by ultrasonic standing wave enhanced latex agglutination tests (USELATs) of clinical samples. In addition, to determine USELAT enhancement of detection sensitivity for the individual antigens compared with conventional card latex agglutination tests (LATs). METHODS: Wellcogen (Abbott Murex), Slidex meningite kit 5 (bioMerieux), and Pastorex (Sanofi) kits and beads coated in house with antibodies to Y and to W135 alone were tested. Positive control antigens consisted of A and C polysaccharide preparations and the Pastorex Y/W135 kit sample. The limiting concentrations of antigen detection were determined by USELAT and by LAT. Thirty five clinical samples (plasma), previously characterised by the polymerase chain reaction (PCR) and culture, were tested by USELAT and, when sample volume allowed, by LAT. RESULTS: USELAT enhancement of control antigen detection ranged from 16 to 128 fold for the different latex systems. Enhancements for the different control antigens were comparable between kits. USELAT tests of clinical (A/C/Y/W135) samples (n = 15) with the Wellcogen (A/C/Y/W135) and Slidex meningite (A/C/Y/W135) kits showed comparable specificities. A set (n = 22) of Y and W135 samples gave 18, 19, and 17 positive results for Wellcogen (A/C/Y/W135), Pastorex (A/C/Y/W135), and in house beads (Y/W135), respectively. Positive USELAT PCR and culture results were concordant. A typical sensitivity for the commercial kits was 80% (Wellcogen). CONCLUSIONS: USELAT identified serogroups for 80% of samples, whereas LATs identified only 40%. The USELAT detection of the A, C, Y, and W135 antigen serogroups showed comparable enhancement for the kits tested. The commercial availability of latex beads coated with antibody to the Y and W135 serogroups would expedite their identification.     

Correlation of bioluminescent assay of gentamicin in serum with agar diffusion assay, latex agglutination inhibition card test, enzyme immunoassay, and fluorescence immunoassay.

An improved bioluminescent assay of gentamicin in serum, based on the dose-dependent effect of the agent on the accumulation of extracellular ATP in Escherichia coli LU 14 cultures, is presented. The accuracy of the bioluminescent assay of gentamicin, expressed as the mean coefficient of variation over the therapeutic range, was 2.8%. Corresponding figures for an agar diffusion assay, a latex agglutination inhibition card test, an enzyme immunoassay, and a fluorescence immunoassay were 6.4, 17.5, 4.2, and 9.4%, respectively. All of the methods correlated well (r = 0.926 to 0.976), with the possible exception of the card test (r = 0.777 to 0.841). The bioluminescent assay requires only 1 microliter of serum, which allows for capillary sampling, and results are available within 75 min.     

Determination of serum apolipoproteins A1 and B by immunoturbidimetry using the Cobas-Bio centrifugal analyzer. Influence of hyperlipemia

The authors describe an immunoturbidimetric technique for the determination of the serum apolipoproteins A1 and B using a Cobas-Bio centrifugal analyser. This methods is sensitive and reproducible (CV less than 5%) and show a good correlation with electroimmunodiffusion which was taken as method of reference. It may be applied to serum stored at 4 degrees C or frozen at -20 degrees C. No light is transmitted in the case of either turbid hyperlipemic sera or sera extensively icteric or hemolyzed. The physiological values found in a large population of men and women of various ages are in agreement with those reported in the literature. The method described thus provides a suitable means of investigation adapted to large series for screening for coronary disease states.     

Meningitis antigen detection: interpretation of agglutination by ultrasound-enhanced latex immunoassay

Detailed instructions for performance and interpretation of ultrasound-enhanced latex agglutination tests for the rapid identification of bacteria causing meningitis are described. This recently developed technique, which enhances the sensitivity of most latex immunoagglutination assays, has been studied mainly in the context of detection of antigens of meningitis-causing bacteria. The test concentrates on the Wellcogen bacterial antigen kit (Murex Diagnostics Ltd) that contains five latex suspensions specific for Haemophilus influenzae type b, Neisseria meningitidis ACYW135, N. meningitidis B/Escherichia coli K1, Streptococcus group B and Streptococcus pneumoniae. Light photomicrographs of positive agglutination are shown. Particular attention is paid to the appearance of the latex in negative control samples following exposure to ultrasound. Guidance is given on interpretation and assessment in clinical samples.     

Diagnostic and prognostic value of serum elastase 1 in acute pancreatitis

Summary Serum elastase 1 was determined in the serum of 38 patients with acute pancreatitis, using specific radioimmunoassay technique. Serving as controls were 36 healthy people, 33 patients with chronic pancreatitis, 49 patients with various GI-tract diseases, and 6 patients with pancreatic carcinoma. Sensitivity of elastase 1 for the diagnosis acute pancreatitis was 97% after admission to the hospital and 100% within 48 h after onset of acute pancreatitis. The determination of elastase 1 is clearly superior to that of trypsin, pancreatic lipase, or pancreatic amylase, if diagnosis has to be made more than 48 h after the onset of the disease. The specificity is restricted, because there are some cases with chronic pancreatitis and GI-diseases with raised values. There is no possibility to estimate the severity of acute pancreatitis by measuring serum elastase 1.Abbreviations AIP Acute edematous interstitial pancreatitis - CT Computed tomography - ERCP Endoscopic retrograde cholangio-pancreatography - NP Necrotizing pancreatitis - GI Gastrointestinal     

A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma

Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined.     

Measurement of glucagon in human plasma by enzyme immunoassay

In order to investigate the validity of an enzyme immunoassay for glucagon, the glucagon levels of human plasma were determined by both enzyme immunoassay (EIA) and radioimmunoassay (RIA). After a glucose load, plasma glucagon measured by both EIA and RIA fell in 12 normal subjects. The glucagon levels measured by both assays during glucose tolerance test showed good agreement in a group of 10 patients. After arginine infusion, plasma glucagon increased in 6 normal subjects and 3 patients and glucagon values measured by EIA correlated well with those by RIA. The present study demonstrates correlation between glucagon levels measured by RIA and EIA and indicates the usefulness of EIA for determining glucagon in human plasma.     

Comparison of latex agglutination with enzyme immunoassay for detection of rotavirus in fecal specimens

Human rotaviruses are the most important etiologic agents of acquired diarrhea in infants and young children worldwide. Early diagnosis is essentialfor effective patient treatment. The latex agglutination (LA) assays for rotavirus diagnosis are rapid, inexpensive, and the most widely used to screen specimens. The performance of the LA Rotagen (Biokit S.A., Barcelona, Spain) was evaluated for rotavirus detection infecal samples of outpatients with acute gastroenteritis. This assay was compared with the enzyme immunoassay (EIA) EIARA (Bio-Manguinhos, Rio de Janeiro, Brazil). From January to October 2000, 285 fecal specimens were analyzed. Forty-four samples (15.4%) were reactive, 214 (75.4%) were nonreactive, and 27 (9.5%) were indeterminate by LA. All LA-positive samples were positive by EIA, and 2 LA-negative samples were positive by EIA. Of specimens indeterminate by LA, 67% were positive by EIA. The sensitivity, specificity, and accuracy of LA were 69%, 100%, and 93%, respectively. These results indicate that assay is as sensitive and specific as the EIA, and it could be applied on a large scale for screening stool specimens in suspected rotavirus diarrhea. However, the indeterminate results must be confirmed by other methods, such as EIA.