A rapid, sensitive and versatile two-site immunoradiometric assay for insulin

A two-site immunoradiometric assay for insulin is described which is both rapid (processing time 60 min) and highly sensitive (lower detection limit 2 pM). Insulin is bound by a 125I-labelled mouse monoclonal antibody raised against human proinsulin and binding assessed by immunoprecipitation with an immunoadsorbent prepared from guinea pig polyclonal antisera raised against bovine insulin. Human, rat, bovine and porcine insulins (10-600 pM) showed similar reactivities in the assay. The human insulin-like peptides, proinsulin, des-31,32-proinsulin and des-64,65-proinsulin (25 pM) had reactivities which were 44.7%, 63.2% and 73.4% of that of insulin, respectively. The assay was highly reproducible with a coefficient of variation of 2.3% for the highest human insulin standard (1000 pM) and 5.5% for the lowest (2 pM). The assay was suitable for determining the concentration of insulin in plasma of fasting human subjects, in normal and tumour-bearing rats and for in vitro studies of insulin secretion from rat pancreatic islets.     

Newborn's fibrinolytic mechanism: components and plasmin generation

Plasminogen activity and antigen, tissue-type plasminogen activator (tPA) activity and antigen, plasminogen activator inhibitor (PAI) activity, and plasmin generation rates were determined in 32 normal newborn plasmas and 25 normal adult plasmas. The newborns showed reduced levels of plasminogen activity and antigen and tPA antigen, and activity, normal levels of PAI activity, and slower plasmin generation rates. The slower generation was shown to be due to the hypoplasminogenemia. The in vitro plasmin generation studies also showed that the newborn needed 11 times the usual concentration of urokinase and 5 times the usual concentration of tPA to achieve the minimal activation rate of the adult.     

A sensitive and specific assay for 5-methoxytryptophol in plasma.

5-Methoxytryptophol (ML) is found in the pineal gland and is known to have biological activity especially as an antigonadotrophic agent, but methods have been lacking for its measurement in the circulation. A gas chromatography-mass spectrometry assay using a trimethylsilyl derivative has been developed for the routine measurement of ML in plasma. The assay is of great specificity and has a sensitivity of 20 pmol/l. Studies on the levels of pineal indoles in the circulation, however, have been hampered by the possibility that extraneous compounds are being cross-measured. Thus the specificity of the routine assay has been further validated by comparing it with an alternative assay system where all the major parameters were changed, i.e. derivatizing reagent, internal standard and mass number. Results that were obtained using both assay systems were closely comparable.     

Development of a sensitive and rapid chromogenic factor IX assay for clinical use

A chromogenic factor IX assay is developed which requires only two time-dependent steps. Diluted plasma is mixed with a reagent containing factors VIII and X. The reaction is started by addition of a reagent containing factor XIa, thrombin, CaCl2, and phospholipids. Then factor XIa activates factor IX if present, thrombin activates factor VIII, and subsequently the complete factor X activating complex (factor IXa, factor VIIIa, Ca ions, and phospholipids) rapidly activates factor X. Finally, ethylenediaminetetraacetic acid plus a chromogenic substrate are added to stop the reaction and to measure formed factor Xa. Factor Xa formation is proportional to the plasma factor IX concentration (from 0 to 140%). The two reagents needed for the assay are stable at room temperature during a whole working day and for 3 h at 37 degrees C. A new isolation procedure for factor VIII is described. Factor VIII is purified from bovine plasma in a few steps with a yield of 20% and a 8,000-fold purification.     

Effects of porcine plasmin on the coagulation and fibrinolytic systems in humans.

Pig plasmin (Lysofibrin) was given to 11 patients with phlebographically verified venous thrombosis, 2 of whom were treated two and three times, respectively. The effect on coagulation and fibrinolytic parameters was studied. The platelet count, Owren's P&P (prothrombin plus factors VII and X), plasminogen, factor XIII, and antithrombin III did not change during the treatment. All patients developed a proteolytic activity demonstrable on both unheated and heated fibrin plates. The fibrinogen decreased successively to very low levels, and parallel to this an increase in fibrin/fibrinogen degradation products was found. The factor VIII and factor V activities decreased immediately after each Lysofibrin infusion but normalized rapidly again. The factor VIII molecule, however, retained its reactivity to rabbit antiserum against factor VIII. Immediately after the plasmin infusion a decrease of both alpha2-macroglobulin (alpha2-M) and the rapidly reacting alpha2-antiplasmin was observed. alpha2-M decreased successively and in several of the patients values were unmeasurable for a period of some days. A complex formation between pig plasmin and the alpha2-antiplasmin was demonstrated in crossed immunoelectrophoresis. The complexes were rapidly cleared from the circulation. No interaction between the pig plasmin and the inhibitor of the plasminogen activation, alpha1-antitrypsin or inter-alpha-inhibitor, was found.     

A standard for human plasmin.

An international collaborative study involving ten laboratories was undertaken to assess the suitability of a preparation of human plasmin in 50% glycerol to serve as a single standard for the bioassay of plasmin activity. The study included examination of the stability of the proposed standard and comparisons between assay methods. The results suggest that the human plasmin preparation coded 72/379 is suitable to serve as a standard. It is sufficiently stable to define the unit of plasmin activity for several years at or below 4 degrees C and for short periods at about 20 degrees C. The preparation has been assigned a potency of 8 units per ml. The International Committee on Thrombosis and Haemostasis (Basle, 1974) has recommended the use of this material as a standard.     

Membrane potential fluorescence: a rapid and highly sensitive assay for nicotinic receptor channel function

Seven cell lines expressing native and transfected nicotinic receptor subtypes were evaluated functionally by using fluorescent assays based on membrane potential and calcium dynamics with "no-wash" dye systems. Both assays provided the same rank orders of potency for (+/-)-epibatidine, 2S-(-)-nicotine, 7R,9S-(-)-cytisine, and 1,1-dimethyl-4-phenylpiperazinium in a cell line expressing rat alpha 3 beta 4 receptors. Nicotinic antagonists mecamylamine and dihydro-beta-erythroidine inhibited responses in both assays. Both agonist and antagonist activity were assessed within the same experiment. Agonists seemed more potent in the membrane potential assay than in the calcium assay, whereas the converse was true for antagonists. The membrane potential assay afforded robust responses in K-177 cells expressing human alpha 4 beta 2 receptors, in IMR-32 and SH-SY5Y cells expressing human ganglionic receptors, and in TE-671 cells expressing human neuromuscular receptors. These lines gave weak to modest calcium responses. Moreover, membrane potential responses were obtained in cell lines expressing rat alpha 4 beta 2 and alpha 4 beta 4 receptors, which were devoid of calcium responses. Thus, membrane potential serves as a sensitive measure of nicotinic activity, and the resulting depolarization may be as important as calcium in cell signaling.     

A histochemical study of fibrinolytic activity and inhibition of plasmin in the lungs of some animal species.

Determined by the histochemical fibrin slide technique, the fibrinolytic activity of lungs decreased in the following order of species: rat, mouse, pig, guinea pig, rabbit, cattle. Examined by the fibrin slide 'sandwich' technique, inhibition of plasmin showed the opposite mode of decrease. The possible role of these differences in the pathogenesis of various disorders of the lungs in the different species is discussed.     

A rapid,sensitive bioassay for luteinizing hormone-like gonadotropins

This report describes a simple and sensitive bioassay for gonadotropins having luteinizing hormone (LH)-like activity. The procedure depends on the ability of the gonadotropins to stimulate testosterone synthesis in testis interstitial cell suspensions prepared by collagenase digestion of immature rat testes. Although a similar procedure has been described previously, the method described here provides two modifications which (1) allow the chemically related gonadotropins LH and human chorionic gonadotropin (HCG) to be distinguished biologically and (2) increase the speed and reduce the cost of the assay. As described, the assay can be utilized to measure gonadotropin activity of avian, reptilian, amphibian, and, possibly, piscine materials as well as that of the well-characterized mammalian hormones. The simplicity and sensitivity (1 pg of HCG, 20 pg of LH) of this assay should greatly facilitate the isolation and purification of gonadotropins from nonmammalian vertebrates and, thus, enable comparative studies which were heretofore nearly impossible.     

Colony-forming cell proliferation: a rapid and sensitive assay system for murine granulocyte and macrophage colony-stimulating factors

A microculture assay for murine granulocyte-macrophage colony- stimulating factor (GM-CSF) has been developed using fetal liver GM colony-forming cells (CFC) isolated by fluorescence-activated cell sorting. These GM-CFC are free of mature hemopoietic cells, such as granulocytes and macrophages, which may interfere with direct assays for GM-CSF. The assay procedure allows the quantitation of GM-CSF within 48 hr by measuring the number of cells produced from 50 GM-CFC in microcultures (15 microliter). The assay is particularly simple to set up and score and yet, because of the reduced volumes, this assay is still capable of detecting 0.2 pg (i.e., 0.2 U) of GM-CSF within 48 hr, i.e., 100 times less GM-CSF than the conventional soft agar assay. By allowing the microcultures to develop for 7 days, the extra proliferation allows a further tenfold increase in the sensitivity of CSF detection. The time and cost of setting up hundreds of GM-CSF assays for fractions from chromatographic columns, e.g., reverse phase high performance liquid chromatography, is reduced by at least five- fold. Enough GM-CFC can be isolated and stored frozen in one afternoon to provide sufficient cells for the daily assay of 200 samples of GM- CSF for several months. Microassay results for several sources of GM- CSF at different stages of purification are compared to the results obtained from the soft agar assay.     

A sensitive,versatile microfluidic assay for bacterial chemotaxis

We have developed a microfluidic assay for bacterial chemotaxis in which a gradient of chemoeffectors is established inside a microchannel via diffusion between parallel streams of liquid in laminar flow. The random motility and chemotactic responses to L-aspartate, L-serine, L-leucine, and Ni(2+) of WT and chemotactic-mutant strains of Escherichia coli were measured. Migration of the cells was quantified by counting the cells accumulating in each of 22 outlet ports. The sensitivity of the assay is attested to by the significant response of WT cells to 3.2 nM L-aspartate, a concentration three orders of magnitude lower than the detection limit in the standard capillary assay. The response to repellents was as robust and easily recorded as the attractant response. A surprising discovery was that L-leucine is sensed by Tar as an attractant at low concentrations and by Tsr as a repellent at higher concentrations. This assay offers superior performance and convenience relative to the existing assays to measure bacterial tactic responses, and it is flexible enough to be used in a wide range of different applications.