Hannele Tölö

Long-term persistence of selective IgA deficiency in healthy adults

A follow-up study of 204 healthy blood donors with IgA deficiency, identified between 1971 and 1980, was carried out. Sera were initially screened by a double diffusion method and 182 were retested by a more sensitive haemagglutination inhibition method. A reexamination was performed in 1990 and, again, in 1991–1992 using an enzyme immunoassay (EIA) developed for the measurement of very low concentrations of IgA. The median follow-up period was 19 years, and in 159 (78%) subjects no serum IgA could be detected in any of the measurements. In 42 (21%) subjects, serum IgA was detectable (>0.18 mg/L), but the level was below the lower limit of the reference range for adults (800 mg/L) and remained relatively constant. Three subjects showed minute amounts of IgA by EIA (0.2–3 mg/L) in one of the follow-up samples in 1990–1992, but the level was below the detection limit of the EIA (0.05 mg/L) in the other sample. Thus, not only does primary IgA deficiency appear to be permanent, but also lower than normal serum IgA levels remain the same in healthy adults.

Long-term follow-up of anti-IgA antibodies in healthy IgA-deficient adults

A follow-up study of anti-IgA antibodies in 159 healthy blood donors with severe deficiency of serum IgA (<0.05 mg/L) and in 45 donors with decreased serum IgA levels (0.05–799 mg/L), identified in 1971–1980, was carried out. Initially anti-IgA antibodies were determined by a hemagglutination (HA) method and two reexaminations were done in 1990–1992 by an enzyme immunoassay. The median follow-up period was 19 years, during which anti-IgA level was changed considerably in only four persons, increased in two, and high level antibodies (>1/1000 by HA) appeared in two. In reexaminations anti-IgA antibodies were found in 30 (19%) subjects with severe IgA deficiency and the antibody levels remained relatively constant in those who had high and medium antibody levels. Anti-IgA antibodies were not found in subjects with decreased, but detectable serum IgA. Thus it seems that only those healthy adults who have severe IgA deficiency develop anti-IgA antibodies and their anti-IgA levels remain fairly constant Of the 159 subjects with severe IgA deficiency, 66 had a history of IgA exposure, but no correlation to anti-IgA development was noted.

A sensitive enzyme immunoassay for the measurement of low concentrations of IgA

A microtitre plate enzyme immunoassay (EIA) for determining low concentrations of IgA is described and validated for serum and plasma products. The measuring range of the assay was 3.3-150 micrograms/l. The predilution requirement of samples was matrix dependent, ranging from 1/16 for serum to 1/2 for 4% albumin. Dilute protein solutions required no predilution. The limits of detection were 50 micrograms/l for serum, 25 micrograms/l for intravenous immunoglobulin, 13 micrograms/l for 20% albumin, 7 micrograms/l for 4% albumin and 3 micrograms/l for washing solutions of blood cell components. Interassay coefficients of variation over the range of 3.4 mg to 1.5 g IgA/l ranged from 3.8 to 5.7%. Respective values for two low-level sera, containing 309 and 512 micrograms IgA/l, were 15.5% and 11.1%. Comparison of the EIA with a commercial radial immunodiffusion (RID) method showed that the results of the two assays correlated well ([EIA] = 0.877 x [RID] + 0.401 mg/l, r = 0.996, n = 20). This assay is also suitable for the large-scale screening of blood donors.

An enzyme immunoassay for the determination of anti-IgA antibodies using polyclonal human IgA.

An enzyme immunoassay (EIA) for screening and quantitation of serum anti-IgA antibodies of IgG class is described. This method is based on the use of purified polyclonal human serum IgA as the coating antigen and a commercial alkaline phosphatase-conjugated anti-human IgG as the detecting antibody. Nonspecific reactions were minimized by blocking vacant protein binding sites with bovine serum albumin and by using individual sample blanks. The IgA specificity of a positive antibody finding was confirmed by testing inhibition: pooled normal human serum inhibited the binding of specific antibodies by over 80%. The same degree of inhibition could also be demonstrated by a commercial myeloma IgA preparation and by the IgA used for coating but not by IgA-deficient serum (< 0.05 mg/l). On the basis of the mean anti-IgA antibody titre in EIA, a value of 12,000 arbitrary units of anti-IgA per litre (AU/l) was assigned to a patient serum used as standard in the assay. Anti-IgA results obtained by EIA and haemagglutination correlated well, which makes it possible to compare earlier HA results with those obtained now by EIA. The measuring range of the assay was 0.6-27 AU/l and the lowest quantifiable concentration 7 AU/l. The dilution requirement for serum was 1/16. The interassay coefficients of variation for control sera with antibody levels from 35 AU/l to 3770 AU/l varied from 9 to 12%.

AN OPTIMIZED ASSAY FOR PREKALLIKREIN ACTIVATOR ACTIVITY IN HUMAN PLASMA PRODUCTS

Prekallikrein activator (PKA) assay is described. PKA was measured indirectly by allowing it to generate kallikrein from prekallikrein and then measuring the amidolytic activity of kallikrein by using the synthetic chromogenic substrate S-2302. It was ensured that sufficient amounts of substrates were included in both enzymatic phases of the assay, and the pH and ionic strength which seemed to be very important in the assay were carefully controlled. Both a kinetic assay and a simple endpoint assay are described. Other amidolytic enzymes probably present in plasma products are also estimated and a correction is made to exclude their role in the assay result. The precision of the kinetic assay is characterized by the coefficient of variation of 3.1% within an assay and the reproducibility by that of 4.8% between assays. For the endpoint assay both values are 4.8%.

Development of a Highly Purified Multicomponent Leukocyte IFN-α Product

A purification process was developed to obtain a human interferon- α (IFN-α) product that contains all major IFN-α subtypes produced by human leukocytes. The purification was accomplished by immunoaffinity chromatography using two monoclonal antibodies (mAb) and gel filtration. The process comprised two effective virus inactivation steps, solvent detergent treatment, and incubation at low pH, and the purified product was filtered with a 15-nm pore size virus removal filter. The overall yield of IFN-α in the process was about 60% when starting from the culture supernatant of Sendai virus-induced human leukocytes. The specific activity was about 1.0 × 108 IU/mg. The level of DNA and protein impurities including mouse IgG was very low. The product contained seven main subtypes: IFN-α 1, IFN-α 2, IFN-α 8, IFN-α 10, IFN-α 14, IFN-α 17, and IFN-α 21. The subtypes IFN-α 4 and IFN-α 7 were minor components. Reverse-phase HPLC indicated a constant subtype composition for the product from batch to batch. Stabilizat...