ATP-insulin conjugates and their use for immunofactor analysis

A method resulting in ATP-insulin conjugates by covalent binding of ATP modified at C(6) amino group of the adenine residue with insulin was developed. The modified ATP was bound to insulin by means of metha-p-toluene sulfonate-N-cyclohezyl Nf [2-morpholinyl(4)ethyl]-carbodiimide. The ATP analogs and ATP-insulin conjugates possess the coenzyme activity in a reaction of luciferin oxidation by luciferase from the fireflies Luciola mingrelica. the catalytic properties of soluble and immobilize on CNBR-activated. Sepharose enzymes in reactions with native ATR, its modified derivatives and ATP--insulin conjugates were compared. The bioluminescence reaction involving ATP--insulin conjugate is inhibited by antibodies against insulin. This effect can form a basis for insulin detection in solution, which is based on competitive binding of free and antibody-labelled ATP--insulin conjugates.     

Immunoenzyme method of sequential saturation for determining antigens using the model of insulin

The method for the determination of insulin by means of the enzyme immunoassay, based on the use of insulin-peroxidase conjugates, has been developed. In this assay the scheme of the successive saturation of the active sites of antibodies is used. The antigenic properties of two conjugates differing in the method of their preparation are compared. The conjugates were obtained by the covalent binding of peroxidase, oxidized in its carbohydrate component, with insulin (conjugate 1) or hexamethylene-diamine-modified insulin (conjugate 2). The conjugates represented a mixture of oligomers differing in their molecular weight. Conjugate 1 possessed higher affinity to antibodies and higher enzymatic activity than conjugate 2. The method for evaluating the quality of antisera to insulin used in the assay has been proposed. The time of the insulin assay is 5-16 hours, the limit of insulin detection is 5 microU/ml, the variation factor is 3-12%.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes

A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen-polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen-polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen-polyelectrolyte complex was less damaging, suggesting that binding to the antigen-polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.     

Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor.

Anticardiolipin antibodies (aCL) found in sera from patients with SLE react with cardiolipin (CL) in the presence of a 50-kDa serum cofactor. The cofactor, which was identified to be beta 2-glycoprotein I by sequencing the N-terminal amino acids, not only enhances CL binding by antibodies in SLE but also depresses it by antibodies associated with syphilis. Cofactor-dependent binding of aCL in SLE to solid phase CL was competitively inhibited by the simultaneous addition of fluid phase CL but was unaffected by either prior or simultaneous addition of a high excess of the cofactor. Binding of aCL in syphilis to solid phase CL was competitively inhibited by either addition of the cofactor or fluid phase CL. aCL in SLE reacted with CL, PS, and PA in the presence of cofactor. In contrast, biotinyl-cofactor bound directly to these anionic phospholipids (PL) and also to PG. These results show that the cofactor-CL complex bears an epitope that confers recognition specificity for aCL in SLE, in contrast with direct CL recognition by syphilitic aCL. The direct binding of the cofactor to PL suggests that the cofactor dependence of aCL binding to PL is due to recognition by aCL of a unique epitope generated upon the formation of the cofactor-CL complex.     

Determination of insulin antibodies.

Insulin antibodies were determined as percentage binding of 125I-insulin in the sera of normal persons and of diabetic subjects treated and untreated with insulin. The effect of the dilution of the serum, circulating insulin and extraction of free and total insulin was evaluated. The determination of insulin antibodies in samples at a final dilution of 1:10 clearly discriminated between insulin-treated and untreated subjects. In insulin-treated subjects, the determination of insulin antibodies in samples at a final dilution of 1:100 gave false-negative results in 28 per cent. However, the determination of insulin antibodies at a final dilution of 1:100 discriminated between insulin-resistant and non-resistant diabetic subjects. Extraction of total insulin at pH 3.0 using 0.1 N HCl increased the percentage of 125I-insulin binding significantly. Extraction of free insulin by charcoal from the samples did not increase the binding of 125I-insulin. The injection of crystalline insulin 4 hours prior to withdrawing the samples did not decrease binding of 125I-insulin.     

A case of insulin resistant diabetes with possible antibodies to insulin receptors.

A case of a 19-year-old, non-obese female with insulin resistant diabetes mellitus and polycystic ovary syndrome was reported. The maximal insulin requirement attained 360 units per day, but a satisfactory control of diabetes did not follow. The patient's serum contained not only anti-insulin antibodies, but also possible anti-insulin receptor antibodies which were demonstrated by the 125I-insulin binding test using insulin receptors derived from human placental plasma membrane. The insulin resistance in this case was assumed to be caused primarily by possible blocking antibodies to insulin receptors and partly by anti-insulin antibodies because of the following observations. First, high serum free insulin (165 microunits/ml) without hypoglycemia indicates the presence of insulin resistance due to other factors than antiinsulin antibodies. Second, the titer of 125I-insulin binding capacity of serum was not unusually higher than those seen in chronically insulin-treated diabetics. Third, immunologically heterospecies insulin (fish insulin) was also ineffective. The clinical features such as absence of ketoacidosis and association with polycystic ovary syndrome resemble those of an unique diabetic syndrome reported previously though acanthosis nigricans and endogenous hyperinsulinemia were not found in this case. Her insulin resistance remitted spontaneously and over the next 18 months' observation, her diabetes remained regulated without insulin therapy.     

Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain

Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)–bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.     

Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples

Homogeneous assays are attractive because they are performed in only one phase, namely, the liquid phase, and thus, they do not require separation of phases as their heterogeneous counterparts do. As opposed to heterogeneous assays, the signal generation in a homogeneous assay is a direct result of analyte binding, which allows the multiple washing and incubation steps required in an indirect heterogeneous assay format to be eliminated. Moreover, homogeneous assays are usually fast and amenable to miniaturization and automation. In this article, we describe the development of a homogeneous assay for the hormone cortisol using the bioluminescent photoprotein aequorin as a reporter molecule. A cortisol derivative was chemically conjugated to the lysine residues of a genetically modified aequorin in order to prepare an aequorin-cortisol conjugate capable of binding anticortisol antibodies. The binding of anticortisol antibodies to the aequorin-cortisol conjugate resulted in a linear response reflected in the emission of bioluminescence by aequorin. A competitive binding assay was developed by simultaneously incubating the aequorin-cortisol conjugate, the anticortisol antibodies, and the sample containing free cortisol. Dose-response curves were generated relating the intensity of the bioluminescence signal with the concentration of free cortisol in the sample. The optimized homogeneous immunoassay produced a detection limit of 1 x 10 (-10) M of free cortisol, with a linear dynamic range spanning from 1 x 10 (-5) to 1 x 10 (-9) M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 x 10 (-7) M to 7.5 x 10 (-7) M and 1.0 x 10 (-8) M to 2.5 x 10 (-8) M, respectively), thereby making this assay attractive for the analysis of this hormone in biological samples. To that end, it was demonstrated that the assay can be reliably used to measure the concentration of free cortisol in saliva without significant pretreatment of the sample.     

Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai virus envelopes and neuraminidase-treated cells

Anti-human erythrocyte antibodies or insulin molecules were covalently coupled to the glycoproteins (the hemagglutinin/neuraminidase and the fusion polypeptides) of Sendai virus envelopes with N-succinimidyl 3-(2-pyridyldithio)propionate and succinimidyl 4-(p-maleimidophenyl)butyrate as cross-linking reagents. Reconstituted Sendai virus envelopes, bearing covalently attached anti-human erythrocyte antibodies or insulin molecules, were able to bind to but not fuse with virus receptor depleted human erythrocytes (neuraminidase-treated human erythrocytes). Only coreconstitution of Sendai virus glycoproteins, bearing attached anti-human erythrocyte antibodies or insulin molecules with intact, untreated viral glycoproteins, led to the formation of fusogenic, targeted reconstituted Sendai virus envelopes. Binding and fusion of reconstituted Sendai virus envelopes, bearing anti-human erythrocyte antibodies or insulin molecules, with neuraminidase-treated human erythrocytes were blocked by the monovalent fraction, obtained after papain digestion of immunoglobulins, made of anti-human erythrocyte antibodies or free insulin molecules, respectively. The results of this work demonstrate an active role of the viral binding protein (hemagglutinin/neuraminidase polypeptide) in the virus membrane fusion process and show a novel and efficient method for the construction of targeted, fusogenic Sendai virus envelopes.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

Insulin-cholera toxin binding unit conjugate: a hybrid molecule with insulin biological activity and cholera toxin binding specificity

The polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond. This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes. Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor. In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin-CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency. This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity. Furthermore, native cholera toxin increased intracellular levels of cAMP by 20-fold, whereas the conjugate had no effect on cAMP levels. The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not. Finally, the binding to thymocytes of insulin-CTB conjugate, but not insulin, was inhibited by gangliosides. Thus, a hybrid hormone molecule has been constructed which has insulin-like biological activity with the receptor specificity of cholera toxin in poorly responsive cells.     

Gold nanoparticles based dipstick immunoassay for the rapid detection of dichlorodiphenyltrichloroethane: An organochlorine pesticide

Gold nanoparticles (GNPs) based dipstick competitive immunoassay was developed to detect organochlorine pesticide such as DDT at nanogram level (ppb). GNPs of definite size were synthesized and conjugated to anti-DDT antibodies (IgY), which served as the detecting reagent. DDA-BSA conjugate (antigen) was immobilized on to nitro cellulose (NC) membrane containing strip. GNPs conjugated anti-DDT antibodies were treated with different concentrations of free DDT ranging from 0.7 ng mL⁻¹ to 1000 ng mL⁻¹ to form an immunocomplex. This immunocomplex solution was further reacted with DDA-BSA conjugate immobilized NC membrane containing strips by dipping the strip in the immunocomplex solution. The free GNPs conjugated anti-DDT antibodies present in the immunocomplex solution were targeted for competitive binding with immobilized DDA-BSA on NC membrane containing strip. Depending on the concentration of free DDT in the sample the binding of GNPs conjugated anti-DDT antibodies to the immobilized DDA-BSA varied and was detected by the development of red color (due to gold nanoparticles) in the detection zone of NC membrane containing strips. The intensity of color development was inversely proportional to the DDT concentration with maximum intensity at zero DDT concentration. The lowest detection limit of DDT was determined to be 27 ng mL⁻¹ with the optimized conditions. The dipstick technique based on GNPs is suitable for the detection of several toxins in food and environmental samples and can be applied for rapid on-site testing of pesticides.     

Use of different hapten-protein conjugates immobilized on nitrocellulose to screen monoclonal antibodies to abscisic acid

The dot-immunobinding method for screening antibodies to proteins on sheets of nitrocellulose has been modified to allow monoclonal antibodies (McAb) to the hapten abscisic acid (ABA) to be screened. Several methods for conjugating ABA to proteins using new bifunctional coupling reagents, specific for hapten keto groups, are described. Hybridomas secreting McAb with a defined specificity for the hapten can be identified by screening supernatants against the carrier protein and other hapten-protein conjugates with different conjugation bridges or modified hapten structure. Inhibition of binding to conjugates by free hapten is used to determine the relative avidity of the McAb for free and bound hapten. All of these tests could be done with no more than about 50 microliter of antibody solution. Dot immunobinding is a useful alternative to radioimmunoassay for screening McAb to haptens.     

Synthesis, characterization, and thermodynamic study of a polyvalent lytic peptide-polymer conjugate as novel anticancer agent

We designed and synthesized a new polyvalent lytic peptide-polymer conjugate as a novel chemotherapeutic agent capable of overcoming multidrug resistance. A hexapeptide (KWKWKW or (KW)?) was designed and conjugated to dextran in multiple copies to afford a polyvalent conjugate. A robust synthesis procedure involving click chemistry and the detailed characterization of the conjugate were reported here. The conjugate Dex-(KW)? exhibited significantly enhanced anticancer potency in vitro by up to 500-fold compared to monomeric (KW)?. The LC?? value was comparable to that of conventional lytic peptides which have more than 20 residues. No hemolytic activity was shown by the conjugates up to 300 μM. Thermodynamic study indicated that the binding of conjugates was predominantly entropy-driven while the binding of free peptides was mainly enthalpy-driven, implying a deeper penetration of conjugate into the core of lipid bilayer. The binding affinity of conjugate to neutral membrane is much higher than that to free peptide (K(conj) ≈ 8822.9 M?1, K(pep) ≈ 1884.7 M?1). In binding to negatively charged membrane, the conjugate surpassed free peptides at high concentrations when the binding of free peptides became saturated. The higher binding capability, attributed to the high local concentration of peptides mounted on a polymer backbone, explains the superior anticancer activity of polyvalent Dex-(KW)?.     

A conjugate of 5-fluorouridine-poly(L-lysine) and an antibody reactive with human colon carcinoma

The ribose moiety of 5-fluorouridine (FUR) was oxidized with periodate and the product was bound through a poly(L-lysine) bridge to monoclonal antibodies, denoted SF25MAb, reactive with a human colon carcinoma LS180. The antibody was linked via its polysaccharide (previously oxidized with periodate) to the poly(L-lysine)-drug conjugate. The linking of FUR-poly(L-lysine) to the antibody markedly increased the latter's binding to the tumor cells. A relatively lower increase was also observed with conjugates of nonrelated antibodies, such as anti-hepatitis B surface antigen and anti-epidermal growth factor receptor antibodies. The pharmacological activity of the specific conjugate FUR-poly(L-lysine) -SF25MAb was higher than that of the drug-substituted polymer alone. The poly(L-lysine) bridge caused toxic effects in vivo, even though substituted both by FUR and by antibody. Therefore, the additional unreacted lysyl residues were blocked by succinylation. Partial blocking of free amino groups on the conjugate rendered it nontoxic but decreased its cell-binding capacity, though to a level still higher than that of the original unmodified antibody. The pharmacological activity of the specific conjugate after blocking was also reduced and necessitated prolonged incubation periods or higher concentrations. Following periodate oxidation and reduction, FUR was as effective as the clinically preferred compound 5-fluoro-2'-deoxyuridine in vitro and in vivo, against the LS180 colon carcinoma. Experiments in nude mice, with LS180 tumor subcutaneous xenotransplants, showed that FUR-poly(L-lysine)-SF25MAb (blocked by succinylation) was not toxic and was effective in the retardation of tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the affinity characteristics of monoclonal antibodies by solid-phase immunoenzyme analysis

An approach is proposed for measuring the binding constant (Kb) for monoclonal antibodies (MA) interacting with an immobilized antigen in indirect ELISA. This approach allows the measurement of optical density (A405) in the peroxidase reaction initiated by the conjugate at different concentrations (C0) of antibodies. Using the Scatchard plots, the dependence of A405/C0 = f (A405) for the whole range of MA concentrations was examined, and the tangential of the slope (tg alpha = Kb) of the linear portion of the antigen molecule was calculated. Analysis of MA affinity parameters by using this approach may find wide use in immunodiagnostic studies aimed at measuring antigen and antibody concentrations in biological fluids as well as for estimating the efficiency of vector drugs in which the diagnostic or therapeutic component is conjugated with the vector (MA or F(ab) fragment) responsible for the drug transport to target cells. The method proposed was used for testing mouse (BALB/C) monoclonal antibodies (IgG1) to pig insulin produced by various hybridomas as well as for estimating the effect on MA of pH, temperature and hydrophobization. The minimal detectable concentration (method sensitivity) was found to depend on Kb.     

Preparation of an insulin conjugate with beta-galactosidase from E. coli for immunoenzyme assay

A modified procedure has been worked out for preparing a conjugate of porcine insulin with E. coli beta-galactosidase employing a heterobifunctional reagent, N-hydroxysuccinimidyl m-maleimidobenzoate. Optimal conditions for insulin acylation and subsequent coupling with beta-galactosidase were selected that afforded the conjugate in a high yield. The ability of the modified antigen to react with antibody was evaluated in the reaction of conjugate binding with immobilized monoclonal antibody to insulin. The conjugate almost completely retained the enzymatic activity and reacted with high specificity with the antibody to insulin. The conjugate can be used in competitive ELISA of insulin.     

Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles.

Vitamin K-dependent protein S is an anticoagulant plasma protein serving as cofactor to activated protein C in degradation of coagulation factors Va and VIIIa on membrane surfaces. In addition, it forms a noncovalent complex with complement regulatory protein C4b-binding protein (C4BP), a reaction which inhibits its anticoagulant function. Both forms of protein S have affinity for negatively charged phospholipids, and the purpose of the present study was to elucidate whether they bind to the surface of activated platelets or to platelet-derived microparticles. Binding of protein S to human platelets stimulated with various agonists was examined with FITC-labeled monoclonal antibodies and fluorescence-gated flow cytometry. Protein S was found to bind to membrane microparticles which formed during platelet activation but not to the remnant activated platelets. Binding to microparticles was saturable and maximum binding was seen at approximately 0.4 microM protein S. It was calcium-dependent and reversed after the addition of EDTA. Inhibition experiments with monoclonal antibodies suggested the gamma-carboxyglutamic acid containing module of protein S to be involved in the binding reaction. An intact thrombin-sensitive region of protein S was not required for binding. The protein S-C4BP complex did not bind to microparticles or activated platelets even though it bound to negatively charged phospholipid vesicles. Intact protein S supported binding of both protein C and activated protein C to microparticles. Protein S-dependent binding of protein C/activated protein C was blocked by those monoclonal antibodies against protein S that inhibited its cofactor function. In conclusion, we have found that free protein S binds to platelet-derived microparticles and stimulates binding of protein C/activated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical verification of the insulin receptor's specificity in the nuclear envelope of Tetrahymena. Comparison with receptors of the plasma membrane

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.