Bo Dinesen

Enzyme immunoassay: an improved determination of urinary albumin in diabetics with incipient nephropathy

An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from normoalbuminuria to overt clinical nephropathy. Intra-assay variation was 2.1% and interassay variation 8.3%. Recovery of added albumin to urine was 95%-106% and dilution of urine was linear. The correlation to urinary albumin determined by immunodiffusion was excellent (n = 80, r = 0.99). Intraindividual variation of the 24 h urine albumin excretion of different days was high in patients with incipient diabetic nephropathy (51.5%) and was only slightly reduced by taking the variation of creatinine excretion into account (39.5%). No correlation was found between albumin excretion, and HbA1c or urine glucose excretion, indicating that minor metabolic variations are not responsible for the huge intraindividual day-to-day variations of UalbV. The study shows that more than one UalbV measurement must be done before classifying patients into groups with or without incipient diabetic nephropathy.

ELISA for human proinsulin

A micro enzyme-linked-immunosorbent-assay (ELISA) for monitoring circulating human proinsulin (hPI) was developed. A micro test plate was coated with guinea pig anti-insulin antibody. As labelling system peroxidase-labelled F(ab1)2-fragments of a guinea pig anti-human-C-peptide was used. The detection limit in buffer (95% level) was 0.6 pmol/l corresponding to 0.06 fmol/incubation well and to 1.2 pmol/l in serum, since samples were diluted 50%. Standard operating range was from 0-160 pmol/l. Interassay variation was 9% estimated from two human control materials (assayed within the range 6-9 pmol/l and 9-14 pmol/l, respectively). Insulin in samples did not interfere in concentrations below 400 pmol/l. Human C-peptide, porcine, and bovine proinsulins did not cross-react even at 10 000 pmol/l. In 38 healthy fasting subjects a reference range less than 1.2-13 pmol/l with a median of 4.1 pmol/l was found. Serum from total pancreatectomised patients showed values below the detection limit. The value from a patient with an insulinoma was 263 pmol/l. When stored at -20 degrees C human proinsulin appeared stable in serum or plasma for at least 9 mth. This ELISA, although among the most sensitive immunoassays for human proinsulin, is still not sensitive enough to measure the concentrations expected in samples from IDDM patients in the fasting state. In spite of this the method is useful in characterising beta-cell function in stimulated situations, as well as in the diagnosis of insulinoma.

Elisa for Human IgG and IpM Anti-Lipopolysaccharide Antibodies with Indirect Standardization

A new attempt to standardize ELISAs for the quantitation of human IgM and IgG anti-lipopolysaccharide antibodies (anti-LPS), without the use of a specific standard material, is described. Sandwich ELISAs for total IgG and IgM were combined with indirect ELISAs for anti-LPS IgG and IgM antibodies on a 96-well microtest plate using identical assay conditions. The concentration of specific IgG or IgM anti-LPS was read on the respective standard curve for total IgG or IgM. The results were corrected for residual immunoreactivity remaining unbound in the wells after one sample incubation in the combined assays. The quantitative results of IgG anti-LPS correlated well with results obtained using an ELISA with direct standardization (r = 0.969). 28 mg/l of IgM anti-LPS was found as median value among 121 blood donors using the described ELISA principle. Binding studies demonstrated a lower apparent affinity of donor anti-LPS IgM than anti-IgG.

Beta2-microglobulin in urine and serum determined by a micro-ELISA technique

An enzyme-linked immunoadsorbent assay for the determination of beta 2-microglobulin in serum and urine using microtest plates as solid phase is described. All reagents are commercially available. The assay has a high capacity and it is inexpensive using only 3% of the amounts of antibodies required in a previously described ELISA based on tubes as solid phase.

Proinsulin immunoreactivity in recent-onset IDDM: the significance of insulin antibodies and insulin autoantibodies.

OBJECTIVE To study the natural history of fasting proinsulin immunoreactivity (PIM) during the first 30 months of IDDM and its relationship to fasting C-peptide and insulin antibodies. RESEARCH DESIGN AND METHODS An incidence cohort of 204 consecutive newly diagnosed IDDM patients were followed prospectively, having blood drawn for measurements at diagnosis and at 1, 3, 6, 9, 12, 18, 24, and 30 months. A sensitive enzyme-linked immunosorbent assay was used for the determination of PIM. RESULTS All patients had detectable fasting PIM in plasma at diagnosis, with a median value and interquartile range of 3.5 pmol/l (2.2–6.2). The median PIM level increased during the first months of IDDM to reach a peak at 9–12 months (9.9–10.3 pmol/l). PIM then declined gradually to 5.6 pmol/l (1.9–13.5) at 30 months without reaching baseline. PIM at each time point was widely scattered in a skewed log-normal distribution without signs of bimodality. After the onset of insulin treatment, median insulin antibody level increased and declined in a similar pattern. Both PIM and antibody level were significantly higher in children and adolescents compared with adults. However, stepwise multiple regression analysis showed that age was only of minor importance for the PIM variation during the study period. Insulin antibody level and fasting C-peptide were the major determinants at 3–30 months, accounting for ∼40% of the variation (R2). Blood glucose was of minor importance, and insulin dose, HbA1c, and BMI were of no importance. The correlation between fasting PIM and fasting C-peptide improved (R2 doubled) if the insulin antibody level was accounted for. Further, the slope of the correlation curve between PIM and C-peptide increased threefold when antibody binding was > 4%. At diagnosis, insulin autoantibodies could be detected in 19% of the patients. Their presence predicted higher proinsulin at 1–3 months, a higher insulin dose the 1st year, and higher levels of insulin antibodies later in the study. CONCLUSIONS Circulating insulin antibodies may affect the level of PIM in IDDM, probably by adding a pool of IgG-bound PIM thereby increasing half-life and plasma concentration. This may explain why C-peptide and PIM levels do not change in concert during the 1st years of IDDM. Unlike C-peptide, PIM can not therefore quantitate β-cell secretion unless the presence of insulin antibodies is ruled out.

Monitoring the production of biosynthetic human growth hormone by micro enzyme-linked immunosorbent assay

Abstract A micro enzyme-linked immunosorbent assay (e.l.i.s.a.) has been developed for monitoring the production of human growth hormone (hGH) in E. coli . The method is unusually flexible, as it is possible to modify its conditions to give a sensitive (detection limit 4 ng l −1 ) or a fast (6 h) version. The assay is reproducible; the between-assay relative standard deviation is 6%. The effects of temperature, incubation time and the concentrations of protein and the most important reagents (the solid-phase antibody and the labelled antibody) are described. The utility of the method is demonstrated in experiments optimizing the conditions for production of hGH in E. coli .

Immunochemical aspects of growth hormone assays.

To construct an immunochemical assay of human growth hormone (hGH) is difficult due to the complicated nature of the hormone. Several forms coexist in the circulation with a 22,000 molecular weight form dominating. Related hormones with up to 93% homology are also present. hGH with its 191 amino acids presents more than 10 epitopes. Furthermore, the existence of two binding proteins adds to the complexity of the assay. Despite intensive efforts involving establishment of international standards, analytical consensus has not yet been reached. It is concluded that more specific immunoassays should be established.

The development of a rapid ELISA for IgE utilizing commercially available reagents

An enzyme-linked immunosorbent assay (ELISA) for human immunoglobin E (IgE) has been developed. To coat the polystyrene tubes (the solid phase) an antibody concentration of 6 mg/l in sodium carbonate buffer, pH 9.8, at 37 degrees C for 24 h was found superior to other conditions. The maximum amount of globulin adsorbed to the polystyrene surface was estimated to be 2.7 mg X m-2. This is consistent with a monolayer being adsorbed. While some of the anti-IgE molecules are inactivated during adsorption, the remaining molecules, once adsorbed, appear to retain activity for extended periods, and the coated tubes were stable for several weeks. Kinetic studies of the association of analyte, of antibody-enzyme conjugate and enzymic catalysis were used for the optimisation of the assay and allowed us to reduce assay time to 6 h. The association of analyte to the coated tube and the association of anti-IgE-peroxidase conjugate to bound IgE, in the present design, had T1/2 of approximately 0.5 h. Equilibrium is not obtained. The dissociation of analyte, surprisingly, was nearly undetectable. At least one of the two antibodies employed in the sandwich assay, preferably the anti-IgE-peroxidase conjugate, should be free of non-specific antibodies in order to eliminate non-specific reactions. The influence of serum matrix was eliminated by diluting serum samples and standards 1:21 in buffer. For the assay of very low concentrations (i.e. less than 10 kIU/l) standards produced in IgE-free serum are required to compensate the influence of matrix. Comparison with a commercial kit (Hoechst-Behringwerke, Frankfurt/Main, FRG) showed good agreement. All reagents are commercially available and the assay is well suited for routine use.

Biosynthetic Human Growth Hormone Identical to Authentic Material

The major component of human growth hormone (hGH) is a protein with 191 amino acid residues, and a molecular weight of approximately 22,000 D (22K-hGH). A minor component which constitutes about 5% of the more abundant form has a molecular weight of approximately 20,000 D. The minor form is derived from the major by deletion of 15 amino acid residues (32–46 of the 22K-isomer). Both molecules are single stranded, and two disulfide bridges stabilizes the structure. The N-terminus as well as the C-terminus is phenylalanine (Chawla et al., 1983).