Purification of murine IgG3 and IgM monoclonal antibodies by euglobulin precipitation.

We describe a simple and efficient non-chromatographic method for the purification of murine IgG3 and IgM monoclonal antibodies (MAbs) which takes advantage of their euglobulin properties. Following filtration, ascitic fluid is dialysed against demineralized water and centrifuged at 22,000 X g for 30 min. The resulting precipitate is dissolved in a high salt buffer (0.1 M Tris-HCl, 1 M NaCl, pH 8). A second cycle of dialysis and centrifugation yields a product of high purity. Nine IgG3 MAbs and eight IgM MAbs were purified by this procedure. Recovery was greater than 90% for seven of nine IgG3 MAbs. It was less reproducible for IgM MAbs and ranged from 40% to greater than 90% depending on antibody and batch. Purity was assessed by SDS-polyacrylamide gel electrophoresis. The purified immunoglobulin was essentially free of albumin, transferrin, and other mouse ascites proteins. No loss of antibody function was observed.

An anti-human mu chain monoclonal antibody: use for detection of IgM antibodies to Toxoplasma gondii by reverse immunosorbent assay.

A precipitating anti-human mu chain monoclonal antibody (designated Tibi 82 McAb) was produced by the cell fusion technique. This McAb (isotype: IgG1 kappa) reacted by radioimmunoassay with all 10 human IgM proteins tested. In contrast, no reactivity was observed with IgG, IgA, IgE, lambda and kappa chains. 19 S IgM proteins were precipitated by Tibi 82 McAb using the Ouchterlony method under standard conditions. Hence specificity of this McAb for the C mu 2 domain was characterized by inhibition of precipitin reactions using human IgM fragments. Despite its narrow specificity for the C mu 2 domain, such a McAb could be used for IgM capture in the detection of specific IgM to Toxoplasma gondii employing the IgM immunosorbent agglutination assay (IgM-ISAGA). Tibi 82 McAb was compared with 3 anti-human IgM polyclonal reagents in the routine analysis of 117 sera. With 2 of them, a correlation coefficient of 0.976 was obtained and Tibi 82 McAb was more sensitive than the third polyclonal reagent tested. The IgM-ISAGA technique was shown to be reproducible using Tibi 82 McAb and similar anti-human mu chain McAbs could permit the wider development of reverse immunosorbent methods for the detection of specific IgM in various infectious diseases.

The cellular composition of the granulocyte series in the normal human bone marrow according to the volume of the sample.

The cellular composition of the granulocytic series in normal human bone marrow was studied in aspirates of different volume. An increase in the proportion of polymorphonuclears was observed in samples of more than 1 ml, thus indicating a dilution of bone marrow by peripheral blood. Certain data from the literature, referring to the differential count in the bone marrow granulocytic series, are discussed. The differences between these data can be explained by the dilution of bone marrow cells by mature granulocytes in blood.

[Clinical use of the assay of the thyroxin-binding-globulin (TBG) and of the calculated index: T4/TBG (author's transl)]. / Intérêt clinique du dosage de la "thyroxin-binding-globulin" (TBG) et du calcul de l'index T4-TBG.

The level of TBG in normal adults is 2.21 +/- 0.4 mg/100 ml, without any sex difference. TBG levels are high in the neonatal period (3.26 +/- 0.49 mg/100 ml) and in children under the age of 1 year (2.61 +/- 0.66 mg). Estrogens induce a significant increase (3.96 +/- 1.0 mg) and corticoids a significant decrease of TBG (1.44 +/- 0.32 mg). TBG is also decreased in thyrotoxicosis (1.88 +/- 0.34 mg) but normal in hypothyroidism. There is a strong correlation between T4 and TBG levels, and the T4/TBG ratio appears to be a useful index, which remains constant in normal adults, and under treatment by estrogens and corticoids.

Antitumor response against myeloma cells by immunization with mouse syngenic dendritoma.

In order to generate an immune response against myeloma cells in an homogenous murine model, a stable hybrid cell line (DCSp) was established through the syngenic fusion between mouse dendritic cells (DC) and mouse Sp2/0 myeloma cells. DCSp cells behaved as potent T cell stimulators and were able to induce Sp2/0 specific cytotoxicity. When mice were immunized with irradiated hybrids before SP2/0 injection, they exhibited a significantly higher rate of survival as compared with controls. When tumors were detected, their emergence was not delayed, and time elapsed between tumor clinical perception and death remained unchanged. A humoral immune response was also always associated. We assume that this stable dendritoma cell line can be considered a valuable tool for myeloma studies in an homogenous mouse model. The efficiency of dendritoma as a weapon against tumor cells and the benefit of syngeny in experimental models are discussed.

[Comparative study of carcino-embryonic antigen and beta2-microglobulin. Methodological study and clinical interpretation (author's transl)]. / Etude comparative de l'antigène carcino-embryonnaire et de la beta2-microglobuline. Etude méthodologique et interprétation clinique.

beta2m was determined by radioimmunoassay in 43 serums from healthy blood donors. Serum concentrations varied from 0.67 to 1.9 mg/l with a mean of 1.29 mg/l. Reproductibility and sensitivity of the method were evaluated. 66 patients with advanced neoplasia were studied. Serum beta2m was greater than 2 mg/l in 70% of the cases and carcinoembryonic antigen (CEA) in 54%. In 26 documented cases with tumor progression and 14 with regression, associated variations of CEA were formed more frequent and of greater magnitude than those of beta2m.

[Beta 2 microglobulin in normal populations and populations at risk]. / Etude de la beta-2-microglobuline dans les populations normales et à risque.

Quantitative analysis of beta-2-microglobulin (beta-2-M) may be a useful test for early diagnosis of acquired immuno-deficiency syndrome (AIDS). Even if beta-2-M is an unspecific marker, 96% of 45 patients with confirmed AIDS and 82% of 45 patients with lymphadenopathy have excessive (3 mg/l) beta-2-M value. beta-2-M is also useful in epidemiological studies, especially in subjects in close contact with patients, and in polytransfused hemophiliacs. At last, beta-2-M may be useful as a screening method in blood banks: from a study of 929 sera, excessive beta-2-M value was observed in 16 cases, which were excluded as blood donors.

[Dosage of serum folates by a radio-assay. Comparison of the results obtained by this method and by microbiological method (author's transl)]. / Dosage par radio-essai des folates sériques. Etude méthodologique et comparaison des résultats avec ceux obtenus par la méthode microbiologique

A radio-assay, using a lactoglobulin fraction of milk proteins as a folate binder and 125-I pteroyl glutamate as a competitor, has been studied in 130 cases. The results have been compared to those obtained with microbiological methods. A good correlation has been observed. The radio-assay allows the measurement of folate in the presence of agents inhibiting the bacterial growth (methotrexate, trimethoprime). It can be used for assay in the cerebro-spinal fluid. This method appears to be useful for clinical investigations.

Detection of herpes simplex virus in direct specimens by immunofluorescence assay using a monoclonal antibody.

A monoclonal antibody (MAb), designated CHA 437, was developed against herpes simplex virus (HSV). This MAb (isotype, immunoglobulin G2b K) reacted with HSV type 1 and HSV type 2. It showed no cross-reactivity with varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. Direct detection of HSV antigen in clinical specimens using indirect immunofluorescence with this MAb was compared with tissue culture isolation. For the 682 specimens tested, the direct specimen test gave a sensitivity of 84.6% and a specificity of 95.7%.

[Radio-immunometric determination of the ferritin for evaluation of the iron storage pool (author's transl)]. / Dosage radio-immunométrique de la ferritine pour l'appréciation des réserves martiales.

Seventy-three patients with several haematological diseases have been studied with radio-iron-kinetic, cyto-chemical measurement of iron in the bone marrow, and radio-immunometric determination of the ferritin in the serum. This method gives results which correlate significantly with other methods which evaluate the iron storage pool, in normal or iron-deficient patients. In some cases, an excess of serum ferritin, contrasting with normal serum iron, is confirmed by cytochemical and/or Fe-kinetic studies. In myeloproliferative diseases however, secretion of ferritin by immature cells may induce an excess of serum ferritin. In such cases, the dosage of ferritinaemia cannot be considered as an index of the iron storage pool. Future development of specific dosage of isoferritins could enable to measure the true iron stores, as well as to give an index estimating the evolution of the abnormal cell population.

[Analysis of various isoferritins with monoclonal antibodies]. / Analyse de différentes isoferritines par des anticorps monoclonaux.

Balb/c Mice were immunized with splenic isoferritin. Spleen cells from immunized Mice were fuzed with SP2O myeloma cells. Four monoclonal antibodies designated respectively M29, M211, M386 and B8 were selected. Various isoferritins were analysed by immunodiffusion (Ouchterlony technic). With these monoclonal antibodies and with Rabbit polyclonal sera: human basic and acidic isoferritins and Horse spleen ferritin. Ferritin could be precipitated by these monoclonal antibodies. These results confirm that the ferritin molecule consists of repeating antigenic sites. No immunoreactivity difference could be detected between acidic and basic human ferritine. Species specificity was recognized. The high affinity constant of the M29 monoclonal antibody allowed development of a standardized radioimmunossay of ferritin.

An anti-human immunoglobulin M monoclonal antibody for detection of antibodies to Toxoplasma gondii.

An anti-human mu-chain monoclonal antibody, Tibi 82, was produced and tested for specificity by radioimmunoassay. Its reliability in detecting IgM antibodies to Toxoplasma gondii was tested by two reverse immunosorbent methods (IgM-ISAGA and IgM-SPIHA) and the IgM fluorescent antibody test (IgM-IFA) on 400 sera. Whereas the results obtained with Tibi 82 and with two polyclonal reagents were highly correlated, the third commercial polyclonal reagent provided many false negative results. By standardizing IgM binding, Tibi 82 allowed the comparison of IgM-ISAGA with IgM-SPIHA on 100 sera: 17% of the sera tested showed discrepancies due to the different toxoplasma antigens used. Although Tibi 82 facilitated the reading of results and enhanced sensitivity and specificity of the double-sandwich IgM-IFA method, the latter was still less sensitive than IgM-ISAGA with Tibi 82. Tests with the monoclonal antibody were consistently superior to tests with polyclonal antibodies.

Optimization of a rapid test by using fluorescein-conjugated monoclonal antibodies for detection of Chlamydia trachomatis in clinical specimens.

A mixture of two fluorescein isothiocyanate-conjugated monoclonal antibodies (MAbs) was used to optimize a direct specimen test (Chlamydia Direct Specimen Test IF; Clonatec, Paris, France) for detection of chlamydial elementary bodies in clinical specimens. One MAb reacted with a subspecies-specific epitope of the major outer membrane protein (molecular weight 43,000) of Chlamydia trachomatis, whereas the other reacted with the periodate-sensitive genus-specific antigen (molecular weight 11,000) of Chlamydia spp. Nonfat dry milk was the most efficient additive at suppressing the fluorescent background and was included in the antibody preparation. Fc-dependent binding of fluorescein-conjugated MAbs to protein A-containing Staphylococcus aureus was inhibited by addition of purified rabbit immunoglobulin. The Chlamydia Direct Specimen Test IF was compared with tissue culture isolation by using 309 genital specimens. The sensitivity and specificity were 77.4 and 98%, respectively.