Particle Counting Immunoassay Research Articles

6] Particle counting immunoassay (PACIA)

Publisher Summary This chapter discusses the various aspects of particle counting immunoassay (PACIA). The PACIA is based on the principle that the number of free particles decreases during the agglutination reaction. It is found that, by counting the unagglutinated particles, with a blood cell counter, it is possible to evaluate the extent of the reaction in a much more sensitive and precise way, than by the naked eye. The PACIA is automated on the basis of the continuous-flow system. The rather strong agitation required to accelerate the agglutination process can be obtained in a mixing coil submitted to continuous vibration. The intra- and interassay precisions were determined, using four or five samples, with concentrations, covering most of the range of the calibration curve. The Ab content of a rabbit antimyelin basic protein (MBP) antiserum used as a calibrator for both PACIA and a solid phase radioimmunoassay was estimated, by the Farr precipitation technique, using 125I-labeled MBP. The sensitivity of PACIA to the small changes in the levels of immune complexes is described, by the results of a study performed on 486 samples of human cord blood, using only rheumatoid factor (RF) inhibition.

Automated particle-counting immunoassay for alpha-fetoprotein.

PACIA, a homogeneous non-radioimmunoassay, has been adapted to the determination of serum alpha 1-fetoprotein. This technique is based on the agglutination of latex particles coated with antibodies to the antigen to be determined. The agglutination is measured by using an optical cell counter designed to count blood cells, to determine the reduction in the number of non-agglutinated particles. Interferences by serum constituents are avoided by coating the particles with the F(ab')2-fragments of th immunoglobulin G fraction of the antiserum. The system is automated, with a sampling rate of 50/h and an incubation time of 26 min. Concentrations used in preparing the standard curve ranged from 1 to 50 microgram/L; analytical recoveries were 93.5 to 98.4%; the correlation coefficient of PACIA with radioimmunoassay, calculated from results on 127 samples, was 0.98; maximum within- and between-assay CVs were 7.4% and 9.6%, respectively.

Particle counting immunoassay (PACIA) V- its application to the determination of human placental lactogen

This article has no abstract.

Particle counting immunoassay (PACIA). III. Automated determination of circulating immune complexes by inhibition of an agglutinating factor of mouse serum

A factor capable of agglutinating human IgG coated particles (latex) has been found in mouse serum. This factor (MAG) was used in an unpurified form to detect circulating immune complexes in the particle counting immunoassay (PACIA) system, which allows measurement of agglutination with great precision. MAG did not react with monomeric IgG, nor with reduced and alkylated aggregated IgG. It was inhibited by immune complexes in small antigen excess. Among the various subclasses of IgG, IgA and IgM, only IgG1 and IgM when coupled to Sepharose beads displayed an inhibitory activity towards MAG. That the inhibitory factors detected in serum were immune complexes or aggregated Ig was suggested by the correlation obtained with the amounts of ‘heavy’ IgG found in the serum samples by Ultrogel chromatography and by the polydisperse distribution of the inhibitory factors in the heavy fraction of gradient ultracentrifugation.

Particle counting immunoassay. IV. The use of F(ab′) 2 fragments and Nϵ-chloroacetyl lysine N-carboxyanhydride for their coupling to polystyrene latex particles

Improved performance with the latex agglutination immunoassay, PACIA, is possible when the F(ab′) 2 fragments of antibody are chemically coupled at their hinge region to a protein-coated latex using a new coupling reagent, N ϵ -chloroacetyl lysine N-carboxyanhydride. This reagent, whether by orientating the complete antibody molecule or its F(ab′) 2 fragment or by preventing antibody denaturation, increased the sensitivity of the PACIA method. The use of F(ab′) 2 fragments eliminated most serum interference with the test.

Particle counting immunoassay (pacia). II. Automated determination of circulating immune complexes by inhibition of the agglutinating activity of rheumatoid sera

Antigen—antibody complexes were detected in patients' sera by inhibition of the agglutinating activity of rheumatoid sera toward IgG coated particles (latex). Precision, sensitivity (1–10 μg/ml equivalents of heat-aggregated IgG), and reproducibility (maximum coefficient of variation of 11%) were obtained by measuring agglutination with an instrument counting the residual free particles. Automation allowed testing of 20 to 40 samples per hour. The inhibitory activity of spontańeously agglutinating sera was determined after inactivating endogenous rheumatoid factor by reduction with dithiothreitol. Non-aggregated IgG did not significantly interface. The agglutinating activity of 6 rheumatoid sera was tested after incubation with the various immunoglobulin classes and subclasses polymerized by coupling to agarose, and all were found to be readily absorbed by IgG1, but poorly by IgG3, IgA1 and IgM. Reactivity with IgG2, IgG4 and IgA2 clearly differed for different rheumatoid sera. Among 70 sera from blood donors, 7 had abnormally high inhibitory activity. Six of these had also an abnormal protein profile, suggesting existence of latent disease. High inhibitory activity in 15 sera out of 18 from patients with systemic lupus erythematosus, and in 23 sera out of 46 from patients with breast cancer suggested that the rheumatoid factor inhibition test has a discriminatory capacity comparable with that of more sophisticated techniques requiring radioisotopes and/or cellular material.

Particle counting immunoassay (Pacia). I. A general method for the determination of antibodies, antigens, and haptens

By using a device designed for counting blood cells, it is possible to measure the agglutination of polystyrene beads (0.8 μ) with accuracy and great sensitivity, the agglutination resulting in a reduction in the number of particles. The latter coated with antigen can be used for determining IgM or IgG antibodies e.g. human rheumatoid factor or rabbit anti-bovine serum albumin antibodies. Macromolecules with multiple antigenic determinants agglutinate particles carrying specific antibodies. This system has been applied for determining HPL and α 1-fetoprotein with a threshold of sensitivity of about 10 μg/l. However the agglutination was decreased by serum factors which led to a 10-fold loss of sensitivity. The interference of rheumatoid factor which agglutinated the particles coated with rabbit or goat immunoglobulins could be avoided by reduction of the serum to be analyzed with 5 mM dithiothreitol for 5 min. Haptens, i.e. DNP-lysine and T 4, were determined by their inhibitory activities toward their specific antibodies, the agglutinator being a hapten-macromolecule conjugate or the antibodies themselves.