Monoclonal antibodies against rat T-kininogen: application to radioimmunoassay and immunohistochemistry

Monoclonal antibodies to rat T-kininogen were produced and 9 hybridomas were selected. Radioimmunoassay (RIA) was developed using 125I-labeled T-kininogen and cell walls of Staphylococcus aureus (Zysorbin) for the separation of bound from free ligand, when IgG2a and IgG2b were used. In the case of IgG1 monoclonals, a second antibody (goat anti-mouse IgG) and Zysorbin were used. By this RIA, 1-16 ng T-kininogen/tube showed a linear inhibition curve, and cross reactivities to rat purified LMW- and HMW-kininogens were less than 0.5%, respectively. These monoclonal antibodies were also used for the immunohistochemical staining of the liver to detect T-kininogen in hepatocytes. By using the RIA and immunohistochemical staining, the T-kininogen levels in rat plasma and liver following carrageenin-induced inflammation were estimated. At 3-5 h after the carrageenin injection, when the paw swelling was at its peak, the plasma level of T-kininogen and staining of the liver were slightly increased. T-Kininogen levels in plasma and liver peaked on the 2nd day, when the paw swelling had already decreased. The result indicates that the increase of T-kininogen level in the liver and plasma occurs with a time lag and T-kininogen is not directly involved in the increase of vascular permeability in carrageenin paw edema.     

Adenosine induces initial hypoxic-ischemic depression of synaptic transmission in the rat hippocampus in vivo

The present study was designed to investigate the role of adenosine in the hypoxic depression of synaptic transmission in rat hippocampus. An in vivo model of hypoxic synaptic depression was developed in which the common carotid artery was occluded on one side in the urethane-anesthetized rat. Inspired oxygen levels were controlled through a tracheal cannula. Rats were placed in a stereotaxic apparatus for stimulation and recording of bilateral hippocampal field excitatory postsynaptic potentials. The percent inspired oxygen could be reduced to levels that produced a reversible and repeatable depression of evoked synaptic transmission restricted to the hippocampus ipsilateral to the occlusion. Further reduction in the level of inspired oxygen depressed synaptic transmission recorded from both hippocampi. The adenosine nonselective antagonist caffeine and the A(1) selective antagonist 8-cyclopentyltheophylline prevented the initial depression in synaptic transmission. We conclude that the initial depression of synaptic transmission observed in the rat hippocampus in vivo is due to endogenous adenosine acting at neuronal adenosine A(1) receptors.     

A radioimmunoassay for the initial metabolites of the F prostaglandins

Antibodies to the F metabolite 9α, 11α-dihydro-15-keto-prostanoic acid (I), produced in the rabbit, do not cross react with any of the primary PGs. There is a 50% cross reaction with the metabolite 9α, 11α-dihydroxy-15-keto-prost-5-enoic acid (III), and a 23% cross reaction with 9α,11α,15-trihydroxy prostanoic acid (F₀α). No cross reactivity resulted with this antiserum when tested against 9α,11α,15-trihydroxy-5-enoic acid (VII) or with 9α,11α-dihydroxy-15-ketoprost-5,13-dienoic acid (VIII). Utilizing this antibody in a radioimmunoassay, some preliminary data are presented on levels of these F metabolites (I and III) for human adult male samples of plasma, urine and seminal plasma.     

The application of robust calibration to radioimmunoassay.

The minute concentrations of many biochemically and clinically important substances are currently estimated by radioimmunoassay (RIA). Traditionally, the most popular approaches to the statistical analysis of RIA data have been to linearize the data through transformation and fit the calibration curve using least squares or to directly fit a nonlinear calibration curve using least squares. Estimates of the hormone concentration in patients are then obtained using this curve. Unfortunately, the transformation is frequently unsuccessful in linearizing the data. Furthermore, the least squares fit can lead to erroneous results in both approaches since the many sources of error which exist in the RIA process often result in outlier observations. In this paper, an approach to the analysis of RIA data which incorporates robust estimation methods is described. An algorithm is presented for obtaining the M-estimates of nonlinear calibration curves. The curves to be fitted are modified hyperbolae based on 12 to 16 observations. A procedure, based on the application of the Bonferroni Inequality, is presented for obtaining tolerance-like interval estimates of the concentration of the hormone of interest in the patients. Results of simulations are cited to support the method of construction of confidence bands for the fitted calibration curve. Data obtained from the Veteran's Hospital, Buffalo, New York are used to illustrate the application of the algorithm which is presented.     

Measurement of apolipoprotein A-I in rat high density lipoprotein and in rat plasma by radioimmunoassay.

A double antibody radioimmunoassay (RIA) for rat apolipoprotein A-I is reported. The ApoA-I isolated from delipidated HDL by gel filtration yielded a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), and its amino acid composition resembled that reported by others. ApoA-I was iodinated by lactoperoxidase and the resulting 125I-apoA-I was purified by gel filtration. Up to 93% of 125I-apoA-I was precipitable by antibody and greater than 99% of bound 125I-apoA-I was displaced by "cold" apoA-I. Other rat lopoproteins and apolipoproteins did not react in this system. Human plasma were also not reactive, nor were dog, goat, and sheep plasmas.     

The characterization of radioimmunoassay for rat pancreatic polypeptide in serum.

A radioimmunoassay for the measurement of rat pancreatic polypeptide (RPP) in serum or plasma has been developed and characterized using a new guinea-pig anti-rat-PP antibody. The assay provides a high degree of sensitivity and lacks cross-reactivity (CR less than 0.01%) to neuropeptide Y and peptide YY. It also does not interact with PPs of other species or peptide hormones namely, amylin, glucagon, human insulin, human-PP, human-proinsulin, rat C-peptide and rat insulin. The assay employs synthetic rat PP as standards from concentrations of 21-2100 pg/ml (i.e., 5-500 pM) and produces a sensitivity limit of 19 pg/ml (4.5 pM) PP at +/- 3 S.D. The intra- and interassay % coefficient of variations are 6.4% and 5.9%, respectively. The % recovery of RPP added to rat serum samples ranges from 98% to 103%. Assay of serum volumes ranging from 25 microliters to 100 microliters does not significantly alter the expected RPP level. The migration patterns of rat serum PP and that of a synthetic RPP are identical by Sephadex G-50 chromatographic analysis. The mean values of fasting and a 2 h post-feeding plasma RPP levels in normal rats are 40 +/- 2 and 80 +/- 10 pg/ml (9.5 pM and 19.0 pM), respectively. Rat-PP release during insulin induced hypoglycemia in conscious rats rises from 38 +/- 5 pg/ml to 261 +/- 34 pg/ml (9.0 to 62.1 pM, P less than 0.005) by 30 min. Additionally, the antibody used in this study cross-reacts well with mouse-PP as determined by linear serum dilution curves, thus making it useful in the measurement of murine-PP. In conclusion, we have developed and validated a sensitive and specific rat-PP assay. This assay provides a new tool for the reliable measurement of PP in physiologic studies using rat and mouse animal models.     

Standardization and clinical application of a radioimmunoassay for human calcitonin.

A radioimmunoassay for the measurement of Human Calcitonin (HCT) is described. Serum levels of HCT in normal subjects and in individuals under different pathological conditions have been studied with this method. HCT labelling is performed following the chloramine T method of Hunter and Greenwood. Adding successively Quso G-32 (a finely powdered silica) and an anion exchange resin (AGI-X10 resin), to the tracer, reduces both the damaged fraction and the free isotope which originate during storage. Purification of the labelled hormone is carried out through a Sephadex G-50 gel column. Sera stored at --20 degrees C preserves its immunoreactivity up to 4 months after extraction. The mean basal HCT levels in 110 fasting normal persons is 277 +/- 123 pg/ml (undetectable 8.28%). No significant correlation between HCT levels and various serum ions has been observed. Basal HCT values in seven patients with medullary thyroid carcinoma (MTC), oscillated between 5 and 110 ng/ml, while in six other patients with non medullary thyroid carcinoma the values remained within normal range. Both a calcium infustion and a pentagastrin injection are used to stimulate HCT secretion. The increase of HCT basal levels produced by the latter in normal controls and in patients with MTC, is faster and more intense. Calcium infusion produced a significant correlation between calcium and the increased HCT levels only in patients affected with MTC.     

The development and application of a radioimmunoassay for 18-hydroxy-corticosterone.

A sensitive and specific radioimmunoassay has been developed for 18-hydroxy-corticosterone (18-OH-B) and applied to the measurement of this steroid in peripheral plasma. High specific activity label (3H-18-OH-B) was prepared using the incubation of 3H-corticosterone with duck adrenal mitochondria. Antisera were produced by immunisation with 18-OH-B gamma-lactone 3-oxime conjugated to bovine serum albumin. The antibodies examined showed 100% cross-reactivity with 18-hydroxy-deoxycorticosterone gamma-lactone (18-OH-DOC gamma-lactone), but minimal cross-reactivity with other steroids. Paper chromatography was used to separate 18-OH-DOC gamma-lactone from 18-OH-B gamma-lactone. The interassay precision was 7.6% and the intra-assay precision 11.0%. The accuracy of the method was confirmed by showing a linear relationship between amounts of 18-OH-B added and amounts of 18-OH-B gamma-lactone measured (y = 0.854 X +15.1, r = 0.9. p less than 0.001). The mean plasma level in normal subjects on an ad libitum sodium intake was 225 +/- 92.7 (SD) pg/ml (n = 17) when standing, and 99 +/- 38.3 (SD) pg/ml (n = 6) after lying down for 30 minutes.     

Measurement of T-kinin in rat plasma using a specific radioimmunoassay.

T-kinin (Ile-Ser-Bradykinin) has been isolated only from the plasma of the rat and it is unclear whether the peptide, or its biosynthetic precursor, T-kininogen, circulates in the human. An NH2-terminally directed antiserum to T-kinin was raised in rabbits using an immunogen prepared by coupling the free -SH group of T-kinin extended from its COOH-terminus by a cysteinyl residue to an -NH2 group on human serum albumin. A radioimmunoassay was developed using this antiserum and 125I-labelled [Tyr10]T-kinin as tracer that was sensitive (least-detectable concentration 3 fmol/tube) and relatively specific for T-kinin (cross-reactivity with bradykinin and kallidin less than 1%). Treatment of rat plasma with an excess of trypsin in the presence of a kininase inhibitor generated T-kinin immunoreactivity equivalent to 455 +/- 71 pmol/ml (mean +/- S.E.M.; n = 9) and this immunoreactivity was eluted from a reversed-phase HPLC column as a single peak with the same retention time as synthetic T-kinin. In contrast, treatment of plasma from healthy human subjects (n = 8) and from patients (n = 8) with inflammation due to acute or chronic gastrointestinal disease under the same conditions did not generate any detectable T-kinin immunoreactivity. It is concluded, therefore, that T-kininogen, the biosynthetic precursor of T-kinin in the rat, is either absent from the plasma of human subjects or is present in a concentration less than 30 fmol/ml. Similarly, T-kininogen is probably not an acute phase reactant in humans.     

Direct radioimmunoassay of progesterone in rat serum

A direct method has been described which makes possible a specific assay of progesterone in rat serum without extraction. Anti-progesterone serum was prepared in our laboratory by the immunization of three rabbits with 4-pregnen-3, 20-dione-3 CMO:BSA. This antiserum (Gunma OGP#1) displayed little or no cross reaction with 20 alpha-dihydroprogesterone (0.38%), pregnenolone (0.44%), 17 alpha hydroxypregnenolone (less than 0.1%), 20 beta hydroxyprogesterone (2.4%), 17 alpha hydroxyprogesterone (2.88%) or deoxycorticosterone (2.19%). The nonspecific inhibitory effect of serum was compensated for by adding progesterone-free serum to the standard curve tubes. The sensitivity of this assay was 1.1 pg/tube and serum progesterone could be measured by using as little as 1 microliter of serum. The working range of the standard curve was 1.25-2560 ng/ml. Under the conditions of this assay (1 microliter of serum per tube), interference from steroid binding proteins did not affect the sensitivity, precision or reliability of the assay. The intra-assay and inter-assay coefficients of variation were 5.5% and 8.7%, respectively, and the assay values correlated well with those obtained by the extraction method (R = 0.997, P less than 0.001). Analytical recovery indicates a close correlation between added and recovered progesterone concentrations (R = 0.992, P less than 0.001), and the recovery rate averaged 96%. Compared with the extraction method, the direct progesterone assay has the advantage of speed, precision and simplicity. The method described is particularly suitable for routine assays of progesterone in rat serum.     

A radioimmunoassay for corticosterone and its application to the measurement of stress in poultry.

A radioimmunoassay for corticosterone was developed using an antibody to corticosterone-21-hemisuccinate:bovine serum albumin. The assay possessed good specificity, sensitivity and reproducibility and required minimal sample preparation. Tests of adrenal function showed that stimulation of the adrenal with exogenous ACTH and with dexamethasone caused an increase and decrease, respectively, in plasma concentrations of corticosterone. Exposure to cold environmental temperatures caused an increase in plasma corticosterone. Handling and the removal of blood samples by venepuncture had no effect upon the concentration of corticosterone. It was concluded that this assay would accurately measure the response to stresses which affect the pituitary-adrenal axis.     

Embryonic synthesis of myoglobin in vivo estimated by radioimmunoassay

Chick embryos in ovo incorporated radioactivity from lysine-U-¹⁴C into myoglobin, as measured by an immunoprecipitation technique. The most consistent results were obtained by injection of the precursor into the yolk sac fluid.Incorporation, or apparent myoblobin synthesis, occurred in cardiac and skeletal muscle but not in liver, although incorporation of amino acid into total soluble proteins was equivalent in all tissues studied. Synthesis was highest in cardiac muscle and appeared there first in younger embryos. Myoglobin synthesis was detectable in the heart of embryos as early as 6 days of age and rose with age thereafter. Myoglobin synthesis appeared later and at lower levels in skeletal muscle.In vitro at neutral pH, tissue extracts of liver and muscle possessed only slight properties of myoglobin degradation.Using nonradioactive precipitin techniques, sensitive to 5–10 μg/ml, myoglobin was detected in embryonic heart muscle by week 2 of life and rose in content thereafter. Two of 8 embryos had trace amounts in thigh muscle near the time of hatching, and no embryos possessed measurable amounts of myoglobin in liver tissue or in pectoral skeletal muscle. Adult birds possessed equivalent amounts of myoglobin in heart and thigh muscle while pectoral muscle and liver tissue had no detectable myoglobin content.     

Purification and radioimmunoassay of rat lactate dehydrogenase A and B subunits.

We have developed procedures for purifying lactate dehydrogenase isoenzymes from rat tissues that involve two affinity-chromatography steps and that facilitate the isolation of milligram quantities of highly purified proteins within 2--3 days. Antibodies raised against pure A and B subunits in rabbits and hens were used in radioimmunoassays and showed negligible cross-reactivity with heterologous subunits. The radioimmunoassays provide a sensitive method for measuring nanogram amounts of A-subunit and B-subunit polypeptides in tissue homogenates and were employed to characterize the enzyme purification procedures.