Monoclonal immunoglobulins: affinity blotting for low concentrations in serum

I describe a simple, economical technique for identifying low concentrations of monoclonal immunoglobulins in the presence of excessive amounts of immunoglobulins of other classes. The technique involves binding of specific antibody to nitrocellulose, separating proteins by isoelectric focusing or zone electrophoresis in agarose gels, using capillary transfer to bind proteins to the nitrocellulose via their antibody affinity, and then detecting transferred proteins with enzyme-labeled antibody. A monoclonal immunoglobulin can be completely characterized in 2 h. No expensive equipment is required. Affinity blotting is about 10-fold as sensitive as native blotting, 100-fold as sensitive as silver staining.     

Detection and characterization of high affinity plasma membrane receptors for human interleukin 1

Interleukin 1 (IL-1) is a polypeptide hormone that acts as a central mediator of inflammation. Since IL-1 action is presumably mediated by specific cell surface receptor(s), we have characterized the binding of this hormone to cells. Purified human IL-1 was labeled to high specific activity with 125I, using Bolton-Hunter reagent. The labeled protein binds specifically to LBRM-33-1A5 (a murine T lymphoma line previously shown to produce IL-2 in response to phytohemagglutinin and IL-1) with an affinity of approximately 0.2-2 X 10(10)/M and, at saturation, to approximately 500 receptors per cell, on intact cells at 8 degrees C in the presence of sodium azide. The affinity of unmodified IL-1 for the murine plasma membrane receptor is 0.9-2 X 10(10)/M, as measured by the inhibition of 125I-IL-1 binding. The murine receptor specificity has been confirmed by demonstrating that, among a series of 12 polypeptide hormones, only IL-1 inhibits 125I-IL-1 binding to LBRM-33-1A5 cells. Treatment of surface-bound 125I-IL-1 with bivalent water-soluble crosslinkers identified a membrane polypeptide of Mr 79,500 to which IL-1 is crosslinked. A variety of cell types have been surveyed for the capacity to bind 125I-IL-1 specifically. The presence of specific binding correlates with the capacity of the cells tested to respond to IL-1. Our results indicate that the biological effects of the polypeptide hormone IL-1 are mediated by high affinity plasma membrane receptors. The identification of these receptors should provide valuable insight into the apparently diverse biological activities of IL-1.     

人胰型及唾液型淀粉酶的分离纯化与鉴定

目的 为获得高比活性、高纯度的人胰型及唾液型淀粉酶。方法 用硫酸铵分级沉淀、Ｓｅ ｐｈａｄｅｘ Ｇ－５０凝胶层析及ＤＥＡＥ－纤维素离子交换层析法分别从人胰腺及唾液中分离纯化人胰型及唾液型淀粉酶,并用聚丙烯酰胺凝胶电泳法进行纯度、酶活性及分子量鉴定。结果 获得了高比活性及高纯度的人胰型及唾液型淀粉酶,胰型及唾液型淀粉酶的表现分子量分别为５５ＫＤ和５７ＫＤ。结论 本方法是获得高纯度及高比活性人胰型及唾     

Characterization and species distribution of high affinity GTP-coupled receptors for human rantes and monocyte chemoattractant protein 1

Equilibrium binding studies with recombinant human chemoattractant cytokines Rantes and monocyte chemoattractant protein 1 (MCP-1) on monocytic THP-1 cells have allowed the functional identification of two distinct receptors for C-C chemokines. One is a novel oligospecific receptor with high affinity for Rantes (50% maximal inhibitory concentration [IC50], 0.68 nM) and low affinity (IC50, 35 nM) for MCP- 1, while the other is the previously described specific receptor for MCP-1 (IC50, 0.5 nM). Receptor affinity for Rantes is enhanced on preparation of isolated membranes with a 12-fold decrease in receptor Kd. The basis of this enhancement is not understood. The Rantes receptor appears to be G protein linked, as binding activity is abolished by guanosine 5'-O-(3-thiotriphosphate) (IC50, 7.3 nM). In contrast to the consequences of MCP-1 binding, we were unable to demonstrate ligand-dependent calcium fluxes on binding of Rantes to human monocytes or THP-1 cells. The binding of Rantes and MCP-1 to mononuclear cells from dog, rabbit, and rat were tested. While high affinity binding could be demonstrated in dog and rabbit, differences in ligand-induced Ca2+ fluxes could be shown between species. This suggests that receptor-ligand interactions and receptor coupling is best examined with autologous receptors and cytokine.     

Exploring high affinity monoclonal antibodies for drug invention

Antibodies can have a medicinal role because of their high specificity in molecular recognition. Although monoclonal antibodies specific for an antigen can be obtained relatively easily, it is difficult to acquire ones with high affinity. Recently, antibody engineering using an in vitro evolutionary process has been put to this task. However, little progress seems to have been made. Our previous study revealed that antibodies have low or high evolvability. An antibody with a poor ability to evolve would also not attain high affinity in in vitro maturation. In this review, we provide information for the improved design of the antigen-combining site and that this will contribute to the engineering of antibodies using an in vitro evolutionary process.     

Identification of high affinity folate binding proteins in human erythrocyte membranes.

Mature human erythrocyte membranes contained specific, high affinity (Kd 3.3 X 10(-11) M) folate binding moieties. Folate binding was pH, time- and temperature-dependent, saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with Mr greater than or equal to 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins), or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging.     

Density and agonist-promoted high and low affinity states of the beta-adrenoceptor on human B- and T-cells

beta-Adrenoceptor binding on peripheral blood mononuclear cells (PBMC) of healthy adult volunteers was investigated using the radioligand 125iodo-cyanopindolol (ICYP). Saturation binding studies were performed with nine different concentrations of ICYP. Receptor density and affinity were calculated by Scatchard plots. Resolution of beta-adrenoceptors into those with high and low affinity state of the beta-adrenoceptor was obtained from inhibition curves with salbutamol using Hofstee plots. Receptor density on enriched B-cells ('B-cells') was two-fold higher than on enriched T-cells ('T-cells') (P less than 0.025). Affinity (KD values) of beta-adrenoceptors did not differ for B- and T-cells. However, when two distinct binding states for beta-adrenoceptor agonists were identified using salbutamol displacement curves, beta-adrenoceptors on T-cells presented more receptors in a high affinity state than those on B-cells (P less than 0.01). Since the ability of an agonist to activate adenylate cyclase is closely correlated with the ratio of low to high affinity states formed in the presence of the agonist, increased intrinsic activity for the beta-adrenoceptor agonist on T-cells may be postulated. In conclusion, determination of the B/T ratio is a prerequisite for interpretation of beta-adrenoceptor changes on peripheral lymphocytes in various diseases.     

Correlation of low and high affinity thiol methyltransferase and phenol methyltransferase activities in human erythrocyte membranes

Human red blood cell (RBC) membranes have been reported to contain both high and low affinity 'forms' of the drug metabolizing enzyme thiol methyltransferase (TMT). The biochemical characteristics of the two 'forms' of human RBC TMT were compared. Apparent Km constants of the high affinity activity for 2-mercaptoethanol and S-adenosyl-L-methionine, cosubstrates for the TMT reaction, were 0.38 mumol/l and 2.6 mumol/l, respectively. These constants may be compared with values of 20 mmol/l and 43 mumol/l, respectively, previously reported for the low affinity form of RBC TMT activity. The properties and regulation of the two forms of TMT were then compared with each other and also with those of two 'control' enzymes, phenol methyltransferase (PMT) and beta-glucuronidase. When high and low affinity TMT, PMT and beta-glucuronidase activities were measured in RBC membranes from 22 individual subjects, there were highly significant correlations among all three methyltransferase activities (all r values greater than 0.95), but beta-glucuronidase activity did not correlate significantly with any of the methyltransferase activities (all r values less than 0.40). The thermal stabilities of the three methyltransferases were very similar. They were all inactivated approximately 50% by incubation at 48 degrees C for 15 min. beta-Glucuronidase activity was approximately 50% inactivated by incubation at 76 degrees C for 15 min. PMT and both TMT activities had similar subcellular distributions and similar responses to ions and to enzyme inhibitors. These results suggested that high and low affinity TMT and PMT activities might be catalyzed by the same enzyme. Alternatively, these three RBC membrane methyltransferase activities might be regulated in a parallel fashion.     

A case-by-case protocol of membranous nephropathy treatment with endovenous infusion of high doses of human immunoglobulins

The treatment of membranous nephropathy is a highly controversial issue. As some patients may have spontaneous remission, in about 50% of cases the risk of treating patients with drugs that may have severe side effects is higher than the potential benefit of arresting disease progression. Some authors therefore propose exclusively symptomatic treatment; other authors use steroids and immunosuppressive drugs, alone or in association with high risk of adverse effects and often uncertain benefits. The intravenous administration of high doses of human immunoglobulins (IVIg) has been also extended to a growing number of kidney diseases including membranous nephropathy. The mechanisms through which IVIg carry out their therapeutic effect are still unclear. The present study is a retrospective and uncontrolled trial, the aim of which was firstly to verify if some patients could respond to extremely short treatment protocols, stopped when they appear to have a stable remission, thereby avoiding expensive continuation of treatment. Secondly, we aimed to verify if some patients, judged as nonresponders to a classical protocol of IVIg therapy, could respond to a more prolonged treatment.     

Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor

Lipoxin A4 (LXA4) triggers selective responses with human neutrophils that are pertussis toxin sensitive and binds to high affinity receptors (Kd = 0.5 +/- 0.3 nM) that are modulated by stable analogues of guanosine 5'-triphosphate (GTP). Here, we characterized [11,12-(3)]LXA4 specific binding with neutrophil granule and plasma membranes, which each display high affinity binding sites (Kd = 0.7 +/- 0.1 nM) that were regulated by GTP gamma S. Since functional LXA4 receptors are inducible in HL-60 cells, we tested orphan cDNAs encoding 7- transmembrane region receptors cloned from these cells for their ability to bind and signal with LXA4. Chinese hamster ovary (CHO) cells transfected with the orphan receptor cDNA (pINF114) displayed specific 3H-LXA4 high affinity binding (1.7 nM). When displacement of LXA4 binding with pINF114-transfected CHO cells was tested with other eicosanoids, including LXB4, leukotriene D4 (LTD4), LTB4, or prostaglandin E2, only LTD4 competed with LXA4, giving a Ki of 80 nM. In transfected CHO cells, LXA4 also stimulated GTPase activity and provoked the release of esterified arachidonate, which proved to be pertussis toxin sensitive. These results indicate that pINF114 cDNA encodes a 7-transmembrane region-containing protein that displays high affinity for 3H-LXA4 and transmits LXA4-induced signals. Together, they suggest that the encoded protein is a candidate for a LXA4 receptor in myeloid cells.     

Radioimmunoassay for erythropoietin using anti-recombinant erythropoietin antibody with high affinity

A sensitive radioimmunoassay (RIA) for the detection of erythropoietin (EPO) was developed using anti-recombinant EPO antibody with high affinity. The sensitivity was 100 amol/tube (5 mIU/ml) and it was possible to detect a serum EPO level between 5 and 200 mIU/ml. This method enabled us to measure native EPO as well as recombinant EPO. With this method we determined serum EPO levels in healthy individuals and patients with chronic renal disease, rheumatoid arthritis and iron deficiency anemia. Values in patients with chronic renal disease were lower than those in healthy individuals, while values in patients with rheumatoid arthritis, or iron deficiency anemia were significantly higher than those in healthy individuals.     

A saturable high affinity binding site for transcobalamin II-vitamin B12 complexes in human placental membrane preparations.

Studies were designed to evaluate the binding of binding of vitamin B12 to cell membrane preparations from human placenta. The transcobalamin II-vitamin B12 complex (TCII-B12), which has a much greater affinity for the membranes than vitamin B12 alone, binds to a single saturable binding site with an approximate Ka = 7.2 mM-1. The binding requires a divalent cation and is temperature-dependent. Free TCII can compete with TCII-B12 for the binding site but has somewhat less affinity than does TCII-B12. Rat TCII-B12 has an affinity constant that is less than one-fifth that of human TCII-B12; human TCI-B12, bovine TCII-B12, hog intrinsic factor-B12 (IF-B12), and human IF-B12 do not bind to the membranes. Pretreating the membranes with trypsin causes a marked decrease in subsequent binding; this suggests the binding site includes a relatively exposed membrane protein. These data suggest that a specific cell surface receptor for the TCII-B12 complex exists in placenta. This TCII-B12 receptor can be solubilized with Triton X-100.     

Unusual low affinity of human liver fibronectin for gelatin

Fibronectin extracted from the human livers was shown to have very low affinity for gelatin unlike plasma fibronectin. It was demonstrated that this unusual property of liver fibronectin was not due to its proteolytic fragmentation.     

Low density lipoproteins and immunoglobulins in human pleural effusions.

Pleural effusions in patients with bacterial pleurisy contain low density lipoproteins (33 per cent of the blood serum LDL-concentration on average) but almost no very-low density lipoproteins (about 1 percent of the blood serum VLDL-concentration on average). Immunoglobulins are present in large amounts forming a series declining from the IgG (70 per cent of the blood serum concentration, found in effusions) to the IgM (50 per cent of the blood serum concentration). The observations are discussed in relation to morphological aspects of molecular transport through the capillary wall, and the consequences of the LDL- and IgL-accumulation in pleural fluids are briefly discussed.     

Multifactorial treatment of hypertensive men at high cardiovascular risk and low-density lipoprotein cholesterol affinity to human arterial proteoglycans

The purpose of this work was to examine in an open, randomized parallel-group study whether an intervention programme directed towards hypercholesterolaemia, smoking and diabetes mellitus in treated hypertensive men was associated with less complex formation between low-density lipoprotein (LDL) and human arterial proteoglycans than was the case with usual care. The intervention consisted mainly of non-pharmacological treatment, but drug therapy could be instituted to achieve the treatment goals in the intervention group. The intervention programme was associated with a significant reduction in body mass index, and 46% of the patients were on lipid-lowering medication at the follow-up examination. The net differences were (intervention − usual care): change in serum LDL-cholesterol, −0.48 mmol L⁻¹ (95% confidence interval −0.84 to −0.11 mmol L⁻¹), precipitated LDL-cholesterol, −5.5 μg (95% CI −9.0 to −1.1 μg). The latter remained after adjustment for the difference in serum LDL-cholesterol between the groups. Our conclusion is that the multifactorial risk factor treatment programme was associated with a reduced tendency of LDL to form complexes with human arterial proteoglycans.