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.

The extracellular domain of the human erythropoietin receptor: expression as a fusion protein in Escherichia coli, purification, and biological properties.

We developed an efficient production system of the soluble extracellular domain of the human erythropoietin receptor (sEPO-R) and characterized the binding of erythropoietin (EPO) with the purified recombinant protein. The sEPO-R, fused to the maltose binding protein (MBP), was expressed as a soluble protein in the periplasm of Escherichia coli (E. coli) and did not accumulate in inclusion bodies. After lysis of the bacteria by an osmotic shock, the fusion protein was purified by affinity chromatography on amylose followed by size exclusion chromatography (SEC). Specific binding of 125I-labelled EPO to the sEPO-R was demonstrated by competitive and saturation binding assays. A single affinity class (Kd = 0.25 nM) of the binding site was evident by Scatchard analysis. This value is similar to the Kd observed between EPO and the EPO-R of high affinity present on human erythroid progenitors. The complex has a molecular size corresponding to a 1:1 complex of EPO and the fusion protein.

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.

Chromatographic separation of recombinant human erythropoietin isoforms.

Erythropoietin (EPO) is the main regulator of erythropoiesis. The human glycoprotein hormone is heterogeneous when analyzed by isoelectric focusing (IEF). We investigated the possibility of fractionating EPO isoforms using different chromatographic methods. A recombinant human EPO (rhEPO) was obtained from the culture supernatants of a human B-lymphoblastoid cell line transfected by the human EPO gene. Highly purified rhEPO preparations were obtained by immunoaffinity purification. More than fourteen isoforms were observed after IEF. Among the different methods developed for isoform fractionation, the most reproducible results were obtained by DEAE-Sephacel chromatography. Seven fractions of decreasing isoelectric point (pI) were obtained. The specific activity of these fractions measured by an immunoradiometric assay was not equally distributed.