[Quantitative estimation by ELISA of IgG anti-D (RH1) antibodies in immunoglobulin preparations and in the sera of immunized donors]. / Dosage par une technique ELISA des anticorps anti-D (RH1) dans les préparations d'immunoglobulines et dans le sérum de donneurs immunisés.

Immunoglobulin preparations of anti-D (RH1) are injected to prevent haemolytic disease of the newborn. Such preparations are obtained by the fractionation of plasma from immunized donors. Measurement of the concentration of IgG anti-D is required to estimate the potency of anti-D preparations and sera from immunized donors. We have developed an ELISA method for the quantification of IgG anti-D. This method included the following steps, sensitization of red cells by anti-D, solubilization of red cell membranes by Triton, and eventually, measurement of IgG anti-D concentration by ELISA. The international reference preparation of anti-D (68/419) was used as a reference. With this method, we measured IgG anti-D concentrations in 5 immunoglobulin preparations of anti-D and in the sera of 10 donors immunized by D antigen. The ELISA results were compared with those obtained by automated hemagglutination. A mean anti-D concentration of 56.2 micrograms/mL was found by ELISA in immunoglobulin preparations. Similar results were obtained by automated hemagglutination (mean 52 micrograms/mL). In the sera of 10 D-immunized donors, anti-D IgG concentration varied from 2.2 to 59.8 micrograms/mL. A good correlation between ELISA and automated hemagglutination was observed in these sera (r = 0.98, p < 10(-7)). In conclusion, the ELISA technique offers an alternative to automated hemagglutination. It requires only the standard equipment necessary for immuno-enzymatic methods.

Quantitative determination of anti-K (KEL1) IgG and IgG subclasses in the serum of severely alloimmunized pregnant women by ELISA.

BACKGROUND: Severe cases of HDN occur after the immunization of the mother with K (KEL1) antigen. To date, the only means of evaluating the concentration of anti-K in maternal serum is by titration with an indirect antiglobulin test (IAT). A more accurate estimation of the serum anti-K concentration is needed. STUDY DESIGN AND METHODS: An ELISA technique was developed for the determination of the absolute concentration of anti-K IgG and IgG subclasses in the sera of alloimmunized patients. In this technique, after absorption of anti-K on K-positive RBCs and subsequent elution at acid pH, the concentration of anti-K in the eluate was measured with a sensitive and reproducible ELISA. This method was validated with monoclonal and polyclonal anti-K. It was then used to assay the sera of eight pregnant women with anti-K immunization, associated with early fetal anemia (Hct, 7-17%) detected between the 20th and the 31st week of pregnancy. In addition, in most of these cases, the anemia was associated with fetal hydrops. RESULTS: The anti-K IgG concentration measured by ELISA in the sera of the eight women varied from 1.0 to 4.1 microg per mL (mean, 2.2 microg/mL). Therefore, severe and early forms of fetal anemia can be observed with a relatively low concentration of anti-K (as compared to the concentration of anti-D in similar cases of fetal anemia due to anti-D). The mean proportion of each IgG subclass of anti-K in these sera was IgG1, 95.9 percent; IgG2, 2.4 percent; IgG3, 1.3 percent; and IgG4, 0.4 percent. CONCLUSION: A simple method for quantitative estimation of anti-K in human serum has been developed. Low concentrations of anti-K can cause fetal anemia relatively early in pregnancy. This method should lead to a better identification of pregnant women whose fetuses are at risk for severe fetal anemia due to anti-K.

Evaluation by enzyme-linked immunosorbent assay of IgG anti-D and IgG subclass concentrations in immunoglobulin preparations.

BACKGROUND: Anti-D immunoglobulin preparations are injected to prevent hemolytic disease of the newborn. The concentration of IgG anti-D in these preparations is usually determined by an automated hemagglutination technique using as a reference a calibrated preparation of anti-D, but the method requires special equipment and cannot be routinely applied to measure the IgG subclasses of anti-D in these preparations. STUDY DESIGN AND METHODS: Taking advantage of a recently described enzyme-linked immunosorbent assay (ELISA) for the determination of the anti-D concentration in sera of alloimmunized pregnant women, IgG anti-D and IgG subclass concentrations were measured in the international reference preparation (IRP) coded 68/419, 10 anti-D immunoglobulin preparations, and sera of 15 D-immunized volunteers. RESULTS: An IgG anti-D concentration of 61.5 +/- 4.8 microg per ampoule (mean +/- SD) was found by ELISA in IRP 68/419. This result was in agreement with previous determinations obtained by radioimmunoassay (60 microg/ampoule). The IgG subclass concentration of anti-D in this preparation was 48.4 microg of IgG1 (78.6%), 3.0 microg of IgG2 (4.8%), 9.7 microg of IgG3 (15.8%), and 0.4 microg of IgG4 (0.7%). The mean proportion of IgG subclasses of anti-D in 10 immunoglobulin preparations was similar (81.7% for IgG1, 5.0% for IgG2, 12.7% for IgG3, and 0.6% for IgG4). In the sera of 15 immunized volunteers, the IgG anti-D concentration varied from 3.1 to 68.4 microg per mL. The mean IgG subclass composition of anti-D was 79.3 percent for IgG1, 2.2 percent for IgG2, 18.1 percent for IgG3, and 0.4 percent for IgG4. The proportions of IgG3 anti-D in these sera were found to range between 1 percent and 87 percent, as in the sera of D-alloimmunized pregnant women. CONCLUSION: ELISA provides an alternative to the radioimmunoassay and the automated hemagglutination technique. In addition, it allows the evaluation of the absolute concentration of each IgG subclass of anti-D in immunoglobulin preparations and necessitates only the conventional equipment required for an immunoenzymatic assay.

An enzyme-linked immunosorbent assay for the quantitation of IgG anti-D and IgG subclasses in the sera of alloimmunized patients.

BACKGROUND: IgG subclass composition of maternal alloantibodies to the D antigen seems to play a role in the severity of hemolytic disease of the newborn. The subclassing of IgG anti-D is usually performed by hemagglutination techniques, but the results are not quantitative and sometimes are difficult to interpret. Thus, there is a need for quantitative methods. STUDY DESIGN AND METHODS: The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for the quantitation of specific IgG anti-D and IgG subclasses in the sera of alloimmunized patients. Group O R1R2 red cells were sensitized with anti-D. Red cell membranes were solubilized with nonionic detergent. IgG and IgG subclasses were measured by a sensitive and reproducible immunocapture ELISA. A serum calibrated for its IgG subclass content was used as a reference, and the anti-D preparation 68/419 was used as an internal control. Optimal conditions for the detection of IgG anti-D and IgG subclasses by ELISA were studied. The absolute concentration and the proportions of IgG subclasses were determined in the sera of 14 pregnant women. RESULTS: A close parallelism was observed between dilutions of the IgG reference serum and the IgG anti-D solubilized from sensitized RBCs. The sum of IgG anti-D subclass concentrations, determined by the ELISA, correlated well with other quantitative methods. CONCLUSION: The method described is sensitive and can be used routinely for the quantitative determination of specific IgG anti-D and IgG subclasses in sera.

Screening of a large number of dyes for the separation of human immunoglobulin G2 from the other immunoglobulin G subclasses immunoglobulin G2 enrichment on immobilized Procion Yellow HE-4R.

Human immunoglobulins (IgG) are produced on a multi-ton scale for therapeutic applications. There is presently no available method to manufacture IgG preparations enriched with immunoglobulins from the IgG2 subclass although they might be useful for therapeutic purposes. By frontal chromatography, we have screened 69 immobilized dyes, among which, six display a different affinity for IgG2 and other subclasses. One (Procion Yellow HE-4R) was studied further. The screening of various mobile phase conditions allowed us to devise a procedure to prepare IgG2 enriched IgG solutions: The cumulative yield for IgG2 was 43% and IgG2/total IgG ratio in the final product was 67%.

Local production of IgG and IgG subclasses in the aqueous humor of patients with Fuchs heterochromic cyclitis, herpetic uveitis and toxoplasmic chorioretinitis.

Changes in local immunity are important when considering the physiopathology of uveitis. The aim of this study was to measure IgG and IgG subclass concentrations in the serum and the AH of patients with three different types of uveitis and to determine for each of them the presence of a local production of IgG in the intra-ocular compartment. This investigation was extended to IgG subclasses. Serum and AH of 46 patients, including 11 with Fuchs heterochromic cyclitis (FHC), 13 with toxoplasmic chorioretinitis, 11 with herpetic uveitis and 11 with senile cataract (taken as controls) were analyzed by ELISA for IgG and IgG subclasses. Three quotients (r1, IgG/albumin in serum; r2, IgG/albumin in AH; and R, r2/r1) were calculated in order to estimate the local synthesis (LS) of IgG and each IgG subclass. In AH of patients with herpetic uveitis, a concomitant and significant increase of all IgG subclasses as well as albumin (with no significant increase of r2 or R) was observed. This finding was interpreted as an indirect consequence of major damage to the blood-aqueous barrier. In patients with FHC, a significant increase of r2 and R involving only the IgG1 subclass was observed, indicating the existence of LS of IgG1 in the majority of these patients. In the AH of patients with toxoplasmic chorioretinitis, no significant modification of IgG subclass or albumin concentrations was observed when compared to controls. In conclusion, it would seem interesting to consider measurement of IgG and IgG subclasses and calculation of the coefficients r1, r2 and R for a better evaluation of the local immunological processes observed in different types of uveitis.

Chromatography of human immunoglobulin G on immobilized drimarene rubine R/K-5BL. Study of mild, efficient elution procedures.

A range of substances were screened to find eluents for human immunoglobulin G (IgG) which are retained with a strong affinity by immobilized Drimarene Rubine R/K-5BL. The strong affinity of IgG for the dye is partly due to the presence of copper in the dye. This was suggested by the effect of substances able to make coordination bonds with metals that elute the IgG and also the effect of metal stripping from the immobilized dye. Several mobile phase conditions were found that allowed desorption of retained IgG on immobilized Drimarene Rubine R/K-5BL without using a protein denaturant. A procedure was also devised for separating IgG2 from other IgG subclasses using chromatography on immobilized Rubine R/K-5BL and column development with an AMP gradient.

Purification of human IgG4 subclass with allergen-specific blocking activity.

Blocking antibodies (bAb) induced by allergen immunotherapy are restricted to the IgG1 and IgG4 subclasses, with IgG1 predominating early and IgG4 coming later. Study of IgG4 bAb has been limited, in part, by the absence of a method to purify IgG4. We describe a rapid immunoaffinity chromatographic method for the purification of that subclass from whole serum. Starting serum (TR) contained 90 micrograms/ml Dactylis glomerata (orchard grass) pollen (DGP)-specific IgG4, measured by indirect ELISA. The blocking activity of TR was assayed in vitro on IgE-sensitized human basophils. Immunoadsorption on a strong-binding anti-IgG4 monoclonal antibody (mAb) removed about 90% of the total and allergen-specific IgG4 and nearly all of the blocking activity from TR. An IgG4-rich fraction was then obtained by absorption of several small volumes of TR on a weak-binding anti-IgG4 mAb column at neutral pH followed by elution with glycine-HCl buffer. The pooled eluates contained 82% IgG4, amounting to a 65-fold purification of the serum IgG4; the yield was approximately 30%. Nearly all the DGP-specific antibody was in the IgG4 component of the eluate. The blocking activity of the eluate was approximately equal to that of TR. Immunoblot patterns with the eluate and with TR on SDS-PAGE of DGP were nearly identical. This method thus provides a fully active, relatively pure IgG4 blocking antibody. Moreover, the results reinforce the importance of using a well-chosen mAb when purifying proteins by immunoaffinity chromatography.

Prospective follow-up of monoclonal gammopathies in HIV-infected individuals.

A cohort of 341 symptomless anti-human immunodeficiency virus (HIV)-1 positive individuals was screened over a 6-year period to detect a serum monoclonal gammopathy (MG) and to approach the prognostic significance of such anomaly in HIV infection. Eleven individuals with a MG were followed-up over a mean period of 50 months from the date of discovery of the MG. At the end of this period, the MG had disappeared in seven individuals, was still present in the four others. The appearance of a second MG was noticed in two cases, and of a third in one case. Immunoglobulin (Ig) typing identified eleven IgG kappa and three IgG lambda. Mean serum concentration of MG of individuals with persistent MG (14.3 g/l) and of individuals without (4.2 g/l) was significantly different (P < 0.05). The discovery of a MG was without prognosis value on the disease progression and did not appear as a primary event in the subsequent development of lymphoma.

[IgG4 levels in polyvalent immunoglobulins and immunoglobulins prepared by precipitation with caprylic acid]. / Taux des IgG4 dans les immunoglobulines polyvalentes et dans les immunoglobulines obtenues par précipitation à l'acide caprylique.

Levels of IgG4 in immunoglobulin preparations obtained either by conventional ethanol fractionation or by ethanol and caprylic acid fractionation (Allergam) were measured by an enzyme-immunoassay (competitive Elisa). In 9 ethanol preparations, the mean percentage of IgG4 was 2.1% +/- 0.5. In 10 preparations of Allergam, the mean percentage of IgG4 was 4.6% +/- 1. The concentration of IgG4 in Allergam preparations is about twice the concentration found in conventional ethanol preparations.