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.     

C reactive protein concentrations in neonates: determination by a latex enhanced immunoassay.

A latex enhanced immunoassay on a centrifugal fast analyser was used to compare serum C reactive protein concentrations in maternal and neonatal blood. In the neonate the C reactive protein concentration at birth was less than 1.0 mg/l; the concentration rose slightly during the first two weeks of life. There was no correlation between C reactive protein concentrations in maternal and neonatal sera. No significant difference was found between the C reactive protein concentrations in blood obtained by either heel prick or venepuncture.     

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.     

Rotavirus detection using ultrasound enhanced latex agglutination and turbidimetry

Application of a non-cavitating ultrasonic standing wave to suspended microparticles brings the particles into close approximation and has been used previously to enhance the performance of several diagnostic agglutination tests. The sensitivity of rotavirus detection by ultrasound enhanced latex agglutination was compared with conventional test-card agglutination. Application of ultrasound gave a 32-fold improvement in the sensitivity of detection of rotavirus antigen in buffer compared with the test card method. A novel turbidimetric approach was used to measure agglutination occurring following the test-card procedure (in place of visual examination) and following exposure of commercial rotavirus latex reagents to a 4.5 MHz ultrasonic field (in place of microscopy). The sensitivity enhancement over the conventional method achievable through ultrasonic exposure was comparable whether agglutination measurements were made visually or turbidimetrically and demonstrates the potential for turbidimetry in combination with the ultrasonic method. Turbidimetry offers an alternative to visual assessment that may be more easily incorporated into automated systems.     

Falsely low MMP-3 by latex turbidimetric immunoassay

We experienced a case with a falsely low value by a blood MMP-3 measuring reagent employing a recently structured new latex immunoturbidity. The case involved duplicate orders for one patient in a single day, and the blood collection amounts and measured values were approximately 6.0 mL and 206.6 ng/mL and approximately 1.0 mL and 107.5 ng/mL. The latter MMP-3 concentration was 48% of the former, showing a low tendency. Therefore, an experiment was conducted by adding serum to the blood collection tubes with or without a serum-separating agent of four different manufacturers (Terumo, Sekisui Medical, Nipro, and Becton Dickinson), and similar results as our experienced case were obtained with the Terumo tube with serum-separating agent, which had been used in this case. The amount of whole blood was obtained by conversion assuming a hematocrit value of 40%, and the addition ratio was calculated relative to the predetermined amount of the tube showing a falsely low value. Falsely low values were observed at < or = 56%, < or = 21%, < or = 20%, and , or = 33% for Terumo, Sekisui Medical, Nipro, and Becton Dickinson, with tubes containing a serum-separating agent, and at < or =10%, < or =8%, < or =19%, and < or =14% for Terumo, Sekisui Medical, Nipro, and Becton Dickinson, respectively, with plain tubes. Falsely low values were not observed with the 10-ml plain tube of Terumo and the 9-ml plain tube of Nipro (untreated tube). Based on these results, care should be taken if samples are below the predetermined amount of the blood collection tube to determine the serum MMP-3 by this method.     

A one-step two-particle latex immunoassay for the detection of Salmonella typhi endotoxin

A simple and rapid test (LIMM, short for latex immunoassay) is described for detecting Salmonella typhi endotoxin. It involves the simultaneous binding of the antigen by two types of reagent particles contained in a micro-tube: an indicator latex particle coated with a monoclonal antibody specific for the O-9 determinant on the endotoxin, and a magnetic bead coated with another monoclonal antibody specific for a different O-determinant. At the end of-the test, the magnetic beads are sedimented by use of a magnet, and the result is read based on the turbidity of the indicator latex suspension. Compared to a similar assay developed previously which uses only a single particle reagent (i.e., a tube agglutination system), LIMM was found to be slightly more sensitive especially when using short (less than 30 min) incubation times, and was at all times easier to read. The sensitivity of LIMM, in fact, increased with increasing time of incubation. When compared to the sensitivity (25 ng/ml) of a conventional slide latex agglutination test performed using the same indicator latex reagent, this parameter was 0-, 4.9-, 12.5- and 28.7-fold better after 5, 15, 30 and 60 min of incubation in the LIMM.     

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.     

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.     

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.     

Collaborative evaluation of antigen detection by a commercial latex agglutination test and enzyme immunoassay in the diagnosis of invasive candidiasis.

The Cand-Tec Candida detection system and enzyme immunoassay for serum mannan were retrospectively compared in a controlled collaborative evaluation of antigen detection in 32 patients with candidiasis proven by biopsy or culture from a normally sterile site and with sera drawn within 7 days of inclusion. With a threshold titer of 1/8, which excluded false-positive results in 17 hospitalized patients without candidiasis, sensitivities for all 32 patients with candidiasis were 44% for the Cand-Tec assay and 17% for the enzyme immunoassay. Both assays provided greater sensitivity when sera were drawn within 24 h of inclusion in the study and in the category of patients with invasive candidiasis (57% by Cand-Tec and 33% by enzyme immunoassay). The Cand-Tec assay gave false-positive results (titer, greater than or equal to 1/8) in 4 of 6 patients with transient candidemia, in 1 of 20 otherwise healthy patients with rheumatoid factor, and in 1 patient with a positive cryptococcal latex agglutination test. Three serum specimens from 3 of 32 patients with candidiasis contained rheumatoid factor and gave titers of greater than or equal to 1/8 by the Cand-Tec assay. Detection of serum mannan by enzyme immunoassay was less sensitive but more specific than the Cand-Tec Candida detection system for the diagnosis of invasive candidiasis.     

The detection of leptospires by a chemiluminescent immunoassay

Rabbit serum hyperimmune to Leptospira interrogans var icterohaemorrhagiae serovar icterohaemorrhagiae, reference strain RGA was conjugated to the chemiluminescent label ABEI (6-[N-(4-aminobutyl)-N-ethyl amino]-2,3-dihydrophthalazine-1,4-dione) in the presence of the coupling agent, EDC (1-ethyl-3(3-dimethyl amino-propyl)-carbodiimide HCl). The luminescent conjugate was incubated with homologous and heterologous antigens. The results indicate that chemiluminescence may provide an accurate and rapid method of detecting leptospires in biological fluids.     

Invasive meningococcal disease in children in Greece: comparison of serogroup A disease with disease caused by other serogroups

Although invasive meningococcal disease caused by serogroup A is not prevalent in developed countries, a considerable number of cases were recently recorded in Greece. In this study, serogroup A meningococcal disease was compared prospectively with meningococcal disease caused by other serogroups, using similar settings of testing and management during a 5-year period between 1999 and 2003. The Neisseria meningitidis serogroup was determined in 262 cases. Serogroup B predominated, accounting for 158 (60%) of the cases. Serogroup A was second most frequent (19%), followed by serogroups W135 (11%), C (8%), and Y (2%). No cases due to serogroup C were recorded during the last year of the study. Patients with serogroup A disease were older and had a milder course compared to patients infected with serogroups B or C. Toxic appearance, purpura, thrombocytopenia, abnormal coagulation tests, and the need for admission to the intensive care unit, fluid resuscitation, inotropic drugs, and mechanical ventilation were less common. Although morbidity and mortality were lower in these patients, the differences were not significant. Serogroup B is predominant in our area, and the introduction of an effective vaccine against it is a priority. Serogroup A has emerged as the second most common serogroup, but the illness associated with it is milder.     

Receptor binding-like specificity of two anti-cholera toxin monoclonal antibodies detected by latex agglutination immunoassay

Six mouse monoclonal IgG antibodies (mAbs) reacting with the cholera toxin B-subunit (CT-B) were obtained from the spleen cells of mice immunized with CT. They were divided into two groups according to their different reactivities with CT in latex (Lx) agglutination. One group of two mAbs was called "non-repetitive" (NR) because they apparently recognized a single epitope on CT, which was unable to agglutinate Lx coated with these NRmAbs, in contrast with the other group of four mAbs that we called "repetitive" (RmAbs). The two NRmAbs, but not the four RmAbs, failed to react with CT when CT was bound first to its GM1-receptor. Thus, the NRmAbs seemed directed at an epitope close to the GM1-binding site of CT-B. These NRmAbs could represent valuable immunogenic candidates for anti-idiotypic immunization against CT and related enterotoxins.     

Study of β‐casein denaturation by microparticle‐enhanced nephelometric immunoassay

Rabbit anti‐native bovine ß‐casein antiserum reacted with native ß‐casein and fragments f( 1–105/7) and f( 106–209) formed during ß‐casein proteolysis by plasmin. Agglutination of ß‐casein‐coated microparticles by anti‐native ß‐casein antiserum was weakly inhibited by ß‐casein f(1–105/7) and ß‐casein f( 106–209) (0·04 and 1·4%, respectively, compared with native ß‐casein). Immunoreactivity of these ß‐casein peptides in microparticle‐enhanced nephelometric immunoassay was more preserved in the whole ß‐casein than in its isolated fragments. The protein concentration producing 50% inhibition of the ß‐casein‐coated microparticle agglutination with anti‐native ß‐casein antiserum increased during ß‐casein denaturation. A microparticle‐enhanced nephelometric immunoassay, quantifying changes of this inhibiting protein concentration, permitted detection of alteration of the immunoreactivity of ß‐casein during its plasmin proteolysis and heat denaturation, providing an adequate test for the integrity of the whole molecule.     

Detection of antibodies to opioid and glutamate receptors by latex agglutination and enzyme immunoassay

We studied adsorption capacity of 5 latexes to synthetic peptide fragments of μ- and δ-opioid receptors and to GluR1 and NR2A subunits of glutamate receptor. Levels of autoantibodies to opioid receptors in the latex agglutination test and enzyme immunoassay were in good correlation. The level of autoantibodies to opioid receptors measured by these methods was increased in patients with opium narcomania, while the content of autoantibodies to the glutamate receptor subunits was increased in epileptics.__________This revised version was published online in July 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 94–97, January, 2005     

Enhanced serological diagnosis of invasive meningococcal disease by determining anti-group C capsule IgM antibody by enzyme immunoassay

AIMS: To describe the development and evaluation of a diagnostic enzyme immunoassay (EIA) for IgM antibody to serogroup C capsular polysaccharide of Neisseria meningitidis (CCPS). METHODS: Purified CCPS was used as the antigen. The optical densities (OD) that determined the cut-off values for the assay were derived using sera from blood bank donors, and the accuracy was evaluated with sera from patients with culture-confirmed serogroup C and non-serogroup C invasive meningococcal disease (IMD), other infectious diseases, culture-confirmed pharyngeal carriage of N. meningitidis and immunoproliferative disorders. RESULTS: In sera collected between days 5 and 20 following positive culture, the assay had a sensitivity of 92%. The two negative sera were from a single patient but showed a significant rise in OD. The sensitivity declined to 80% on days 21-40 then to 75% on days 41-70. No positive reactions were detected in five samples collected after 71 days. The specificity of the assay was at least 97%. CONCLUSIONS: An EIA for IgM antibody to CCPS was evaluated for local conditions and had acceptable sensitivity and specificity. Use of the assay, based on a single blood sample, provided clinically and epidemiologically relevant information for individual and public health management of IMD.     

Comparison of an enzyme immunoassay and latex agglutination test for detection of galactomannan in the diagnosis of invasive aspergillosis

Aspergillus antigenemia was followed up in 215 consecutively observed bone marrow transplant (BMT) patients over a period of two years, using both a latex agglutination test and a sandwich immunocapture enzyme immunoassay (EIA) with a rat antigalactomannan monoclonal antibody as capture and detector antibody. For each patient, sequential sera (3 to 20) were obtained before and after BMT. No positivity was observed before BMT. After BMT, the EIA and latex agglutination test were positive in 19 and 4 patients respectively of 25 patients with confirmed aspergillosis and 14 and 7 of 15 patients with probable aspergillosis. In 19 of 25 patients with confirmed aspergillosis and 9 of 15 patients with probable aspergillosis, the EIA was more sensitive and detected infection earlier than the latex test. In all positive cases, antigenemia rapidly increased in sequential samples and remained strongly positive. In 31 of 169 (19%) BMT patients without clinical signs of aspergillosis, the EIA was occasionally positive in samples taken within the first month after BMT, giving a specificity of 81 % in these patients. In non-BMT patients suffering from other diseases (n=77), the specificity was 98.7%. The overall positive and negative predictive values for the EIA were 54% and 95% respectively. These results favour the use of EIA for early diagnosis and monitoring of aspergillosis in BMT patients, although the predictive value of transient positivity remains to be ascertained.     

Comparison of the PREMIER cryptococcal antigen enzyme immunoassay and the latex agglutination assay for detection of cryptococcal antigens.

A new enzyme immunoassay (EIA), PREMIER Cryptococcal Antigen, was compared with latex agglutination (LA) for the detection and quantitation of circulating capsular polysaccharide antigen from Cryptococcus neoformans. The clinical evaluation of PREMIER EIA as a screening assay, including 475 specimens with 120 LA and EIA positives, resulted in 99% sensitivity and 97% specificity. The clinical specimens included sera and cerebrospinal fluids as well as 10 rheumatoid factor-positive and 20 anti-nuclear antibody-positive serum samples. This monoclonal antibody-based assay detects serotypes A to D at 0.63, 0.63, 7.8, and 62 ng/ml, respectively. With three different known positive specimens, the assay was found to yield coefficients of variation of 2 to 12% for intra- and interassay comparisons of precision and reproducibility. The primary use for semiquantitative values derived with the LA or EIA is to follow the course of disease and monitor drug therapies. The present data suggest that the PREMIER EIA will be a valuable method for this purpose. We conclude that the PREMIER Cryptococcal Antigen EIA provides a rapid, convenient, and reliable antigen detection method for screening and semiquantitative determination of antigen levels.