Low molecular weight plasma proteins isolated from preparations of human immunoglobulin.

Low molecular weight proteins co-purified with IgG constitute 0.22% of the total protein purified from human plasma by ion-exchange chromatography on DEAE-cellulose. We have found that these low molecular weight proteins were obtained free of immunoglobulin by ultrafiltration in 5 M guanidinium chloride. Electrophoresis and isoelectric focusing in polyacrylamide gels demonstrated that this fraction of low molecular weight proteins is remarkably heterogeneous. Chromatography of an Mr 6000 to 12 000 fraction on hydroxyapatite resolved fourteen discrete protein peaks. Three of the peaks contained proteins which appeared to be homogeneous on acid-urea polyacrylamide gels. Two of these proteins were similar in composition to B2 globulin and may represent degradation products of some larger protein. The third protein was found to have an amino-terminal sequence identical to C3a. This population of low molecular weight plasma proteins has previously been shown to contain the cystic fibrosis mucociliary inhibitor and is here shown to contain two proteins similar to B2 globulin, C3a and many proteins remaining to be characterized. The presence of these low molecular weight proteins in measurable concentrations may be insufficiently appreciated in studies using 'purified' immunoglobulins as biological or chemical probes.

Elucidation of non-parallel EIA curves.

Quantitative determinations by EIA can be only obtained by reverse regression when linear portions of sample and standard curves are parallel. However, analysis of complex biological fluids often yields sigmoid curves displaying lower slopes, thus invalidating any quantitative interpretation. We hypothesized that this phenomenon was due to a competition effect between the target (for example an antigen) and related molecules for the binding sites (for example a capture antibody) immobilized onto the solid phase. This has been confirmed experimentally using various target-to-competitor ratios and formulated as a mathematical model. The slope decrease in target detection was related to the proportion of competitor, not in a linear, but in an exponential manner. This mathematical model has been computerized and can be used to correct aberrant sample curves provided the relevant parameters have been previously determined in the same systems.

Dot-immunobinding assay as an accurate and versatile technique for the quantification of human IgG subclasses.

A dot-immunobinding assay on nitrocellulose membranes has been developed for the quantification of human IgG subclasses using subclass-specific monoclonal antibodies. The advantages of this technique can be summarized as follows: (1) possibility of rapid semi-quantitative evaluation and/or precise quantitation from the same dot-pattern; (2) simple procedure with very good reproducibility; (3) sensitivity for nanogram concentrations of individual subclasses, therefore applicable not only to serum but also to other body fluids with a low content of IgG; (4) very small amounts of test material needed; (5) very good correlation of results with other techniques (ELISA, radial immunodiffusion) but without some of the inherent problems of the latter methods.

The effect of IgG aggregate size and concentration on reactivity in immune complex assays.

The reactivity of heat-aggregated IgG of known size, in the Raji cell assay, the C1q binding assay and the C1q solid phase radioimmunoassay as a function of concentration, has been investigated. Marked differences were found in the way that the three assays behave when the IgG concentration and aggregate size are varied. These findings indicate the pitfalls in attempting to express the results of immune complex assays performed on biological fluids in terms of equivalent concentrations of aggregated IgG.

The absolute quantification of human IgM and IgG: standardization and normal values.

Nephelometric, turbidimetric and radial immunodiffusion methods are described, which permit valid, reproducible determination of human IgM and IgG by weight. Subgroups of IgM, 7S-IgM and subclasses of IgG did not interfere with these methods. For IgG the International Unit represented a constant weight quantity in agreement with official WHO estimations. For IgM the IU was not constant and much lower values are found. It is therefore suggested that the IU should be abolished for human IgM and IgG. Absolute values for IgM levels are presented for both sexes and for age groups in the range of 5-80 years. The figures showed significant variation with sex and age.

Two-site immunoenzymometric assays for serum IgG subclass infant/maternal ratios at full-term.

Improvements were made in the range, precision, convenience, automation, and reliability of our previously published two-site immunoenzymometric assays using mouse monoclonal antibodies specific for human IgG and its subclasses. The serum concentration ratios for these immunoglobulin isotypes were measured in neonatal/maternal paired sera from 119 normal full-term deliveries. These ratios are significantly different than 1.0 (P = 0.001) for total IgG, IgG1, and IgG2 (show non-equality of paired neonatal/maternal sera concentrations) but are not significantly different than 1.0 for IgG3 and IgG4. On average, IgG1 is elevated 61%, and IgG2 is depressed 11% in the full-term neonate with respect to its own mother. In some pregnancies, active transport of IgG1 may be selectively enhanced by low material IgG1 concentration and selectively inhibited by high levels.

A new radiometric assay for the quantitation of surface-bound IgG on sensitized erythrocytes.

We have developed a sensitive, straightforward method for the quantitation of surface-bound IgG on sensitized erythrocytes. The assay is based on the consumption by sensitized cells of anti-IgG antiserum. The remaining anti-IgG is quantitated in a second incubation by precipitation with 125I-IgG in the presence of polyethylene glycol. Calibration curves for this assay were constructed using known amounts of unlabeled IgG. The method can be performed in microtitre plates and eliminates the use of purified anti-erythrocyte antibodies, or highly purified specific anti-IgG antisera. The results were completely consistent with those of immunofluorescence assays, but our method was much more sensitive, less than 500 molecules of IgG per cell being detected reproducibly. The technique is not laborious and takes much less time than previously described methods with similar sensitivity. As an example of the applicability of this test, the implications of ligand density for the detection by EA rosetting of Fc receptors on human monocytes are shown. The results suggest that a large variation exists in the affinity of the different types of Fc receptors for their ligands.

Typing of subclasses and light chains of human monoclonal immunoglobulins by particle counting immunoassay (PACIA).

The subclasses of monoclonal IgGs and IgAs were identified by particle-counting immunoassay. The principle of the test is the inhibition of the agglutinating activity of either specific antisera or monoclonal antibodies (for IgA only) on latex particles coated with a monoclonal IgG or IgA of known subclass. The feasibility of assay of polyclonal Ig subclasses was demonstrated. However, the anti-IgG2 antiserum cross-reacted with an allotype (nG4m(b)) of IgG4. The possibility of typing monoclonal Igs for light chains by the same technique was also demonstrated. Results are obtained in 30 min, and the method requires only small amounts of purified immunoglobulins (Igs) and antisera or monoclonal antibodies.

Indirect double sandwich ELISA for the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses.

We have developed sensitive enzyme-linked immunosorbent assays (ELISA) which measure mouse serum heavy chain immunoglobulin isotypes in nanograms per milliliter. In each case the specific isotypic Ig is sandwiched between an isotype-specific antibody used for coating and another isotype-specific antibody coupled to biotin for detection (with alkaline phosphatase coupled to avidin). These methods are simple to perform, specific for each isotype, reproducible with an average coefficient of variation of 5% for IgG1, 3% for IgG2a, 7% for IgG2b, 10% for IgG3, 3% for IgA and 7% for IgM, and at least 100 times more sensitive than radial immunodiffusion. The assays have been used to determine the absolute concentrations of mouse serum heavy chain Ig isotypes.

Protein assays for monoclonal antibodies.

Several techniques were evaluated for the quantitation of the total protein content of an IgG2a monoclonal antibody, KS1/4, and its deacetylvinblastine (DAVLB) conjugate. The UV assay is rapid, but it requires an extensive calibration of the response factor, and impurities may cause a high bias. Amino acid analysis (AAA) is an absolute method that has few interferences, but it requires evaluation of hydrolysis recovery factors. Kjeldahl nitrogen is very sensitive to minor impurities, and it requires a conversion factor to calculate percent protein. The Kjeldahl assay also is less precise (observed RSD of 3-4%) than the UV and AAA assays (observed RSD of 2-3% for both). The bicinchoninic acid assay, a representative of colorimetric assays, inherently requires comparison to a calibrated standard of the same material and tends to be less precise than the other assays. Thus, the UV and AAA assays are the techniques of choice for measurement of the total protein content of KS1/4 and its DAVLB conjugate.

Development of automated immunoassays for immune status screening and serodiagnosis of rubella virus infection.

The fully automated IMx immunoassay analyzer was used to develop a system for the detection of IgG and IgM antibodies to rubella virus for immune status screening and diagnosis of primary infections. Reagents and assay protocol software were developed using rubella virus sensitized microparticles as the solid phase to capture specific antibodies from serum samples. Anti-human IgG or IgM antibody coupled to alkaline phosphatase enzyme followed by methylumbelliferyl phosphate substrate was used to detect the presence or absence of antibodies specific to the antigens on the solid phase. To evaluate the efficacy of the IMx rubella IgG assay, immune status screening was performed with a clinical patient population of 501 sera. When compared to an IgG specific enzyme immunoassay and passive hemagglutination assay the agreement was greater than 99%. The IMx rubella IgM assay was utilized to determine the presence of rubella specific IgM antibodies in 462 sera. These results were compared to IgM specific enzyme immunoassay results and also demonstrated greater than 99% agreement. Seroconversion following rubella vaccination of susceptible individuals was demonstrated by IgG and IgM antibody responses as early as two weeks postvaccination. In addition to automation, the IMx system offers rapid assay times and calibration curve storage without sacrificing clinical efficacy.

Anticardiolipin antibodies and thrombosis: buffer's influence on the detection and quantitation of anticardiolipin antibody measured by ELISA.

The influence of adult bovine serum (ABS) and fetal calf serum (FCS) on IgG anticardiolipin antibodies (ACA) levels measured by ELISA has been investigated. In control subjects the frequency of the optical density (O.D.) distribution was found to be non-gaussian. The O.D. of the 97th percentile was similar with both buffers, although the distribution of O.D. was statistically different (P less than 0.004). The slope of the dilution curves of two house standards as well as the curves obtained with reference standards were found to be very different depending on the buffer. In 55 consecutive patients with thrombotic events, recurrent fetal loss or auto-immune disorders, ACA positivity was also dependent on the way to express the data and the choice of the buffer: when O.D. values were read with their respective standard curves, IgG ACA positivity was 98% with FCS and 20% with ABS (P less than 0.001), but when the 97th percentile O.D. was considered, 40% were positive with FCS and 51% with ABS (n.s.). Furthermore 10 patients were found to be negative with FCS and positive with ABS and 4 others showed the inverse picture. Levels of positivity were also dependent on the buffer. These differences arise from the solution used to dilute the sera and not of that to block the plates. These methodological problems may explain some conflicting data found in the literature about the prevalence of ACA positivity.

Simple radioimmunobinding assay for quantitation of sperm antibodies of IgG immunoglobulin class.

The immunobead test (IBT) is an excellent test for initial sperm antibody screening and evaluation but is impractical when used to quantitate antibody levels using a twofold dilution series. The aim of this study, therefore, was to develop a relatively simple radioimmunobinding assay that would allow quantitation of sperm antibodies of IgG immunoglobulin class, which predominate in male sera. [125I]-Protein G was chosen as the radioligand because it binds to all IgG subclasses, but not to IgA or IgM. The results of the investigation indicate that the [125I]-protein G assay (PGA) allows efficient quantitation of sperm antibodies, as evidenced by a highly significant (P less than .0001) correlation (Spearman's, Rs = 0.94) between the PGA results and IgG-IBT titres.

The viral safety of intravenous immunoglobulin.

Human immunoglobulin for intravenous (IV) use has an established safety record with regard to transmission of hepatitis B virus. The bulk of available evidence also suggests that the human immunodeficiency virus (HIV) is not transmitted by IV immunoglobulin. There has been one report, however, of isolation of HIV from two patients with hypogammaglobulinaemia who had been treated with several immunoglobulin products. Certain IV immunoglobulin products have transmitted non-A, non-B (NANB) hepatitis but careful clinical assessment of recipients of other products suggests that non-infective preparations can be made. Interpretation of available data most likely to be correct is that contamination with NANB is reduced but not eliminated by cold-ethanol fractionation and that the use of further virucidal procedures in the finishing of immunoglobulin products will confer a higher degree of safety.

[ELISA diagnosis for Mycoplasma pneumoniae infection with human normal immunoglobulin products as control sera].

It was found that the human normal immunoglobulin G product, prepared from pooled plasma of at least 1,000 normal human donors, contained some specific IgG antibodies to Mycoplasma pneumoniae by Western blotting. M. pneumoniae-ELISA OD values increased linearly from 25 to 800 micrograms/ml in the concentration of the product, there were no differences in the M. pneumoniae-ELISA OD value of the products among lots. Then we used this product as control serum for measurement of IgG antibodies to M. pneumoniae in human sera by ELISA and compared the results with those obtained by complement fixation (CF) and indirect hemadosorption (IHA) tests. ELISA titers determined by this method had a mutual relation with CF and IHA titers.

Anticardiolipin antibodies by an enzyme-linked immunosorbent assay: fundamental studies on the conditions for antigen-application and specificity of the assay.

We have established an enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (aCL) using standard sera obtained from the Rayne Institute, St. Thomas' Hospital (London, U.K.). In this study, we compared several fundamental requirements for the assay with the standard assay as a reference, such as conditions for antigen application and test samples using our patients' samples. In addition, the specificity of our assay and cross-reactivities of aCL were also evaluated. In the standard assay, the concentration of antigen was optimal in the range of 30-100 micrograms/ml. The antigen-coating temperature was optimal at 4 degrees C for 16 hours. The method based on rapid evaporation of CL-ethanol solution can be used instead of the standard method. On the other hand, there was no significant difference in the results between physical conditions of the antigen (CL-ethanol solution vs. CL-micelles), between washing solutions (saline vs. PBS containing 0.05% Tween-20) and between test samples (sera vs. plasma). The aCL activity in our patients' samples was almost completely inhibited by pre-incubation of sera with either CL or phospholipid reagent for activated partial thromboplastin time (APTT). Interestingly, the aCL activity of the lupus anticoagulant was negative, but aCL-positive samples were also absorbed by the reagent for APTT. No inhibition of the aCL activity, however, was observed when patients' sera were preincubated with ss-DNA.

[Level of total serum IgG4 using ELISA]. / Dosage des IgG4 sériques totales par méthode Elisa.

We report on an indirect competitive immunoenzymatic assay (ELISA) for the determination of serum total IgG4 levels. This procedure allows accurate and sensitive measurements. Normal values vary according to age and sex, with mean normal levels of 22.3 +/- 14.6 mg/dl in adults. Serum IgG4 levels observed in atopic subjects are not significantly different from those in age and sex matched normal subjects. However, IgG4 levels are significantly increased in hay fever and honey bee sensitization. Measurement of serum total IgG4 levels clearly needs to be combined with the evaluation of specific anti-allergen antibodies of this subclass in investigations of the possible reaginic or blocking activity of IgG4 antibodies.

[Molecular composition of the fractions of immunoglobulin preparations studied by gel filtration]. / Izuchenie molekuliarnogo sostava fraktsii immunoglobulinovykh preparatov metodom gel'-fil'tratsii.

The method of gel filtration in columns permitted the separation of aggregated fractions into polymers whose content did not exceed 10% and dimers, their content ranging from 3.6% to 22.11%. The preparations were also found to contain fractions of monomers and fragments, Fab-fragments being detected in 10 out of 20 batches under study (4.04-27.36%). The shelf life of all preparations did not exceed 8-12 months. The use of spectrophotometric techniques ensured obtaining the most objective results in the calculation of the percentage of fractions contained in immunoglobulin preparations. The evaluation of the molecular composition of immunoglobulin preparations by the method of gel filtration is conducive to the improvement of their quality.