Ingvar Larsson

Radioimmunoassay of delta sleep-inducing peptide using an iodinated p-hydroxyphenylpropionic acid derivative as tracer

A highly sensitive radioimmunoassay for delta sleep-inducing peptide (DSIP) has been developed. A p-hydroxyphenylpropionic acid conjugate of DSIP was used for radioiodination. Using reversed-phase high performance liquid chromatography the labelled DSIP derivative was isolated in a high yield and with a high specific activity. The assay allows measurement of DSIP-like material in body fluids with a minimum detectable concentration of 0.1 ng/ml standard DSIP (10 pg/tube).

Characterization and application of a radioimmunoassay for β-endorphin using an antiserum with negligible cross-reactivity against β-lipotropin

Abstract High avidity antisera against β-endorphin (β h - EP ) were obtained in two of five rabbits immunized with unconjugated synthetic human β h - EP . One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with β-lipotropin (β h - LPH ) and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/l. The cross-reaction with methionine-enkephalin (β h - LPH 61–65) was 9%, while that with α-endorphin ( β h - LPH 61–76) was 69%. This implied that the specific recognition site was in the amino-terminal region of β h - EP . Although this sequence is present in β h - LPH it was poorly recognized by the antiserum, suggesting that the free amino-terminal is essential. This interpretation was supported by the finding that α-N- acetyl -β h - EP was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/l. β h - EP was not detectable ( β h - EP was detectable ( 5 ± 3 pmol/l ; mean ± S.D.) after metyrapone. β h - EP was elevated in Addisons disease (23, 54 and 76 pmol/l), Nelsons syndrome (37, 39 and 109 pmol/l), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushings disease (7 pmol/l). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human β h - EP . The specificity of the antiserum K-7762 was such that the β h - EP concentration in plasma extracts could be reliably estimated by radioimmunoassay without prior chromatography.

A new sensitive immunosorbent radioassay for the detection of circulating antibodies to polypeptide hormones and proteins.

A solid-phase immunosorbent radioassay for the detection of circulating antibodies to protein hormones is described. The assay is based on the binding of the homologous 125I-labelled antigen to the antibodies which are then bound to anti-IgG antibodies covalently coupled to Sepharose. It can easily be applied as a complement to any radioimmunoassay for the detection of circulating antibodies to the ligand measured. The assay system avoids falsely elevated values due to interference of high serum concentrations of the antigen. The assay was applied to measure antibodies to FSH, LH, TSH, GH, prolactin, insulin and thyroglobulin (Tg). Among patients with chronic thyroiditis Tg antibodies were found in 100% of the sera. In diffuse toxic goitre 73% of the patients had detectable Tg antibodies. Insulin antibodies were present in 82% of the sera from patients with insulin treated diabetes. No antibodies were found against the other protein hormones tested.

Radioimmunoassay of human sex hormone binding globulin: improved radioiodination procedure

Sex hormone binding globulin (SHBG), purified by affinity chromatography from retroplacental blood plasma, was reacted with 3-(p-hydroxyphenyl) propionic acid N-hydroxysuccinimidyl ester (PHPPS, Bolton-Hunter reagent). The derivative of SHBG obtained (parahydroxyphenylpropionyl-SHBG; PHPP-SHBG) was stable and could, in contrast to underived SHBG, be efficiently 125I-iodinated with a lactoperoxidase technique. The PHPP-SHBG labelled with 125I had good antiserum binding and stability properties and was used for radioimmunoassay (RIA) of SHBG in serum. The RIA requires a total incubation time of 3 h. It has been standardized with purified SHBG and has a sensitivity of 5 micrograms/l, giving a lowest detectable concentration in the routine procedure (samples diluted 1:40) of about 0.2 mg/l. Variation within and between assay was 4.1% and 7.2%, respectively, for samples with values within the normal range. Values obtained by this RIA procedure correlate well with those obtained by a dihydrotestosterone binding method and by an electroimmunoassay technique. The mean serum concentration of SHBG in healthy, regularly menstruating women (n = 42) was 3.7 +/- 1.0 (SD, standard deviation) mg/l and in healthy men (n = 100) 2.0 +/- 0.9 mg/l.

Purification and storage of thyroglobulin. Two important factors influencing the radioimmunoassay for thyroglobulin

The effect upon the assay of the quality of the thyroglobulin (Tg) used as standard and tracer was evaluated by comparison of two preparations, one purified with protease inhibitors added (Tg-PI) and the other without (Tg-O). Tg-PI proved more stable than Tg-O. After freezing in phosphate-buffered saline almost all 125I-Tg-O was found to have dissociated into 12 S Tg, while only about half the 125I-Tg-PI had done so. Storage in glycerol, 500 g/l, at -20 degrees C or freezing in goat serum improved the quality of the 125I-Tg markedly, but Tg-PI still remained more stable than Tg-O. In addition, the two antisera tested gave different results in the radioimmunoassay with Tg-PI and Tg-O. With one antiserum a gradual loss of Tg immunoreactivity occurred parallel to the dissociation of Tg, while no such effect was noted with the other antiserum. This difference is believed to depend on varying proportions of conformational antibodies in the antisera, the binding sites for the conformational antibodies being distorted by the dissociation of the Tg molecule, while the binding sites for the sequential antibodies remain intact.

A Scintillation Camera Technique for Quantitative Estimation of Separate Kidney Function and Its Use before Nephrectomy

A scintillation camera technique was used for measuring renal uptake of [131I]Hippuran 80-110 s after injection. Externally measured Hippuran uptake was markedly influenced by kidney depth, which was measured by lateral-view image after injection of [99mTc]iron ascorbic acid complex or [197Hg]chlormerodrine. When one kidney was nearer to the dorsal surface of the body than the other, it was necessary to correct the externally measured Hippuran uptake for kidney depth to obtain reliable information on the true partition of Hippuran between the two kidneys. In some patients the glomerular filtration rate (GFR) was measured before and after nephrectomy. Measured postoperative GFR was compared with preoperatively predicted GFR, which was calculated by multiplying the preoperative Hippuran uptake of the kidney to be left in situ, as a fraction of the preoperative Hippuran uptake of both kidneys, by the measured preoperative GFR. The measured postoperative GFR was usually moderately higher than the preoperatively predicted GFR. The difference could be explained by a postoperative compensatory increase in function of the remaining kidney. Thus, the present method offers a possibility of estimating separate kidney function without arterial or ureteric catheterization.

Hydra head activator-like immunoreactivity in human brain astrocytomas grade III–IV and the surrounding brain tissue

We have developed a specific and sensitive radioimmunoassay for the hydra head activator neuropeptide (HHAP). The antibody recognizes the C-terminal portion of HHAP and shows no cross-reactivity with other neuropeptides. The assay allows measurement of HHAP-like material in tissue extracts with a minimum detectable concentration of 22 fmol/ml standard HHAP (2 fmol/tube). The concentration of immunoreactive (IR) HHAP in histopathologically characterized tissues was determined in 43 specimens from astrocytomas grade III-IV and surrounding brain tissue from 18 patients. Twenty-two control specimens of gray and white matter and five from hypothalamus were taken from 5 brains at autopsy. The concentration of IR-HHAP in the brain tumors, including the actively growing tumor front, is lower than in normal brain, and thus appears not to act as a growth factor. High performance liquid chromatography (HPLC) of extracts of hypothalamus and tumors revealed two major peaks of IR-HHAP; one eluted with the same elution volume as synthetic HHAP. HPLC of cerebral cortex extracts revealed only one major peak of IR-HHAP eluting close to the void volume, which may indicate a posttranslational processing variability of HHAP in different brain regions. By immunocytochemistry HHAP immunoreactivity was localized to the cytoplasma of neuroectodermal cells.

Determination of Urinary Fibrin/Fibrinogen Degradation Products by Radioimmunoassay

Glomerular fibrin/fibrinogen related material is often found in the early stage of especially proliferative types of glomerulonephritis and during rejection of renal grafts after transplantation. The presence and concentration of urinary fibrin/fibrinogen degradation products (FDP) have been found to be correlated to the extent of glomerular fibrin deposits. Most methods for FDP determination hitherto available are not sensitive enough to detect such small amounts of the fibrin/fibrinogen related material in the urine as are found in early glomerulonephritis. A radioimmunoassay for determining urinary FDP is described, in which the high molecular weight degradation products (HMWDP) are first converted into end degradation products (D and E) by addition of plasminogen and streptokinase to the urine sample. In the test an antiserum against purified D-products is used, which avoids the influence of varying binding properties and therefore makes the method more specific.

Methods of reducing the effect of spontaneous dissociation of antigen-antibody interactions in radioimmunoassays with special reference to internal sample attenuator counting (ISAC)

A time dependent decrease of sample counts was observed in an adsorption radioimmunoassay with internal sample attenuator counting. The drift caused a bias in the estimate of the amount of antigen in the samples. The size of this deviation was dependent upon the length of time after the calibration curve was made that the samples were measured. This was essentially due to dissociation of the antigen-antibody complexes as the adsorber can act as a second receptor to the antigen. Addition of slowly sedimenting starch microspheres or starch particles inhibited the drift by forming a diffusion barrier on the attenuating pellet.

Characterization of specific antisera to native human luteinizing hormone.

Antisera against highly purified native hLH (10 000 IU/mg = 0.5 mg hLH/mg protein) were raised in 4 rabbits. The antisera were invariably of high titre and high avidity. They were purified by affinity chromatography on a column with hCG-Sepharose 4 B. Before and after adsorption the antisera were tested in a double antibody radioimmunoassay. Before adsorption hCG cross-reacted in all antisera, but in none were the inhibition curves identical. No such cross-reaction remained after the affinity chromatography. These purified antisera made it possible to develop highly specific and sensitive assays for measuring native hLH. We conclude from studies of the effect of native glycoprotein hormones and their subunits that there are probably differences in the antigenic sites of the complete hLH molecule and in the isolated beta-subunit of hLH. The investigation also indicates that even highly purified preparations of hTSH may be contaminated with hLH and that the preparations of hLH-subunits may contain a certain amount of the native hormone.