Forces acting on particle-enhanced immunoassays

The aim of the present work is to study the role of the different forces involved in the agglutination of immuno gamma-globulin (IgG) covered latex particles due to antigen-antibody reaction. An experimental investigation on the adsorption of IgG molecules on three latexes with different surface charge densities is described. Photon correlation spectroscopy was used to determine the hydrodynamic layer thickness of the IgG molecules adsorbed on the latexes. In order to get an insight into the forces acting between two antibody-covered particles approaching each other, the colloidal stability and immunoreactivity of these biocomplexes were studied. They can be stabilized by electrostatic or hydration forces. The immunological agglutination of IgG-immobilized latex particles due to the addition of the antigen was quantified through scattered light intensity measurements. The immunoresponse increases with ionic strength of the medium until a maximum value is achieved. Above this maximum, the immunoreactivity decreases.     

Preparation and sensitization of polystyrene latex beads by some antigens and antibodies. Factors affecting sensitivity and specificity of latex agglutination tests

Polystyrene latex beads were polymerized at two different pH (7.3 and 9.5) and their ability to create latex agglutination systems were studied. Sensitization of these latexes by DNA, antibodies to C-reactive protein, myoglobin, and human IgG. was conducted. Sensitivity and specificity of these systems were compared. Practical recommendations are given for preparation of such systems.     

PREPARATION AND SENSITIZATION OF POLYSTYRENE LATEX BEADS BY SOME ANTIGENS AND ANTIBODIES. FACTORS AFFECTING SENSITIVITY AND SPECIFICITY OF LATEX AGGLUTINATION TESTS

ABSTRACT

Polystyrene latex beads were polymerized at two different pH (7.3 and 9.5) and their ability to create latex agglutination systems were studied. Sensitization of these latexes by DNA, antibodies to C-reactive protein, myoglobin, and human IgG. was conducted. Sensitivity and specificity of these systems were compared. Practical recommendations are given for preparation of such systems.     

Small-angle X-ray scattering on latexes

Small-angle X-ray scattering (SAXS) is a tool which allows the study of the structure and the interaction of polymer latexes with great accuracy. The low electron density of the polymers used for the synthesis of latex particles as e.g. polystyrene allows the matching of the contrast by adding sucrose to the serum. Thus, scattering intensities measured at different contrast, i.e., at different excess electron densities can be evaluated (contrast variation). This yields precise information on the radial electron density of the particles. In this article recent SAXS-investigations on latex particles are reviewed. It is demonstrated that core-shell latexes can be analyzed precisely by contrast variation. The same method can be applied to swollen latex particles to examine the polymer concentration near the boundary to the water phase. Here it is shown that the depletion of the polymer molecules near this boundary is very small which points to a minute wall-repulsion effect. Since the excess electron density of polystyrene latex particles in water is very small, the scattering from adsorbed layers of surfactants dominates the measured intensity in this particular system. Therefore the adsorption equilibrium of a given surfactant as well as the competitive adsorption of two different surfactants on a polystyrene latex can be assessed by SAXS.     

Fundamental study on latex reagents for agglutination tests

Competitive adsorption of Fab and Fc fragments on to particles revealed that the main driving forces for the adsorption of Fab and Fc fragments are ionic and hydrophobic forces, respectively. Latex particles were sensitized with antihuman C-reactive protein-antibody under a condition where ionic binding force was suppressed, and hence antibody was supposed to attach to the particles predominantly at the Fc site. The resulting latex indicated a high efficiency for the determination of C-reactive protein. Among the latexes used, a partially hydrolysed styrene-acrylamide copolymer latex was the best with respect to test efficiency and storage stability.     

Particle counting immunoassay (PACIA)--VI. The determination of rabbit IgG antibodies against myelin basic protein using IgM rheumatoid factor

PACIA, which is based on latex agglutination and instrumental counting of residual non-agglutinated particles, is a practical method for the determination of IgG antibodies against myelin basic protein (MBP). These antibodies agglutinated latex to which MBP had been covalently bound by carbodiimide. The agglutination was greatly enhanced by rheumatoid factor, the final reaction being directly proportional to the amount of IgG antibody attached to the latex bound MBP. To avoid interference by endogenous IgM rheumatoid factor each sample was reduced with dithiothreitol. The antibody content of a rabbit antiserum used as a calibrator for both PACIA and a solid phase radioimmunoassay (RIA) was estimated by the Farr precipitation technique using ¹²⁵I-labelled MBP. The sensitivities of PACIA and RIA were similar i.e. 10 ng/ml IgG anti-MBP antibodies. However, the presence of serum proteins tended to decrease the agglutination reaction. The correlation coefficient between methods was 0.93. The worst coefficient of variation achieved by PACIA over daily analyses during 5 days was 23%. Despite this imprecision, which for antibody titration is still acceptable, PACIA seems an attractive method for large epidemiological studies because of its sensitivity, its short incubation time (30 min instead of 24 hr for RIA), facility of application and stability of reagents.     

A review of factors affecting the performances of latex agglutination tests

The present review describes the different strategies followed to improve the performance of latex agglutination tests. The analysis is mainly focused on the diverse parameters that affect the final colloidal stability of the immunoprotein-latex system. These parameters include: the surface properties of polymer carriers; the different kind of antibodies usually employed; the use of BSA as stabilizer; the co-adsorption of various macromolecules (BSA, surfactants and lipids) and antibodies; recent approaches to colloidal stability at high ionic strengths due to hydration forces; and the covalent coupling of antibodies on functionalized latex particles. Special emphasis is given to the relation between electrophoretic mobility and the colloidal stability of the sensitized particles and how this knowledge can be utilized for a better understanding of the immunoagglutination kinetic.     

A review of factors affecting the performances of latex agglutination tests.

The present review describes the different strategies followed to improve the performance of latex agglutination tests. The analysis is mainly focused on the diverse parameters that affect the final colloidal stability of the immunoprotein-latex system. These parameters include: the surface properties of polymer carriers; the different kind of antibodies usually employed; the use of BSA as stabilizer; the co-adsorption of various macromolecules (BSA, surfactants and lipids) and antibodies; recent approaches to colloidal stability at high ionic strengths due to hydration forces; and the covalent coupling of antibodies on functionalized latex particles. Special emphasis is given to the relation between electrophoretic mobility and the colloidal stability of the sensitized particles and how this knowledge can be utilized for a better understanding of the immunoagglutination kinetic.     

Amino, chloromethyl and acetal-functionalized latex particles for immunoassays: a comparative study

Latex particles with different functionalized surface groups (amino, acetal and chloromethyl) for the covalent linking of protein molecules were synthesized and characterized. Immunopurified anti-ferritin antibodies were then covalently coupled with a mean efficiency rate (protein covalently bound to latex particles with respect to the total amount of protein added) of 60%. The reagents developed were applied to the measurement of serum ferritin concentration in a turbidimetric procedure, showing a good measuring range and a lowest detection limit of 3.5 ng/ml in the case of the amino-modified particles. These immunological reagents were compared with a commercial nephelometric method, showing a good linear correlation in all cases but no transferability in the acetal and chloromethyl latex with additional carboxyl groups, probably due to interference with other serum components. The differences among latex found in this study indicate that it would be necessary to optimize the assay conditions for each type of particle, in order to achieve a maximum immunoreactivity.     

Chicken antibodies: a tool to avoid false positive results by rheumatoid factor in latex fixation tests

19 rheumatoid factor (RF)-positive sera (Waaler-Rose test) and ten control sera were tested in latex agglutination. All RF-positive sera agglutinated latex particles coated with the different mammalian IgGs (cow, horse, human, mouse, sheep and rabbit) and with chicken anti-human C3 (which was used as a positive control), but none of them with non-specific chicken IgG. The control sera were all negative except when tested with chicken anti-human C3. Chicken antibodies should therefore be useful in assays were RF could interfere and give false positive reactions (e.g., nephelometry, latex agglutination or ELISA).     

Effect of particle size on phagocytosis of latex particles by guinea-pig polymorphonuclear leucocytes

The effect of the size of foreign particles on phagocytosis by guinea-pig polymorphonuclear leucocytes (PMNs) was investigated using latex particles having approximately the same surface potentials but different sizes. Two types of latexes with different negative surface potentials, those having amino groups (Ami-latexes) and those having carboxylic acid groups (Ca-latexes), were used in this experiment. Since Ca-latexes had different surface potentials for different particle sizes, the carboxylic acid groups were modified by the carbodiimide method to make the surface potentials almost equal. Phagocytosis was estimated by the rate of oxygen consumption. It was found that the phagocytosis reached its maximum when the latex particle size was about 0.5 microns. This finding was plausibly explained by considering the two probabilities that a latex particle comes into contact with a PMN surpassing a potential barrier and the contact takes place at certain receptor areas favourable to phagocytosis on the PMN surface.     

Symposium on the immunodiagnosis of rheumatic and related diseases, Part II. Rheumatoid factors

Human rheumatoid factors are antibodies of IgG, IgA, or IgM class that show reactions with antigenic determinants present on other immunoglobulin molecules. The most commonly measured rheumatoid factor relates to the 19S IgM type, which reacts by agglutination of latex particles coated with 7S IgG and is often measured in the standard latex fixation test. Approximately 65 to 70 per cent of patients with rheumatoid arthritis show positive serologic tests for rheumatoid factor; however, a number of other chronic disease conditions are also associated with positive rheumatoid factor reactions, including infective endocarditis, sarcoidosis, leprosy, and other hyperglobulinemic conditions. Although extensive serologic and immunochemical studies have identified a number of specific antigenic structural sites on immunoglobulin molecules that react with rheumatoid factors, recent studies have shown that a certain proportion of such antibodies may show cross-reactivity with DNA-histone complexes as well. It is still not entirely clear how rheumatoid factors fit into the pathogenesis of rheumatoid arthritis itself.     

The laboratory identification of serum rheumatoid factor in the dog

A modified Rose-Waaler test with sheep red blood cells coated with canine IgG was found satisfactory for detecting rheumatoid (antiglobulin) factor (RF) in dog serum. Low titres of RF were found in some normal dogs. Most dogs with rheumatoid arthritis were positive for RF at titres of 1 in 40 or greater. A small proportion of dogs with diseases other than polyarthritis were also positive for RF. A commercial slide agglutination test used for detecting human RF was unsatisfactory for the dog, giving false positive and negative reactions. A latex tube agglutination test with latex particles coated with dog IgG was developed but non-specific agglutination was a constant technical problem.     

A new method, distribution-analyzing latex immunoassay (DALIA), to measure specific immunoglobulin G against mite and wheat allergen in human sera

A distribution-analyzing latex immunoassay (DALIA), based on the agglutination of latex particles coupled with mite or wheat allergen, was developed to determine allergen-specific IgG in human sera. The immune complex between chemically coupled-allergen latex and specific IgG was agglutinated specifically and efficiently by employing an IgM-type monoclonal second antibody with strong amplification activity. The extent of agglutination was evaluated by determining the relative ratio of volumes (RV) of agglutinates to residual nonagglutinating particles with a particle counter. This method exhibited a high sensitivity (detection limit ≤ 5 munits/ml) in the determination of allergen-specific IgG, and no influence of inhibitory factors such as competitive antibodies (specific-IgA, -IgM) and nonspecific IgG (≤320 mg/ml) was observed. The concentrations of specific IgG against mite allergen in the sera of 130 allergy patients with atopic dermatitis and 52 normal subjects were 22.3 ± 12.3 and 16.5 ±4.2 units/ml, respectively, and the concentrations of specific IgG against wheat allergen in the same two groups were 5.4 ± 4.2 and 2.1 ± 2.2 units/ml, respectively. The coefficients of variation of intra- and interassay ranged from 3.4% to 11.2% in both cases. The present method is an excellent homogeneous immunoassay which may be used as a routine assay that can measure 50 samples per hour without prior treatment.     

Direct agglutination test and other assays for measuring antibodies to Toxoplasma gondii.

The performance of a direct agglutination test for the detection of toxoplasma specific IgG immunoglobulin was compared with that of the latex agglutination test. The direct agglutination test was less sensitive but more specific than the latex agglutination test. Quantitative results were not directly comparable, reflecting the different antibody profiles detected in each assay. The direct agglutination test represents an alternative to the latex agglutination test as a screening test for toxoplasmosis. Patients at risk of life threatening infection require detailed serological examination using additional assays.     

Kinetics of the Agglutination of IgG-coated Latex Particles by Clq: the Influence of Heat-labile Serum Components

Interaction between human C1q and IgG coated latex particles has been studied by means of a standard aggregometer equipment. A dose-dependent agglutination was observed and 100 ng of C1q were readily detected. The kinetics of the agglutination was also monitored. Serum, partially purified C1, and high molecular weight fractions from Sephadex G-200 fractionated serum produced agglutination only in the presence of EDTA. In the absence of this chelator these products disintegrated preformed C1q-IgG-latex particle agglutinates. This disagglutinating principle is heat-sensitive and tentatively macromolecular C1 dependent. The most probable basis of the activity is the competition between C1, with a high affinity for IgG particles, and C1q. The inability of C1 to induce particle agglutination might be caused by the C1 subunits C1r and C1s sterically inhibiting the subunit C1q to bridge between the particles.     

Antibodies against macaque monoclonal immunoglobulin G in rheumatoid arthritis

The presence of rheumatoid factors (RF) in the serum of rheumatoid arthritis (RA) patients is commonly evidenced by agglutination tests: the Waaler-Rose assay, based on the use of human red blood cells (RBCs) coated with rabbit anti-RBC antibodies, and the latex test, which uses latex particles coated with denatured human immunoglobulin G (IgG). The aim of the present study was to characterize the RF able to agglutinate human RBCs coated with macaque antihuman RBC IgG antibodies secreted from macaque-mouse heterohybridomas (two from rhesus monkey and one from crab-eating macaque). Human RBCs coated with macaque monoclonal antibodies (MacMoAbs) were used for agglutination tests and these were carried out in parallel with standard tests (Waaler-Rose and latex agglutination tests) on sera from 82 RA patients, 86 patients with other forms of inflammatory chronic arthritis and 47 healthy human subjects. MacMoAb-coated RBCs identified RF in the sera of 66% patients with RA. By contrast, the frequency of positive sera in other inflammatory diseases was 5% and all 47 healthy controls were negative. Antimacaque IgG antibodies were found to be more specific for RF than standard tests, in the sera of patients with RA.     

Chloroactivated latex particles for covalent coupling of antibodies. Application to immunoassays

The aim of the present work is to prepare and characterize a functionalized latex with chloromethyl groups on the surface and to perform the covalent coupling of anti-human serum albumin (a-HSA) IgG protein. The chloromethyl-styrene latex (CMS) was synthesized by means of a core-shell emulsion polymerization in a batch reactor. The monodisperse-obtained latex was characterized by determining the diameter (TEM and PCS), the surface charge density (conductometric and potentiometric titration), the amount of chloromethyl groups on the surface (hydrolysis reaction), and the stability vs electrolyte concentration (turbidity measurements). Electrokinetic characterization was also performed (electrophoretic mobility versus pH and ionic strength). IgG was chemically bound to the latex particles under different sensitization and block-stabilization conditions. Colloidal stability of complexes was studied to select an immunolatex suitable for the development of latex immunoassays. The final part of this work consists of a study of the immunoreactivity of the IgG-latex complexes at different pH and ionic strength, in particular under physiological conditions. The results show that chemically bound IgG to chloromethyl latex provides an IgG-latex complex suitable for application in immunodiagnosis tests.     

Determination of plasmid-associated hydrophobicity of Yersinia enterocolitica by a latex particle agglutination test.

A quick and simple method was developed to distinguish hydrophobic from hydrophilic cells. The latex particle agglutination test is based on the hydrophobic interactions between cells and latex particles which result in the agglutination of the suspension mixture. There was a direct correlation between the expression of plasmid-associated cell surface properties and latex particle agglutination by Yersinia enterocolitica. Multivalent cation-induced agglutination of suspensions of washed cells of virulent Y. enterocolitica and latex particles is indicative of their amphipathic character. Electrostatic interaction may also play a role in the latex particle agglutination reaction.     

Automatic and manual latex agglutination tests for measurement of cholera toxin and heat-labile enterotoxin of Escherichia coli.

Automated and manual latex agglutination methods were employed to measure cholera toxin (CT), heat-labile enterotoxin (LT) of Escherichia coli, and their subunits A and B. Dow polystyrene latex particles (diameter, 0.22 microns) and polystyrene-chlorostyrene latex particles (diameter, 1 micron) were sensitized by rabbit-specific immunoglobulin for each antigen and used as the reagents of the automated and manual agglutination tests, respectively. Automated agglutination was performed by a nephelometric assay system measuring time-dependent differences of light scattering due to agglutination, and manual latex agglutination was carried out in microtiter plates. As low as 1,000 and 31 pg of CT per ml were estimated by the automated and manual agglutination tests, respectively. Using these methods, the amount of CT and LT was measured in several clinical isolates of Vibrio cholerae and E. coli. Furthermore, it was discovered that cyclic AMP is not essential for the production of CT by measuring the amount of the toxin in numbers of cyclic AMP-dependent mutants of V. cholerae (with the agglutination tests).