The influence of binding capacity and affinity on the improved performance of N-terminally extended hCG peptides, determined by ELISA-based procedures.

The improvement of peptide-ELISA responses by the use of small synthetic peptides elongated at the N-terminus with an Ata-group or a (Lys)7 extension has been analyzed. For this purpose, binding capacity and affinity were evaluated by specific ELISA procedures. The ELISA experiments on binding capacity, performed with saturating antibody concentrations, revealed a difference of more than three orders of magnitude in binding capacity between the parent peptides and the N-terminally linked peptides, in favor of the latter peptides. Antibody affinity values were determined by a liquid-phase equilibrium method as well as by a solid-phase equilibrium method. N-terminal extension of the peptides had almost no effect on the affinity when equilibrium between the peptide and the antibody was reached in solution. In contrast, solid-phase affinity was greatly enhanced when the N-terminally linked peptides were adsorbed to the polystyrene surface. This enhancement was determined by the N-terminal extension and the peptide amino acid sequence (40 to 600 times higher). Thus, the use of N-terminally extended peptides can greatly increase the performance of a peptide-ELISA through improved surface effects, resulting in higher binding capacity and functional affinity.

Adsorption studies of tritium-labeled peptides on polystyrene surfaces.

In this study, three presentation formats of an epitope peptide (hepta-peptide), derived from the human chorionic gonadotropin amino acid sequence, were compared for adsorption to the polystyrene wells of a microELISA plate. The peptides had either a free N-terminus, an Ata-group or a linear (Lys)7-extension at the N-terminal. In order to measure the adsorption properties, all peptides were tritiated by synthesizing an additional 3H-labeled glycyl residue to the N-terminus of their peptide sequence. Over a broad range of peptide concentrations used as coat solution, extension of the peptide by an Ata-group consistently increased adsorption by a factor of 1.5 to 3 compared to the free parent peptide. Of the three peptides studied, the Ata-peptide showed the highest surface coverage of 0.6 mg/m2 when 1.0 mmol/l was offered as the concentration of peptide in the coating solution. The highest surface coverage observed for the parent peptide was 0.4 mg/m2 (at 1.5 mmol/l). The lysyl (K7) peptide showed a maximum plateau value of 0.2 mg/m2, and therefore the lysyl (K7) extension reduced the peptide surface coverage at relatively high coat concentrations (above 0.1 mmol/l) compared to the parent peptide. At lower input concentrations (below 0.1 micromol/l), however, the packing density of the lysyl (K7) peptide was up to 25 times higher when compared to the other two peptide analogs. We conclude that better adsorption as well as improved antibody binding activity and (functional) affinity could explain the higher reactivity observed in ELISA procedures when peptides are N-terminally extended by an Ata-group or lysyl (K7) extension.

Purification of rubella virus E1-E2 protein complexes by immunoaffinity chromatography.

A murine monoclonal antibody directed against the E1 membrane glycoprotein of rubella virus was immobilized on an N-hydroxysuccinimide-activated chromatographic support. The antibody was used to purify rubella virus E1-E2 protein complexes from Tween-80/diethyl ether extracts of cell culture supernatants containing virus particles. The adsorption behaviour of immunosorbents with ligand densities of 2.9, 5.4 and 11.1 mg monoclonal antibody per millilitre of gel was investigated using batchwise conditions. Then the immunoaffinity purification process was optimized with regard to adsorption efficiency by adjusting the flow rate, the bed height and the amount of sample loaded onto the column. The optimized immunoaffinity purification process which is reproducible and relatively simple (one-step) had a yield of 73%, a concentration factor of 5-8 and a purification factor of about 2600. No mouse IgG due to ligand leakage could be detected in the immunopurified product using an enzyme immunoassay. High-performance size exclusion chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, immunoblotting and electron microscopy showed that the immunopurified product contained rosette-like structures formed by complexes of E1 and E2 proteins. The product retained its hemagglutinating activity and proved to be suitable for application in a fluorescent enzyme immunoassay for determination of anti-rubella IgG in human serum.

Assessment of the functional affinity constant of monoclonal antibodies using an improved enzyme-linked immunosorbent assay.

The use of an enzyme-linked immunosorbent assay (ELISA) for the determination of affinity constants implies heterogeneous measurements. Therefore, despite their simplicity, direct solid-phase binding assays are not common. Many investigators have serious, and mostly justified, reservations about the application of solid-phase affinity methods. They refer to problems such as diffusion effects and difficulties in reaching equilibrium due to heterogeneous binding and co-operativity. Accordingly, functional affinity determinations are often described as meaningless. These objections apply to the measurement of the affinity of a monoclonal antibody using the enzyme-linked immunosorbent assay of Beatty et al. (J. Immunol. Methods (1987) 100, 173), which is based on the effect of antibody affinity on the sigmoidal dose response curve. The affinity constant is calculated by mathematical equations, based on the Law of Mass Action and the authors made a number of important assumptions--avoiding the above mentioned problems--in order to justify the use of the Law of Mass Action. By carefully examining these assumptions we have developed an improved ELISA procedure for functional affinity determinations on the basis of a primary coating with the antigen only. the coating conditions were validated by employing gold labelled colloidal particles and physical counting of the bound particles under the scanning electron microscope. Since monovalent binding between human chorionic gonadotropin and its monoclonal antibody could be achieved under equilibrium conditions, the application of the Law of Mass Action and hence of the Beatty formula became possible. We conclude that under these conditions functional affinity determinations are appropriate.

Characterization of monoclonal antibodies physically adsorbed onto polystyrene latex particles.

The physicochemical and immunochemical properties of monoclonal antibodies (MAbs), adsorbed onto polystyrene latex particles, have been investigated. Both native and pH 2 pretreated MAbs were compared before and after immobilization. It was found that the antigen binding capacity of the immobilized, acidic pretreated MAbs was significantly higher than for the immobilized, native IgG molecules. This enhanced antigen binding capacity appeared to be due to an improved molecular orientation following adsorption of the monomeric, pH 2 treated IgG fraction. Additionally, experiments using F(ab')2 fragments demonstrated that the Fc portion of the MAb molecule is of major importance for achieving the enhanced binding capacity. Binding studies showed that the (apparent) affinity of native and pH 2 pretreated MAbs were similar; the K(a) values of the immobilized MAbs were higher than those of MAbs in solution.

Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to protein A Sepharose 4 Fast Flow.

The binding capacity of protein A Sepharose 4 Fast Flow for mouse IgG1 monoclonal antibodies (mabs) appears to be highly dependent on the buffer composition with respect to both concentration and ion type. Depending on the particular mab dynamic binding capacities up to 20 mg mab per ml gel could be obtained, when these mabs were isolated from supernatants of protein free hollow fibre cell culture systems. Variation of linear flow rate from 10 up to 300 cm/h and temperature (4 degrees C versus 25 degrees C) had a slight effect on the dynamic binding capacity, when a high ionic strength buffer was used during adsorption. Applying optimum binding conditions, final IgG fractions with a purity of more than 95% monomeric IgG were obtained. However, as side effect of the use of binding buffers with high ionic strength, the binding of acid proteases was also promoted.

Monitoring of the production of monoclonal antibodies by hybridomas. Part I: Long-term cultivation in hollow fibre bioreactors using serum-free medium.

The long-term cultivation of hybridoma cells in hollow fibre bioreactors using serum-free medium, was monitored with respect to quantitative and qualitative aspects of the produced mAbs, cell viability, LDH and proteolytic activity. During the culture periods of hybridoma cells producing mAb OT-1C and 3A, the mAb concentration showed a decreasing trend with a concomitant increase of IgG fragments. The major IgG fragments did not bind the antigen and the molecular weights were significantly different from the corresponding IgG heavy and light chains. In addition, a good correlation was found between cell lysis, the presence of acid protease(s) and IgG fragments. The physicochemical and immunochemical properties of the "intact" mAbs (such as molecular weights, IEF patterns and affinity) did not change significantly during the culture period.

Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.

An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.

Affinity of monoclonal antibodies. Interpretation of the positive cooperative nature of anti-hCG/hCG interactions.

The interaction of some individual MAbs and human chorionic gonadotrophin (hCG) showed apparent positive cooperativity as observed by equilibrium binding studies. This form of cooperative interaction has now been further characterized. The main results were: (1) the apparent positive cooperativity was strongly dependent upon concentration and temperature; (2) the cooperativity was strongly reduced by using peptic F(ab')2 fragments of IgG and became undetectable when the MAb was replaced by the corresponding Fab fragment; (3) the molecular weight of the complex changed from 226 kDa to 450 kDa upon increasing the hCG/MAb ratio. From these and additional results it is hypothesized that the apparent positive cooperativity results from self (Fc-Fc) associations mediated or facilitated by prior antigen binding.

Application of a sol particle immunoassay to the determination of affinity constants of monoclonal antibodies.

The affinity constants (Ka) of monoclonal antibodies (MAb) for binding to their corresponding antigens (Ag), unlabelled and in buffered solution were determined by the following procedure: 1. Incubation of MAb (fixed concentration) with Ag (concentration dilution series). 2. Rapid bound/free separation by adding immobilized second antibody, followed by centrifugation. 3. Determination of free Ag in the supernatant using a gold sol particle agglutination immunoassay (SPIA) in a microtitration plate format. 4. Calculations and interpretation were based on Scatchard and Sips plots. Ka values found by this procedure were found to be similar to those obtained by a radio-immunoassay (RIA) procedure. The present method avoids possible artefacts in Ka values introduced by the procedure or chemical modification due to labelling of MAb or Ag. It enables rapid, simultaneous screening of a considerable number of different MAbs under non-specialized (i.e. RIA) laboratory conditions.

Some aspects of the chromogen 3,3',5,5'-tetramethylbenzidine as hydrogen donor in a horseradish peroxidase assay.

Enzyme immunoassays frequently incorporate the use of horseradish peroxidase as the enzyme label. This enzyme usually catalyses the oxidation of a chromogen which can be quantified after termination of the enzyme reaction. A chromogen widely used for this purpose is 3,3',5,5'-tetramethylbenzidine. The two electron oxidation of tetramethylbenzidine yields a component with an absorbance maximum at 450 nm. If the enzyme reaction is terminated by lowering of the pH (less than 1.0), an additional increase of the absorbance at 450 nm is observed. It is shown that this additional increase is partly due to a 1.4-fold increase in the molar lineic absorbance of oxidized tetramethylbenzidine, caused by the acidic pH, as well as a quantitative shift of the existing equilibrium between tetramethylbenzidine, oxidized tetramethylbenzidine and their charge-transfer complex. The total absorbance increase upon acidification of the reaction mixture depends therefore on the reaction conditions as well as the reaction coordinate.

Application of scanning electron microscopy and energy-dispersive X-ray analysis to the solution behaviour of Zn-insulin: precipitation phenomena.

Scanning electron microscopy (SEM) has been applied in combination with energy dispersive X-ray analysis (EDAX) to identify and analyse particles or particulate matter, occasionally present in clear neutral Zn-insulin solutions. SEM photographs revealed the existence of three different types of precipitate, consisting of particles with a crystalline, amorphous or gel-like nature, respectively. At present, it is not clear which conditions lead specifically to each of these three types of precipitate. The advantages of the EDAX method are shown. The technique enables semi-quantitative analysis to be performed on a single particle as small as 0.2 microns. It was demonstrated with the EDAX method that the particles occasionally found in clear Zn-insulin solutions contain insulin as well as Zn in roughly the same ratio as in the insulin starting material. It is concluded that the EDAX method has great potential in pharmaceutical technology, inter alia for the analysis of emulsion systems (in the frozen state), as well as suspensions and particulate matter in injection fluids. This technique is particularly useful in the latter case, due to its applicability to extremely small sample sizes.

The influence of gamma-irradiation upon the chemical and biological properties of insulin.

Partially purified insulin preparations of bovine and porcine origin, were subjected to gamma-irradiation with doses ranging from 1.0 up to 25 kGy (0.1-2.5 Mrad) at 0 degrees C or ambient temperature. The susceptibility of insulin to the irradiation was determined by chromatography, electrophoresis and assay of the biological activity. The sterilizing effect of the gamma-irradiation was investigated for Bacillus pumilus as well as for artificial mixtures of lactose and several bacilli. It is concluded that the sterilizing dose for the investigated insulins was greater than or equal to 2.2 kGy. At doses up to 25 kGy at 0 degree C no specific radiolytic products were detectable, whereas the biological activity was fully retained. The content of dimers and the content of related peptides appeared to increase gradually with the irradiation dose absorbed. No effects of long-term storage could be demonstrated on biological and chemical properties of insulin after 2.2, 4.5 and 7.5 kGy.

Pharmacokinetics and miscibility of 4 highly purified porcine insulins: a study in vitro and in healthy volunteers.

In this study 4 new, highly purified porcine insulin preparations (ORGANON) were characterized by their time action profiles and in vitro and in vivo miscibility. These insulin preparations were soluble insulin, and suspensions of amorphous zinc insulin, mixture of 30% amorphous and 70% crystalline zinc insulin and NPH insulin. The time action profiles, assessed with the euglycaemic clamp technique and measurements of plasma insulin levels, in healthy volunteers were very similar to corresponding, already available, insulins of other manufacturers. In vitro miscibility was assessed for the soluble insulin with the 3 intermediate acting insulins in mixing ratios varying from 1:1-1:5. The recovery percentages of added soluble insulin, 75 sec after mixing in a 1:1 ratio with NPH, Tardum and Sub Tardum insulin, were 97.1%, 67.8% and 42.4%, respectively. The recovery of added soluble insulin decreased significantly with time of contact and with lowering of the mixing ratio for all insulins tested. In vivo insulin miscibility was performed for soluble and NPH insulin in a mixing ratio of 2:3, administered immediately after mixing in the syringe. The insulin action profiles were not altered when soluble and NPH insulin were administered after mixing as compared with the separate injections into contralateral thighs. In conclusion, the pharmacokinetics of these highly purified porcine insulins are in agreement with corresponding already available insulins. NPH insulin can be mixed with soluble insulin without affecting the absorption kinetics of either insulin.

Particle-labelled immunoassays: a review.

Up to now, various types of particles have been used as labels in immunoassay. Well known examples are erythrocytes and latex particles. More recently, colloidal gold and dye particles have been introduced as a label. Each type of particle offers one (or more) method(s) of detection which depend(s) on the physical properties of the particles. In this paper, the present state-of-the-art regarding particle-labelled immunoassays will be reviewed.