Iron transfer form transferrin to ferritin mediated by polyphosphate compounds.

We have studied iron transfer from transferrin to ferritin in the presence of ATP, GTP, ADP, AMP and 2,3-diphosphoglycerate. These compounds, with the exception of AMP, can release iron from transferrin at pH 7.4 and form a stable Fe(III)-phosphate complex. From these complexes, only a limited number of Fe(III) atoms can be incorporated into ferritin. Ascorbate enhances iron transfer from transferrin to ferritin at the beginning of the process but subsequently inhibits further iron deposition in ferritin.

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.

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.

Immunological characteristics of human ferritins; consequences for human serum ferritin determination.

Standard curves established with human spleen, liver, placenta and heart ferritins for eight commercial radioimmunological procedures show that liver and spleen ferritins present almost identical responses, whereas three times as much placenta ferritin and six times as much heart ferritin were required to give the same response as liver and spleen ferritins. There are great variations between the kits in the estimation of the ferritin level of a control serum.

Latex allergosorbent test (LAST): a new immunoassay for specific IgE with latex particles.

We describe a two-step latex (Lx) agglutination assay for the titration of specific anti-Dermatophagoides pteronyssinus IgE. The samples are first incubated with allergen-coated Lx of 2.3 microns diameter. Bound IgE is digested by pepsin and then titrated by its agglutinating activity on 0.8 micron Lx particles coated with antihuman Fc epsilon rabbit F(ab')2. This latex allergosorbent test detects 100 pg of specific IgE per milliliter and does not depend on the concentration of total IgE. Owing to a tenfold increase in the allergosorbent surface, no competition with the binding of specific anti-D. pteronyssinus IgG is observed. Pepsin digestion eliminates potential interferences caused by autoantibodies against IgE. A good correlation (r = 0.92) is found with Phadebas RAST on a series of 91 samples. The latex allergosorbent test does not make use of radioisotopes and can be performed in less than 6 hours.

High sensitivity, homogeneous particle-based immunoassay for thyrotropin (Multipact).

We describe the first homogeneous, nonradioactive, high-sensitivity assay for human thyrotropin (TSH). The assay is based on particle immunoassay techniques, wherein 800-nm particles form the basis for the immunochemistry, delivery, and the detection technologies, respectively. Our assay also is the first to involve the use of fragmented monoclonal antibodies (to eliminate serum interferences) covalently coupled to particles without loss of their binding properties. Assays are performed in a semiautomated mode with use of a new modular system (Multipact). Equilibrium is reached in less than 2 h. Precision profile, sensitivity, and clinical studies indicate that the assay is accurate, has good precision at low concentrations, and that detection-limit characteristics compare well with those of a leading commercial high-sensitivity immunoradiometric assay (IRMA) for TSH. Dilution characteristics were satisfactory down to the assay's detection limit for a range of clinical samples. Correlation studies vs a reference IRMA method yielded the regression equation, present method = 0.976 (IRMA) + 0.002 milli-int. unit/L (r = 0.98), for 223 samples with TSH concentrations in the range 0 to 30 milli-int. units/L. For 40 samples with TSH less than or equal to 1.0 milli-int. unit/L it was: present method = 0.94 (IRMA) + 0.005 milli-int. unit/L (r = 0.96).

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.

Overview of the clinical development of a diphtheria-tetanus--acellular pertussis vaccine.

A tricomponent acellular pertussis vaccine containing pertussis toxoid, filamentous hemagglutinin, and pertactin combined with diphtheria and tetanus toxoids (DTPa) was developed as a less reactogenic alternative to the traditional whole cell pertussis (DTPw) vaccine. In studies of DTPa as a primary vaccination and as a booster dose in DTPa- or DTPw-primed children, the vaccine was safe, well-tolerated, and highly immunogenic; it was less reactogenic than DTPw but at least as immunogenic. A three-dose primary vaccination sequence with DTPa vaccine in the first 6 months of life protects against pertussis under conditions of high infectious pressure. These results support the licensing of the vaccine for primary and booster vaccination in a growing number of countries. Combined DTPa-based pediatric vaccines are in clinical development.

Particle-counting immunoassay (PACIA) of pregnancy-specific beta 1-glycoprotein, a possible marker of various malignancies and Crohn's ileitis.

Pregnancy-specific beta 1-glycoprotein (SP1) was assayed by particle-counting immunoassay (PACIA) with a sensitivity of 1 microgram/L. In serum from 50 men, the SP1 concentration was less than 1 microgram/L, whereas three of the specimens from 46 nonpregnant women had values exceeding 1 microgram/L. In 29% of 950 consecutive patients' sera, SP1 concentrations exceeded 1 microgram/L--in sarcoma (six of six), in malignant hemopathies (101/127, 80%) such as myeloma (20/26, 92%) and acute myeloblastic leukemia (23/27, 90%), and in various other types of cancer (11/19, 58%) except for bronchial epithelioma, which did not lead to any significant increase of SP1 in the five patients examined. The concentration of SP1 was also frequently increased in patients with Crohn's ileitis (28/43, 65%) but not in patients with other inflammatory disorders.

Pregnancy-specific beta 1 glycoprotein (SP1) in the cerebrospinal fluid.

Pregnancy-specific beta 1 glycoprotein (SP1) was assayed by Particle Counting Assay in the cerebrospinal fluid (CSF) from 26 non-neurological patients, from 190 patients with various neurological disorders and from 84 patients with malignant hemopathies. With a sensitivity limit of 0.5 microgram/l, SP1 was undetectable in normal CSF. High levels were observed in CSF from one pregnant woman with herpetic encephalitis and from another woman with post-puerperal thrombophlebitis as a result of high serum concentrations and leakage of the blood-brain barrier. SP1 was detected at low levels in the CSF from 1 patient out of 5 with Creutzfeldt-Jakob disease and from a patient with Behçet's disease. Seven patients out of 84 with malignant hemopathies presented cerebral involvement; 3 of them had detectable SP1. However, SP1 was also detected in the CSF of 2 patients in apparently complete remission. The determination of SP1 in CSF appears to be of limited value in the diagnosis of neurological disorders and in the early detection of a cerebral localization of malignant hemopathies.

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.