Antibody-coated Latex Particles Research Articles

Preliminary Evaluation of two New Rapid Immunoturbidimetric D-dimer Assays in Patients with Clinically Suspected Venous Thromboembolism (VTE)

The practical utility and diagnostic accuracy of two new rapid, automated and quantitative immunoturbidimetric D-dimer methods have been evaluated in a population of 123 randomly selected patients with suspected VTE. The STA Liatest D-dimer and MDA D-dimer methods are based on the photo-optical measurement of the rate of agglutination of antibody-coated latex particles. The VIDAS D-dimer automated Elisa was used as the reference method. Diagnosis was confirmed in 51 patients (29 PE, 19 DVT, 3 DVT+PE). The immunoturbidimetric methods compared favorably with the VIDAS Elisa as judged from the correlation coefficients of linear regression lines (r = 0.82, MDA vs VIDAS; r = 0.75, STA vs VIDAS) and areas under the curve of ROC plots (VIDAS 0.83; STA 0.83; MDA 0.81). At a discriminant value of 500 ng/mL, all three D-dimer assays showed high sensitivity (96-98%), high NPV (93-97%) and moderate specificity (39-42%). Reproducibility of results around the cut-off is an important aspect of the diagnostic utility of D-dimer assays. CV's of duplicate determinations in this critical zone showed average values of 5.4% and 17.0% for MDA and STA, respectively. These data demonstrate that such rapid and automated latex-based methods for the quantitative measurement of D-dimer hold promise as reliable and cost-efficient approaches for the exclusion of VTE. Prospective patient management studies will be required to confirm this.

Clinical Evaluation of an Immunoturbidimetric D-Dimer Assay in the Diagnostic Procedure of Deep Vein Thrombosis and Pulmonary Embolism

We investigated 128 patients with suspected deep vein thrombosis and 26 patients with suspected pulmonary embolism. Plasma cross-linked fibrin degradation products were measured instantly by a new rapid and fully quantitative immunoturbidimetric assay (Boehringer Mannheim) which recognizes the D-dimer epitope by antibody-coated latex particles. Diagnosis of deep vein thrombosis was established by either ascending venography (n=105) or colour duplex ultrasound (n=8), whereas for the exclusion of deep vein thrombosis only venography was accepted. The sensitivity/specificity for the diagnosis of deep vein thrombosis was 98% 44% . Patients with suspected pulmonary embolism were examined by pulmonary angiography (n=19) or perfusion lung scanning alone (n=6), if sufficient. One pulmonary embolism was diagnosed at post-mortem examination. For pulmonary embolism, sensitivity/specificity was 100% 50% . These findings indicate that the new immunoturbidimetric technique is as reliable as former ELISA methods and allows to rule out thromboembolic disorders. D-dimers showed a correlation to the extent of the deep vein thrombosis, proximal thrombosis producing higher D-dimer levels. Patients presenting immediately after the onset of symptoms were found to have higher D-dimers than patients examined after a few days. A quantitative D-dimer measurement thus seems to provide precious additional information of the duration and the extent of thromboembolic disease.

Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry

The simultaneous detection is described of cucumber mosaic virus (CMV), potato virus Y (PVY) and tomato mosaic virus (ToMV) by flow cytometry. Extracts from leaves of healthy and CMV or PVY infected plants were incubated with latex particles, each with a diameter of 3 μm. Extracts from ToMV infected or uninfected plants, however, were incubated with particles, each with a diameter of 6 μm. Beads were washed and incubated in succession with primary and secondary antibodies, the latter labeled with phycoerythrin (PE) or fluorescein (FITC). CMV and PVY were distinguished on the basis of the fluorescence emitted by FITC and PE; ToMV was distinguished from CMV and PVY on the basis of the different diameter (6 μm) of the particles on which it was adsorbed. The three viruses were detected also by another approach. Latex particles with a diameter of 3, 6 and 10 μm were separately sensitized with antibodies specific for CMV, PVY and ToMV. An equal number of sensitized particles was mixed and incubated with the plant extracts containing the three viruses and then with anti-CMV, anti-PVY and anti-ToMV antibodies labeled with FITC. The study describes also a virus purification method based on the use of antibody coated latex particles. The method is simple technically and applicable to the purification of large as well as minute amounts of different viruses (CMV, PVY and ToMV).

Candidal Antigenemia: A Prognostic Determinant

We evaluated 98 hospital patients, debilitated by serious illness or major surgery with or without candiduria, for the development of candidal antigenemia with a commercially prepared antibody-coated latex particle assay. Group 1 (41 patients) had persistent Candida albicans candiduria (colony counts of more than 10,000 per ml.), group 2 (25 patients) had candiduria (more than 10,000 colonies per ml.) due to species other than Candida albicans and group 3 (32 patients) had no documented candiduria. Indwelling urinary catheters were present in 78 per cent of the patients in group 1, 80 per cent in group 2 and 66 per cent in group 3. In group 1 elevated candidal antigen titers of 1 to 8 or more occurred in 87 per cent of the patients with candiduria and other positive sites and in 31 per cent of those with candiduria alone (p less than 0.005), compared to 50 and 18 per cent, respectively, in group 2. In group 3 positive candidal cultures subsequently developed from other sites in 6 patients, 2 of whom had elevated titers. Patients with indwelling urinary catheters and candiduria had higher candidal antigen titers than patients without positive urine cultures.Increased mortality was noted in patients with elevated titers of 1 to 8 and urine cultures positive for Candida albicans plus other positive sites than in those with candiduria alone (100 versus 63 per cent, p less than 0.005). In groups 2 and 3 there was a higher mortality rate among patients with candiduria plus other positive sites. The sensitivity of elevated candidal antigen titers in the presence of candiduria and/or multiple positive culture sites was 62.8 per cent, with a specificity of 84 per cent and a predictive value of 68.7 per cent.We conclude that the commercial candidal antigen detection system was a useful laboratory adjunct in the evaluation of the debilitated patient with candiduria.

Rapid identification of bacterial antigen in blood cultures and cerebrospinal fluid.

The ability of antibody coated latex particle suspensions to aid the diagnosis of septicaemia and meningitis in children was assessed in 272 blood culture sets and 85 cerebrospinal fluid specimens. This was also assessed in cases caused by Neisseria meningitidis group B infection. The method was 100% sensitive and 99% specific, with good predictive values (range 63% to 100%) and provided valuable support to the routine bacteriological examination of blood culture and cerebrospinal fluid.

Presence of high concentrations of circulating Toxoplasma antigens during acute Toxoplasma infection in athymic nude mice.

In mice infected with an avirulent strain of Toxoplasma gondii, circulating Toxoplasma antigens were detectable in the sera during weeks 1 to 3 of infection by a simple agglutination test that uses latex particles coated with anti-Toxoplasma antibodies. An infection in athymic nude mice resulted in high agglutination titers in the anti-Toxoplasma antibody-coated latex particle test and the absence of anti-Toxoplasma antibodies in sera during the acute phase, suggesting that the detection of circulating Toxoplasma antigens is a good tool for the diagnosis of acute toxoplasmosis, especially in severely immunocompromised hosts.

Rapid detection of cross-linked fibrin degradation products in plasma using monoclonal antibody-coated latex particles.

Conformational and structural changes on conversion of fibrinogen to fibrin and its cross-linking by Factor XIIIa lead to the development of new antigenic determinants that permit differentiation between their plasminolytic cleavage products. A monoclonal antibody (DD-3B6/22) that is specific for cross-linked fibrin derivatives containing the D dimer configuration has been used in developing a latex agglutination procedure that can detect fibrin degradation products in either plasma or serum. Fibrinogen or its degradation products do not cross-react with this antibody. Results were calibrated with an enzyme immunoassay, which used a purified D dimer standard. Plasmas from 40 normal subjects, all having D dimer levels below 250 ng/mL measured by enzyme immunoassay, were all negative by latex assay. In contrast, positive latex agglutination titers were obtained with 87 of 88 patients with demonstrated deep venous thrombosis, pulmonary embolism, or disseminated intravascular coagulation. Compared to enzyme immunoassay, latex agglutination assay is less sensitive, but this latex procedure provides a rapid and less elaborate test for elevated levels of cross-linked fibrin degradation products in patients with thrombosis. Plasma assays for fibrin degradation products are preferable to those using serum.

Latex immunoassay of human serum Lp(a+) lipoprotein.

A sensitive latex immunoassay for human serum lipoprotein Lp(a+) is based on direct agglutination by Lp(a+) of latex particles coated with specific antibody. The agglutination is quantified by turbidimetry using a photometer at 360 nm. The stabilization of antibody-coated latex particles by bovine serum albumin occurs under well-defined conditions (pH, concentration of bovine serum albumin, and antibody loading of latex particles). The standard curve of serum lipoprotein Lp(a+) ranges from 0.05 to 1.15 mg/l. Inter- and intra-assay coefficients of variation were less than 8% and 3%, respectively. Results were well correlated with those obtained by electroimmunodiffusion (r = 0.98, n = 108).

Reactivity of Small Molecular Forms of Human Factor VIII/von Willebrand Factor with Botrocetin and Anti-Factor VIII-Coated Latex Particles

Two recently developed tests for measurement of factor VIII/von Willebrand factor (FVIII/vWF), i.e. platelet agglutination by botrocetin and a kinetic latex antigen assay, were compared with ristocetin cofactor and electroimmunoassay, in respect with FVIII/vWF size-distribution. FVIII/vWF was measured in six cases of atypical von Willebrand's disease (type II), in gel-filtered fractions of normal cryoprecipitate and in the course of depolymerization of purified normal FVIII/vWF by disulfide reduction. Small molecular forms of FVIII/vWF from normal and variant type II plasma, and those derived by disulfide reduction of high-molecular weight FVIII/vWF, showed remarkably decreased reactivity in ristocetin-, botrocetin- and latex-assay. We conclude that for botrocetin-induced platelet agglutination, as well as for agglutination of antibody-coated latex particles, multiple interactions with repeating subunits of FVIII/vWF are required. As a practical consequence, the combined measurement of FVIII/vWF by the latex test and electroimmunoassay provides a simple tool for discriminating between the classical von Willebrand's disease and its variants.

Improved detection of bacterial antigens by latex agglutination after rapid extraction from body fluids.

Nonspecific agglutination of antibody-coated latex particles, unrelated to the presence of specific bacterial antigens, is a major difficulty with commercial latex particle agglutination tests. Rheumatoid and other factors are known to interfere with latex tests. We studied the use of six chelating, reducing, and anticoagulatory reagents in a rapid extraction of antigen procedure to free heat-stable antigens of Haemophilus influenzae type b and group B streptococcus which had been added to human sera. We also screened sera for the incidence of nonspecific agglutination from the three following groups: 123 patients with positive serology tests, 112 hospitalized patients, and 87 blood donors. The rapid extraction of antigen procedure involved a 1:4 dilution of the sera with each of the six reagents, incubation at 100 degrees C for 3 min, and centrifugation at 13,000 X g for 5 min. Two commercial latex kits were tested (Bactigen and Wellcogen). Nonspecific agglutination was entirely eliminated by each of the six extraction reagents. Sera from 52% of the patients with positive serology tests, 29% of the hospitalized patients, and 28% of the blood donors showed nonspecific agglutination with Bactigen before extraction. Nonspecific agglutination was eliminated in all but one sample after the rapid extraction of antigen procedure. This simple, rapid extraction procedure eliminated nonspecific reactions in cerebrospinal fluids and amniotic fluids and reduced this problem in urines and sera with each commercial kit used on clinical specimens.

Particle-counting immunoassay of human somatotropin.

Human somatotropin was assayed by a novel automated nonradioisotopic technique, "particle-counting immunoassay," that requires a 45-min incubation and only 60 microL of 10-fold diluted sample. The principle of the assay is agglutination of antibody-coated latex particle by somatotropin, the reaction being measured by the (instrumented) counting of residual non-agglutinated particles. The dynamic range in serum extends from 0.2 to 40 micrograms/L. The between-assay CV was 12% for a concentration of 3.3 micrograms/L and 8.5% for 31.9 micrograms/L. The coefficient of correlation (r) with radioimmunoassay was 0.97. Curves for various dilutions of the macromolecular and monomeric forms of the hormone were not parallel.

Monoclonal antibody to streptococcal group B carbohydrate: applications in latex agglutination and immunoprecipitin assays.

Two monoclonal mouse antibodies with specificities for group B streptococcal capsular antigens were evaluated in assays for the identification of group B streptococci (GBS). One of these antibodies (A9) was shown to precipitate group B carbohydrate antigen in reactions with both purified group B antigen and antigen present in autoclave or enzyme extracts of GBS. A9 antibody was also specific for group B antigen in gel diffusion reactions with extracts of Lancefield group A, B, C, D, F, and G streptococci and was a highly sensitive reagent in detecting soluble group B antigen by counterimmunoelectrophoresis. Antigen extracted from all five serotypes of GBS was shown to be precipitated by A9 antibody. A second monoclonal antibody (C8) was reactive with intact GBS but did not precipitate soluble antigen in bacterial extracts. In contrast with what has been shown for polyclonal rabbit anti-group B antiserum, neither antibody was significantly inhibited in binding or precipitation assays by high concentrations of free rhamnose or other monosaccharides of carbohydrates found in group B antigen. Rhamnose, the most abundant carbohydrate of the group B antigen, does not appear therefore to be an immunodominant determinant in the binding of A9 or C8 antibody. The epitopes of both monoclonal antibodies are exposed on the surface of live as well as heat-fixed GBS cells. A9 antibody-coated latex particles were compared with a commercially available polyclonal latex agglutination reagent and shown to be equally sensitive and specific in the detection of soluble group B antigen in urine and cerebrospinal fluid from patients with GBS infections. Because of its uniformity and defined antigen specificity, which includes reactivity with all five serotypes of GBS, A9 antibody offers the potential of an improved immunodiagnostic reagent for the identification of GBS.

Serological grouping of meningococci and encapsulated Haemophilus influenzae strains by latex agglutination.

The latex agglutination method, utilizing antibody-coated latex particles, was adapted for serogrouping of Neisseria meningitidis and serotyping of encapsulated Haemophilus influenzae strains from agar plates. It was found to give more clear-cut results than conventional slide agglutination. A 100% agreement with the antiserum agar method was found for all strains isolated from blood or cerebrospinal fluid. Many meningococcal strains from nasopharyngeal carriers are autoagglutinable, but some of these gave a positive reaction with the group B latex reagent, although they were negative by the antiserum agar method. The latex agglutination method has several advantages over others: the lack of autoagglutination, easy performance, easy interpretation, and very low consumption of antisera.

DETECTION OF BACTERIAL ANTIGENS BY STAPHYLOCOCCAL CO-AGGLUTINATION TEST

Counterimmunoelectrophoresis (CIE) and agglutination of antibody-coated latex particles have been used to detect bacterial antigens in CSF, blood and urine of patients with certain bacterial infections. Antibody-coated staphylococci coagglutinate in the presence of homologous antigen. Earlier studies in our laboratory (J. Clin. Micro. Jan. 1977) have shown that in 36 cases of H. influenzae type b (HIB) meningitis the coagglutination test (CT) detected antigens in 86% of CSF specimens obtained before therapy while CIE was positive in 72%. Antigens could be detected by CT in 46% of sterile specimens obtained from the same cases 1-10 days after therapy. In further studies, staphylococci coated with 4 different antibodies were used to detect antigens in body fluids of patients with meningitis. The following shows the number of positive reactions per total tested for each organism: No cross reactions were noted among the above or in CSFs from 9 cases of meningitis caused by other bacteria. No false positive reactions were noted in 21 control CSFs. CT is at least as sensitive as CIE in detecting HIB antigens and has the advantage of simplicity and rapidity.

A comparison of the TRCII and latex-particle tests for the titration of FR-antigen

The great convenience of a method using a suspension of antibody-coated latex particles for the titration of fibrinogen-related antigens over the tanned red cell haemaglutination immunoassay (TRCHII test) of Merskey, Lalezari, and Johnson (1969) has been demonstrated. The usefulness of Ca-Ancrod for the defibrination of heparinized plasma is presented here.

Latex agglutination test for the diagnosis of Haemophilus influenzae meningitis.

Abstract Capsular antigens of Haemophilus influenzae were detected in cerebrospinal fluid (CSF) from 28 of 30 (93.3 per cent) clinically diagnosed or suspected cases of H. influenzae meningitis with the use of a rapid, sensitive slide test with antibody-coated latex particles. Two specimens were nonreactive. In one of these, no organisms were found on direct microscopic examination of the CSF, and growth in culture was delayed. No cross-reactions occurred among antigens of H. influenzae types a through f, with type 6 pneumococcal antigen, or with CSF specimens from patients with other bacterial meningitides. Heat-labile factors were responsible for low-titered nonspecific reactions in 2.3 per cent of control CSF specimens. This non-specific reactivity was destroyed by heating the specimens at 100 °C. for 15 minutes. Conversely, the reactivity in CSF specimens from cases of H. influenzae meningitis was not altered by heat. Results of this study indicate that the latex agglutination technique is a valuable diagnostic aid. It provides a rapid and reliable indication of H. influenzae infection, even in some cases where organisms are scarce or not found in the CSF and growth in culture is delayed or absent.

Detection of Fibrinogen Degradation Products by Use of Antibody Coated Latex Particles

SummaryA method for rapid determination of fibrinogen split products in human blood basing on agglutination of antibody coated latex particles is described. It’s sensitivity corresponds to that of Ouchterlony plates (17 μg/ml plasma). The result is available within 1 hr after drawing of blood. The method is semiquantitative with a at least as good reproducibility as the haemagglutination inhibition method. The reaction kinetics follow the principle of optimal proportion in a precipitin system.46 healthy people and 36 patients with various diseases were tested with the described method. 3 controls and 12 patients gave positive agglutination reactions that could not be attributed to the presence of split products. The mechanism of these side reactions which could be distinguished from the reactions due to fibrinogen degradation products is discussed. In the plasma of 10 patients split products could be demonstrated by the latex agglutination technic which was also successfully used to control the course of chronic intravascular coagulation in a patient with disseminated cancer of stomach. On account of the obtained results the method can be recommended as a fairly sensitive screening test for rapid bedside detection of fibrinogen degradation products.

Antibody-latex erythrocyte label. A new method for the detection and counting of minor red cell populations.

Summary. A new technique, employing antibody coated latex particles, for the labelling and precise estimation of minor red cell populations is described. The technique is shown to be capable of detecting minor populations diluted to at least 1 in 1,000,000 in the major population. Labelled cells are easily differentiated from unlabelled cells in a blood film and there seems to be no inherent limit to the sensitivity of this technique. Examinations of artificial cell mixtures revealed labelled cells in expected numbers.

Quantitative Estimation of Split Products of Fibrinogen in Human Serum, Relation to Diagnosis and Treatment

Abstract Split products of fibrinogen and fibrin are found in the sera of patients with defibrination syndrome and/or fibrinolysis. They may result from spontaneous (primary) fibrinolysis or secondary fibrinolysis of intravascular fibrin deposits. The split products can be detected by several immunologic methods. Both immunodiffusion and immunoelectrophoresis in agar gel show abnormal bands in high-titer pathologic serum samples (usually more than 12 µg./ml.). One of the lines present on immunodiffusion is closer to the point of application than is the other. The position of the closer band might result from the presence of small amounts of fibrinogen-sized molecules or from moderate amounts of partially polymerized or digested fibrin. A precipitin test in a capillary tube offers a simple and sensitive method for demonstrating split products; immediate precipitin occurs with high-titer products but lesser amounts may require up to 18 hours incubation. The Fi test, agglutination of antibody-coated latex particles, is simple, rapid, moderately sensitive, and commercially available but sometimes yields false-positive results. The precipitin or Fi test on thrombin-treated blood, plasma, or serum may be positive when split products are present in high titer, can be read immediately, and thus provides a rapid bedside test. Neither the precipitin nor the Fi test is as sensitive as the tanned red-cell hemagglutination inhibition immunoassay (TRCHII) for the quantitation of fibrinogen and its split products. This test is sensitive to 2.0-5.0 µg./ml. of fibrinogen or split products and much more reliable than the other methods. For example, 13 of 22 samples with up to 24 µg./ml. of split products yielded negative results with the Fi test and positive results with TRCHII. Because defective and incomplete coagulation may coexist with fibrinolysis in these clinical syndromes, an excess of thrombin must be added to remove thrombin-clottable fibrinogen and establish the presence of nonclottable split products. It was necessary to demonstrate split products to diagnose occult fibrinolysis; throm-treated normal serum was found to contain up to 2.0-5.0µg./ml. of split products. Up to 768 µg./ml. split products were detected in serum from patients with reduced fibrinogen with associated primary fibrinolysis (idiopathic, hepatic disease), induced fibrinolysis (streptokinase, urokinase) or in defibrination syndrome with secondary fibrinolysis (metastatic cancer, abruptio placentae, diffuse allergic vasculitis). In other patients with secondary fibrinolysis, up to 96 µg./ml. were occasionally encountered during and following obstetrical delivery of normal or dead fetus, in pulmonary embolism, myocardial infarction, and rheumatoid arthritis. The actual quantity of split products was of greatest value in assessing clinical progress. Heparin therapy in patients with defibrination syndrome, for example, was associated with a rise in plasma fibrinogen and a fall in the concentration of split products. The data indicate that trace amounts of fibrinolytic split products may occur in normal serum. Larger amounts are found both in primary and secondary fibrinolysis, which are relatively common disorders.

DETECTION OF CRYPTOCOCCUS NEOFORMANS ANTIGEN IN BODY FLUIDS BY LATEX PARTICLE AGGLUTINATION.

SummaryAntigen of C. neoformans was detected in serum and/or CSF from 7 of 9 cases of CNS cryptococcosis by a rapid, sensitive slide test in which antibody-coated latex particles are agglutinated. The procedure yielded few nonspecific reactions, which were of low degree. Decline in reactivity following treatment suggests that the test has prognostic as well as diagnostic value. Lack of reactivity of blood and CSF from 2 patients was shown not to be attributable to serotype variation.