Jacalin: isolation, characterization, and influence of various factors on its interaction with human IgA1, as assessed by precipitation and latex agglutination.

An IgA1-specific lectin, Jacalin, was isolated from dried seeds of the jackfruit, Artocarpus integrifolia, by affinity binding to IgA1-Sepharose and elution with D-galactose. Jacalin is a glycoprotein with two non-covalently bound subunits (15 and 18 K). Interactions between Jacalin and human Igs were studied by precipitation in gel and in solution, and by agglutination of IgA1-coated latex by Jacalin. Jacalin precipitated only with IgA1-containing samples, including monomers, polymers, monoclonal, polyclonal and secretory IgA1, but not IgA2 of both A2m(1) and A2m(2) allotypes, nor with IgG1, 2, 3 and 4, IgM, IgD, and IgE; after neuraminidase treatment, only IgA1 and IgD were precipitated. Jacalin had a relatively broad pH range of activity in both precipitation and agglutination of IgA1-latex. Bivalent metal cations (Ca, Mg, Mn, Cu, Zn, Co, Cd), EDTA, Triton X-100, Tween-20, Na deoxycholate and ionic strength did not influence these reactions. Na dodecylsulphate, guanidine and urea inhibited the reactions whereas NP-40 rather enhanced them. Among 39 types of sugar tested, 10 displayed inhibitory activity, decreasing in the following order: p-nitrophenyl-alpha-D-galactopyranoside, 1-O-methyl-alpha-D-galactopyranoside, D-melibiose, p-nitrophenyl-beta-D-galactopyranoside, GalNAc, stachyose, 1-O-methyl-alpha-D-mannopyranoside, D-galactose, D-galactosamine and 1-O-methyl-alpha-D-glucopyranoside. IgA1, treated with neuraminidase or not, but not the other human Igs, was also an excellent inhibitor of agglutination, being more powerful than the best sugars studied. Only neuraminidase-treated IgD was also inhibitory, but less so than IgA1. Jacalin preferentially bound to alpha-linked non-reducing D-galactose. The configuration of OH-groups at C-2, C-4 and C-6 of D-galactose was important for the reaction. Jacalin recognizes terminal Gal beta 1-3GalNac-, as in the IgA1-hinge, and/or GalNAc-, but not Gal beta 1-4GlcNAc-, nor Gal beta 1-6GlcNAc-, nor their sialylayted extensions. Latex agglutination and its inhibition assay are particularly well suited for the study of these lectin-glycoprotein interactions.

Immunoturbidimetric assay of beta 2 microglobulin using latex particles in microplates.

A latex particle immunoturbidimetric assay in microplates has been developed for the quantitation of beta 2 microglobulin. The assay which involves three pipetting steps and one incubation, has a clinically useful range of 0.4-16 mg/l. A comparison of the method with a commercial radioimmunoassay in the analysis of 125 sera revealed a high degree of correlation. Although the assay was performed manually, it showed considerable potential for full automation.

Latex particle immunoassay of carcinoembryonic antigen.

Immunoassay by particle counting (IMPACT) was used to assay carcinoembryonic antigen. The dynamic range in serum was shown to extend from 1 to 120 ng/ml with a detection limit of 0.3 ng/ml. With a total throughput time of 52 min, the assay displayed intra- and inter-assay coefficients of variation ranging from 5.4 to 9.8%. Good specificity was obtained with the aid of monoclonal antibodies and by peptic digestion of the serum sample. The method was compared with three different RIAs and one EIA test and correlation coefficients of 0.94-0.97 were obtained. CEA levels in normal individuals (smokers and non-smokers) and in various diseases were also measured.

A quantitative C-reactive protein assay using latex agglutination in microtiter plates.

In this quantitative assay, latex particles coated with anti-C-reactive protein (CRP) are agglutinated by CRP following vortex agitation in microtiter plates. A decrease in absorbance at 405 nm is directly proportional to CRP concentration. This 30 min assay is simple and necessitates only two pipetting steps after serum dilution. The linear part of the standard curve ranges from 5 to 150 mg/l and CRP concentrations up to 300 mg/l can be determined without additional dilution of sera. Within-assay reproducibility varies from 4.4% to 7.5% while between-assay reproducibility ranges from 9.3% to 12.2%. Correlation studies performed with 104 sera assayed by automated nephelometer and turbidimeter gave correlation coefficients of 0.96 and 0.97 respectively.

Particle counting immunoassay of choriogonadotropin using monoclonal antibodies.

A latex particle immunoassay has been developed for the quantification of choriogonadotropin in human serum using two monoclonal antibodies specific for the beta-chain of the hormone. The assay, based on optical counting of monomeric particles, was achieved in 40 min and the calibration curve was linear between 10 and 200 IU/l. Intra- and interassay precisions at three different levels of the curve varied between 3.3 and 10.9%. The method was validated by comparison with two different radioimmunoassays and correlation coefficients of 0.97-0.99 were obtained.

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.

C-reactive protein in serum and cerebrospinal fluid in various neurological disorders. Apparent local consumption during bacterial meningitis.

The level of C-reactive protein (CRP) was determined in the cerebrospinal fluid (CSF) by particle counting immunoassay. In non-neurological patients (N = 24), CRP was detectable only in 10 samples at concentrations ranging from 1.5 to 37 micrograms/l. The multiple sclerosis group did not differ from the controls. The highest CRP levels were found in viral and bacterial, including tuberculous, infections of the nervous system, with overlapping results for the various types of infections. However, in serum, the levels of CRP were much higher in pyogenic than in viral meningitis. We compared the CSF CRP/serum CRP ratio to the same ratio for albumin and found a significant correlation between the two ratios in viral, but not in bacterial, infections. These results suggest a local consumption of CRP during bacterial meningitis.

Immunoassay of low concentrations of albumin in urine by latex particle counting.

We describe here a nonisotopic immunoassay, based on particle-counting technology, for the determination of urinary albumin. The assay takes only 35 min and has been fully automated on the IMPACT (Acade Diagnostic Systems, Brussels, Belgium) machine. The system measures albumin within a linear range between 6.25 and 50 mg/L and has a detection limit of 0.4 mg/L. Analytical recoveries at three concentrations ranged between 96% and 102%. Within-run precision ranged from 1.6% to 9.5%. The method was compared with a commercial nephelometric immunoassay system and a correlation coefficient of 0.996 was found for 216 urine samples. No antigen excess affects the shape of the curve in our system, whereas in nephelometry a 3 g/L solution of albumin starts to decrease the dose-response curve.

Immunoassay by particle counting for coagulation testing: application to the determination of antithrombin III, von Willebrand factor antigen (vWF:Ag) and plasminogen.

Based on immunoassay by particle counting, three methods for antithrombin III, von Willebrand factor and plasminogen were developed on an automated IMPACT machine and on a semi-automated MULTIPACT system. Precision of the techniques, measured at low, medium and high level of the calibration curve showed coefficients of variation varying from 4.3 to 13.8%. Accuracy was evaluated by dilution recovery test and by correlation with rocket immunoelectrophoresis and chromogenic substrate techniques. The results show that the proposed methods correlate well with existing techniques and that immunoassay by particle counting is applicable to several coagulation tests.

Turbidimetric latex immunoassay of placental lactogen on microtiter plates.

In this latex immunoassay for human placental lactogen, microtiter plates are used as the reaction vessel and the absorbance at 405 nm is measured to quantify the reactions. This 30-min assay necessitates only one serum dilution and two pipetting steps. The calibration curve extends from 0.5 to 15 mg/L. CVs range from 4.2% to 7.0% for within-run determination and from 7.0% to 11.2% for between-run determinations. A correlation coefficient of 0.949 was obtained for 84 sera when the method was compared with a commercial radioimmunoassay.

Measurement of beta 2-microglobulin in serum by a particle-enhanced nephelometric immunoassay.

A particle-enhanced immunoassay of beta 2-microglobulin in serum is described. It is based on the agglutination of complexes formed between the serum beta 2-microglobulin and latex particles coated with F(ab')2 fragments of polyclonal anti-beta 2-microglobulin antibodies. The analytical range of the method is 0.50 to 16 mg/l; it can be extended by appropriate dilution to 0.12 to 80 mg/l with good precision (CV less than 5% over the whole range). The accuracy and the precision are confirmed by a good correlation with radioimmunoassay (n = 123, r = 0.993). No error due to antigen excess was observed, even up to 292 mg/l. The main advantages of the method are its simplicity, its low cost per test and its high sensitivity (final dilution of the sample at 1/1200) with no known interference. The calibration curve is stable for at least 2 weeks.

Immunoassay by particle counting for coagulation testing: application to the determination of protein C.

Latex particles coated with F(ab)'2 fragments of anti-protein C IgG antibodies are agglutinated by protein C, and the quantity of particles agglutinated is proportional to the concentration of protein C. The reaction can be quantitated by optical particle counting. Based on this system, we designed an immunoassay for protein C. Precision measured at low, medium and high levels of protein C varied from 3.3% to 13.7%. Specificity was evaluated by dilution recovery. A correlation coefficient of r = 0.959 was found when the new method was compared with a chromogenic technique on 131 plasmas.

An automated particle-counting immunoassay (PACIA) for serum IgE.

Total IgE was determined in 107 sera using a novel, automated, non-isotopic immunoassay called PACIA (particle-counting immunoassay) based on agglutination of anti-IgE coated latex particles by IgE. The IgE values ranged from 10 to 50,000 iu/ml and were compared with results obtained by a conventional radioimmunoassay (RIA) which used a fast double antibody separation technique--the coefficient of correlation was 0.985 and the regression line y = 0.82 x +130.00. PACIA had several advantages over the RIA technique: using a sampling rate of 50/hr, results were obtained in 35 min compared to 16-20 hr, no labelled IgE was required and the separation step, which relied on measuring the number of non-agglutinated particles by an optical cell counter, was fully automated. The threshold of sensitivity was 10 iu/ml and the maximal coefficient of variation for between assay precision was 12.9%.

Automated particle-counting immunoassay for digoxin.

We describe a non-radioisotopic automated immunoassay for serum digoxin in the therapeutic range at 50 samples per hour by use of the particle-counting immunoassay technique and a special reagent, rheumatoid factor. Digoxin is determined by evaluating its inhibitory activity on the agglutination of digoxin-coated latex particles. The agglutinating system consists of the anti-digoxin IgG mixed with rheumatoid factor. This anti-IgG autoantibody reacts with IgG when they are aggregated or bound to a surface. Interference by serum proteins was prevented by pepsin digestion at 37 degrees C. Correlation with radioimmunoassay was r = 0.94 for 109 samples with concentrations between 0 and 5.7 micrograms/L. Analytical recovery overall averaged 102%. Within-assay precision (CV) ranged from 12.5% at 0.96 micrograms/L to 4.4% at 2.97 micrograms/L; between-assay precision varied from 8.4% at 0.75 micrograms/L to 5.8% at 2.92 micrograms/L.

Automated particle-counting immunoassay of C-reactive protein and its application to serum, cord serum, and cerebrospinal fluid samples.

The assay for C-reactive protein has been fully automated as a particle-counting immunoassay. For cerebrospinal fluid, cord serum, and adults' serum its range of sensitivity extends from 1 microgram/L to 300 mg/L, with a minimal sample dilution of twofold and a maximal dilution of 50-fold being required. This range is so broad because free antibodies are added to the reaction medium. However, we have used Fab fragments rather than whole antibody to avoid too steep a standard curve and a decrease of agglutination at high concentrations of antigen. For 99 consecutive cord sera examined, the concentrations of C-reactive protein ranged from 7 micrograms/L to 1.750 mg/L. The geometric mean was 50 micrograms/L and the upper normal limit (geometric mean +/- 2 SD of the log values) was established at 525 micrograms/L.

Immunoassay of theophylline by latex particle counting.

We set up an immunoassay by particle counting for theophylline. Theophylline concentration is assayed by its capacity to inhibit the agglutination of theophylline coated latex particles by a specific monoclonal antibody, the agglutination being enhanced by a rabbit anti-mouse IgG antiserum. The dose range is 2-64 mg/L. The cross-reactions observed with caffeine (0.3%), theobromine (0.2%), 3-methylxanthine (0.7%) and 8-chlorotheophylline (2%) are very good when compared with other published methods. Within and between-run precisions measured at low, medium and high level of the calibration curve show coefficients of variation ranging from 3.9% to 9.5%. Our assay was correlated with the Fluorescence Polarization Immunoassay (FPIA) and a correlation coefficient of 0.96 was determined for 89 samples.

Particle counting immunoassay (PACIA) of ferritin.

Particle Counting ImmunoAssay (PACIA) has been adapted to the determination of serum ferritin. Polystyrene particles (0.8 micrometers) were coated with anti-ferritin antibodies and the concentration of ferritin determined by the agglutinating activity of this protein. The agglutination was measured by the reduction of the number of non-agglutinated particles counted in an optical blood cell counter. Non-specific agglutination and inhibition of agglutination were prevented in 99% of clinical samples by the use of F(ab')2 fragments of the antiserum IgG to coat the particles and by using slightly dissociating conditions (pH 9.2, ammonium thiocyanate, EDTA). The system was automated with a sampling rate of 50/h and an incubation time of 25 min. The standard curve ranged from 1 to 100 micrograms/l; recoveries were between 93.6 to 100.2%; the correlation coefficients of PACIA with radioimmunoassays calculated respectively on 99 and 91 samples were 0.974 and 0.984; maximal within- and between assay CV were 11.2% and 7.7%.

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.