Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

Can potential hazard of Creutzfeldt-Jakob disease infectivity be reduced in the production of human Growth Hormone?

Scrapie infectivity is reduced 5-6 logs following filtration through 100,000 MW cut-off filter plus overnight treatment with 6 M urea. These steps, applied to purified human Growth Hormone (hGH), increase the margin of safety of hGH.     

The production and characterisation of monoclonal antibodies against human prolactin and the development of a two-site immunoradiometric assay

Monoclonal antibodies against human prolactin (PRL) have been produced and characterised and used to develop a sensitive two-site immunoradiometric assay (IRMA). Nine anti-PRL monoclonal antibodies were assessed for reactivity in immunoblotting experiments with PRL, hPL, hGH and pituitary gland extract. There was no detectable crossreactivity with hPL or hGH. In liquid phase radioimmunoassay (RIA) studies using three of the antibodies there was no detectable crossreaction from hPL or hGH. Five antibodies were positive in immunocytochemical studies using sections of human pituitary gland. Using FPLC purified monoclonal antibodies, a two-site IRMA was developed that could assay PRL over the range 17.5-3500 mIU per litre and was readily adapted to assaying serum samples from patients. The two-site IRMA could be performed within one day without loss of sensitivity and has potential as a rapid and simple method for screening clinical samples.     

Production and characterization of monoclonal antibodies against human thyroglobulin

Spleen cells of Biozzi-HR mice immunized with human thyroglobulin (hTg) were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty monoclonal antibodies (MAbs) selected by an enzyme immunoassay (indirect ELISA) were produced, purified and characterized. The equilibrium association constant (Ka) of one of the MAbs, determined by Scatchard analysis of the ELISA data, was found to be 2 X 10(9) M-1; the Ka of the other MAb, estimated from titration curves by comparison with the aforementioned MAb, ranged from 8 X 10(9) M-1 to 6 X 10(7) M-1. The reaction between the MAb and hTg was not inhibited by thyroxin (T4), triiodothyronine (T3) and triiodothyropropionic acid (DT3). Species specificity of the MAb was studied using bovine and porcine Tgb. The topology of the MAb was investigated by competitive inhibition immunoassays. Seven distinct antigenic regions were identified.     

Evaluation of the biological activity of human growth hormone by enzyme linked immuno-receptor assay (ELIRA) method

The aim of this research was to study the application and effectiveness of Enzyme Linked Immuno Receptor Assay (ELIRA) method for understanding the bioactivity of human Growth Hormone (hGH) in micro-titer plates. For this purpose, rabbit hepatocyte microsomes which contained hGH receptors were used for coating of ELISA micro-titer plates. Then hGH was interacted with coated receptors. Fractions of bounded complexes were identified by antibodies in an Enzyme-based substrate detection system. Different assay conditions such as: buffers, blocking agents, temperatures and times of incubation were analyzed. Our result indicated that, the carbonate coating buffer was not effective in receptor coating in ELIRA. Overnight incubation of hGH and hGH receptors in HEPES assay buffer and BSA blocking resulted in the lower linearity and correlations (R(2) = 0.46 to 0.85). However, 3 h incubation in Tris-HCl assay buffer at 30°C resulted in higher linearity and correlations (R(2) = 0.95 to 0.97). Finally, the coating of microwells by 250 μg/ml of microsome membranes in Tris buffer at 30°C for 3 hr and blocking by skim milk resulted to the best linearity and higher correlation, (R(2)?=?0.985) and lower detection limit about 2 ng/ml of bioactive hGH.     

Evaluation of the Biological Activity of Human Growth Hormone by Enzyme Linked Immuno-Receptor Assay (ELIRA) Method

The aim of this research was to study the application and effectiveness of Enzyme Linked Immuno Receptor Assay (ELIRA) method for understanding the bioactivity of human Growth Hormone (hGH) in micro-titer plates. For this purpose, rabbit hepatocyte microsomes which contained hGH receptors were used for coating of ELISA micro-titer plates. Then hGH was interacted with coated receptors. Fractions of bounded complexes were identified by antibodies in an Enzyme-based substrate detection system. Different assay conditions such as: buffers, blocking agents, temperatures and times of incubation were analyzed. Our result indicated that, the carbonate coating buffer was not effective in receptor coating in ELIRA. Overnight incubation of hGH and hGH receptors in HEPES assay buffer and BSA blocking resulted in the lower linearity and correlations (R² = 0.46 to 0.85). However, 3 h incubation in Tris-HCl assay buffer at 30°C resulted in higher linearity and correlations (R² = 0.95 to 0.97). Finally, the coating of microwells by 250 μg/ml of microsome membranes in Tris buffer at 30°C for 3 hr and blocking by skim milk resulted to the best linearity and higher correlation, (R²?=?0.985) and lower detection limit about 2 ng/ml of bioactive hGH.     

Quantitative aspects of ouabain binding to human erythrocyte and cardiac membranes.

1. [3H]ouabain binding to human erythrocyte membranes is a time- and temperature-dependent process. The association of ouabain to the membrane-bound receptor follows second-order kinetics, while the dissociation is a monomolecular reaction. An association rate constant of 4-6 x 10(4) M-1 sec-1 and a dissociation rate constant of 1-4 x 10(-4) sec-1 were measured at 37 degrees C. The dissociation constant calculated from these data agrees with that determined from equilibrium binding experiments. There is only one type of ouabain binding sites with high affinity for the drug as reflected by the low dissociation constant of 0-28 x 10(-8) M. 2. The dissociation constants of the ouabain-receptor complexes from human erythrocyte and cardiac membranes are identical. 3. The maximal number of membrane-bound ouabain binding sites was measured from equilibrium binding experiments as 288 +/- 28 per single erythrocyte. Thus one receptor site corresponds to less than 1 mum2 of the membrane, provided the receptors are diffusely distributed on the surface of the membrane. 4. Neither the maximal number of ouabain receptors nor the affinity for the drug changes with the age or sex of the blood donor. 5. A maximal transport capacity for sodium of 5-6 m-equiv/hr.1. is calculated from the number of receptor sites per erythrocyte and from the turn-over number of the (Na+ + K+)-ATPase.     

Determination of intrinsic affinity constants of monoclonal antibodies against carcinoembryonic antigen

A 2-phase method is described for the determination of the intrinsic affinity constants (K-values) for the interaction between monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) and CEA. The Mabs were coupled covalently to CNBr-activated paper discs. MAb coupled discs and a 2-fold dilution series of 125I-CEA were incubated at 20 degrees C until equilibrium was reached. Nonlinear curve-fitting was used for estimation of K-values and different calculation models were thereby tested. The K-values for 12 different anti-CEA MAbs were determined to be 0.3-52 X 10(6) M-1, which is 1-2 orders of magnitude lower than the values obtained in a previous study with some of these MAbs using the ammonium sulphate method to separate free from bound antigen. The K-values obtained by the paper disc method are probably better estimates of the intrinsic association constant than those obtained previously. There are two main reasons for this: (1) free and bound antigen are separated from each other under physiological conditions and (2) the opportunity for multipoint interaction between MAb and CEA is minimized when the MAb is coupled to a rigid carrier substance. Thus, even when the MAb reacts with greater than or equal to 2 epitopes in CEA, as seems to be the case with several of our anti-CEA MAbs, the intrinsic K-value should be obtained. The fundamental validity of 2-phase assays, sometimes questioned in the literature, is demonstrated.     

Binding of fibronectin and albumin to Enterococcus (Streptococcus) faecalis

Binding of human fibronectin (FN) and albumin to Enterococcus (Streptococcus) faecalis was investigated. Scatchard analysis showed that the cells could bind a maximum of 1300 molecules of FN with an association constant of 4.8 x 10(6) M-1. Binding did not appear to involve lipoteichoic acid. Heating the cells or pretreatment with protease or periodate reduced the binding, suggesting the involvement of protein and/or carbohydrate-containing components as the surface receptor. The cells could bind a maximum of 50,000 molecules of albumin but with a much lower affinity than FN (association constant 10.8 x 10(3) M-1). Surface hydrophobicity of E. faecalis was markedly decreased by albumin but not by FN.     

Functional affinity constants of the reaction between 125I-labelled C1q and C1q binders and their use in the measurement of plasma C1q concentrations.

Purified 125I-labelled C1q has been found to react with glutaraldehyde-treated red cells (glut-RBC), with a value for the functional affinity constant, K, of 1-3 X 10(8) M-1, based on measurement of concentrations of bound and free reactants at equilibrium. Values of K obtained for other C1q-binders were as follows: diaminopropane, 2 X 10(2) M-1; monomer IgG, 5 X 10(4) M-1; heat-aggregated IgG, 0-5-2-5 X 10(8) M-1; IgG-anti-IgG complexes, 0-31 X 10(8) M-1. The functional rate constant for association (ka) between 125I-labelled C1q and glut-RBC was 5 X 10(5) M-1 S-1 at 37 degrees in 0-17 M NaC1. The rate of dissociation of the C1q-glut-RBC complex was biphasic with rate constants (kd) of 2 X 10(2) S-1 and 2 X 10(5) S-1. Calculated values of K from the ration ka/kd gave values of 2-5 X 10(7) M-1 and 2-5 X 10(10) M-1. It is suggested that the range of values of K reflects the involvement of 1,2 or more binding sites on the C1q molecule. Reduction of the ionic strength of the medium from 0-17 M to 0-14 M increases the rate of association of C1q and glut-RBC eleven-fold, indicating involvement of ionized groups at the binding site. A method is described for measuring plasma C1q concentrations by saturation assay, using 125I-labelled C1q and glut-RBC. Plasma C1q concentrations fell in the range 170-250 microng/ml.     

Two apparently nonrepeated epitopes on gametes of Plasmodium falciparum are targets of transmission-blocking antibodies.

One-site and two-site immunoradiometric assays have been developed against an antigen on gametocytes of Plasmodium falciparum, using monoclonal antibodies (Mabs) which block transmission of the parasites to mosquitoes. Three such Mabs have been studied, each of which immunoprecipitates a complex of three gamete surface proteins of apparent Mr 260,000, 59,000, and 53,000 from Triton X-100 extracts of the parasites. The assays showed that the Mabs recognized one or the other of two distinct, nonrepeated epitopes on the target antigen(s). In the one-site assay certain combinations of two Mabs interacted at appropriate concentrations to enhance binding of the Mabs to the antigen. The same combinations of Mabs synergize to suppress infectivity of gametocytes to mosquitoes.     

ELISA-based determination of immunological binding constants

An expression for the time-dependent concn of antibody in a hemispherical antigen-coated well is derived by taking the Laplace transformation of the diffusion equation. From this expression, and from antibody adsorption kinetics measured using ELISA, it is possible to evaluate the rate constant of bimolecular association, k1, the rate constant of first order antibody-antigen dissociation, k2, and their ratio, the binding equilibrium constant or affinity, Ka. For the interaction of an anti-arsanilate monoclonal antibody with arsanilate-coupled albumin, analysis yields k1 = 8.8 X 10(3) M-1 sec-1, k2 = 2.5 X 10(-4) sec-1 and Ka = 3.5 X 10(7) M-1, for mean values over 10 experiments. These results are discussed in reference to the conventionally-obtained values for binding constants, including the affinity of the anti-arsanilate monoclonal for the hapten (p-azobenzenearsonate)-N-acetyl-L-tyrosine, determined by equilibrium dialysis.     

Production, characterisation and use of monoclonal antibodies to human interleukin-5 in an enzyme-linked immunosorbent assay

Two mouse monoclonal antibodies (Mabs) against recombinant human interleukin-5(rhIL-5) have been produced, characterised and purified. Both are IgG1 antibodies and neutralised the activity of rhIL-5 in the B13 assay. Neither Mab cross-reacted with mouse IL-5. A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was developed with different combinations of the mouse Mabs and also a rat anti-mouse IL-5 Mab, TRFK5, which also has activity against rhIL-5. The most sensitive assay, with a lower detection limit of 0.5 ng/ml IL-5, used TRFK5 as the capture antibody and the mouse anti-human IL-5 Mab as second antibody. The sensitivity of this assay was increased by an enhanced chemiluminescent reagent and resulted in a lower limit of detection around 40 pg/ml IL-5.     

Kinetic analysis of interactions between bispecific monoclonal antibodies and immobilized antigens using a resonant mirror biosensor

A resonant mirror biosensor (IAsys) protocol is described for the comparative kinetic analysis of the ability of monoclonal antibodies (Mabs) and bispecific antibodies (Babs) to bind immobilized antigens. The protocol has been optimized and validated using the panel of affinity-purified antibodies, including two parental Mabs, one specific to human immunoglobulin G (hIgG) and another specific to horseradish peroxidase (HRP), and a Bab derived thereof by cell fusion (anti-hIgG/HRP Bab). The real-time kinetic analysis of antigen–antibody interactions using this protocol allows to demonstrate the differences in the avidity of bivalently binding Mabs and monovalent Babs. As shown in our previous study [J. Immunol. Methods 261 (2002) 103], the observed equilibrium association constants (K_ass) determined by IAsys using this protocol yield figures almost overlapping with those obtained by solid-phase radioimmunoassay (RIA). The described protocol is suited for the investigation of the effects of valency on the binding properties of antibodies. It also may be applied for the selection of Mabs and Babs with desired features, for different fields of application.     

Specificity and association constants of 33 monoclonal antibodies to human IgA epitopes

We used an immunofluorescent sequential-saturation-of-antibody assay and an interactive computer program for Scatchard analysis to determine association constants (Ka) of 33 murine monoclonal antibodies (Mabs) specific for human IgA epitopes. Ka ranged from 0.37 to 690 x 10(7) liters per mole (an approximate 1900-fold difference). Specificity was validated with a panel of 18 highly purified IgA1 and IgA2 myeloma proteins and secretory IgA using an immunofluorometric assay. Western blots of bacterial IgA protease digests were used to locate the epitopes of IgA specific Mabs in either the Fab, Fc, or hinge region. Mabs specific for unique epitopes on secretory IgA or free secretory component (FSC) were produced and evaluated.     

Binding kinetics of monomeric and aggregated IgG to Kupffer cells and hepatocytes of mice

The binding kinetics of human monomeric IgG and stable heat-aggregated IgG (A-IgG) to Fc receptors of hepatocytes and Kupffer cells isolated from mice was studied. After injection of radiolabelled proteins the 60-70% of hepatic uptake was recovered in parenchymal cells (hepatocytes). In experiments in vitro the A-IgG bound in larger amounts to hepatocytes and Kupffer cells than monomeric IgG. The association rate constants of aggregates were somewhat higher for Kupffer cells than for hepatocytes whereas the percentage uptake of aggregates by Kupffer cells was only 5-15% of that of hepatocytes. The equilibrium constants of aggregates binding to both cells amounted to 0.4-1 X 10(8) M-1 for A-IgG compared with an equilibrium constant for monomeric IgG of 1-2 X 10(7)M-1. The maximum number of IgG and A-IgG molecules bound per cell was higher on hepatocytes (mean 14 X 10(6)) than on Kupffer cells (mean 2 X 10(5)) which is in agreement with the higher binding capacity of hepatocytes for these proteins observed in vivo and in vitro experiments. The ability to compete for receptor binding seemed to reside exclusively in the Fc portion of IgG since F(ab')2 fragments of IgG failed to inhibit labelled monomeric IgG or A-IgG. The receptor seems to be specific for IgG since unlabelled monomeric IgA demonstrated no binding inhibition of labelled IgG or A-IgG on hepatocytes and Kupffer cells. The overall results further suggest that hepatocytes might through Fc receptors play a collaborative role with the mononuclear phagocytic system in the clearance of circulating immune complexes.     

Production and partial characterization of monoclonal antibodies against 3,3',5-triiodo-L-thyronine

Spleen cells of Biozzi HL mouse (selection V) immunized with bovine albumin-triiodothyronine conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Thirteen monoclonal antibodies, selected by an enzyme-linked immunosorbent assay and a radioimmunoassay, were produced in mouse ascites fluid, purified and analyzed. All of them were IgG1 (kappa). The cross-reactivity of all these monoclonal antibodies with thyroxine (T4) was less than 0.2%. The association constant determined by Scatchard analysis ranged from 1.5 x 10(9) M-1 to 2.7 x 10(10) M-1.     

Equilibrium binding characteristics of monoclonal antibodies recognizing melanoma cell surface antigens

The equilibrium binding characteristics of a panel of six monoclonal antibodies (MAb) recognizing melanoma cell surface antigens (125 kdal cell surface melanoma associated glycoprotein antigen, 125kD-MAA; high molecular weight melanoma associated antigen, HMW-MAA; and a non-protein melanoma associated antigen, NP-MAA) were investigated using the cell lines SK-MEL-2, SK-MEL-5, and M21. The MAbs displayed equilibrium association constant (K) values ranging from 10(7) M-1 to 10(10) M-1 and maximum MAb binding values (Qmax) from 2 x 10(4) to 2 x 10(6) MAb molecules bound per cell. High trypsin concentrations were shown to have deleterious effects on Qmax values obtained for antibodies recognizing the 125kD-MAA, and even low trypsin concentrations affected Qmax values obtained for MAbs recognizing the HMW-MAA (although a complete linear recovery of HMW-MAA antigen was observed in 20-25 hours). Significant changes in Qmax were also noted for different cell passages. Except for MAb 43.2, little variation in K was observed when different cell lines were used. Linear Scatchard plots were obtained for all MAbs except 43.2 in which case concave down behavior was observed suggesting the existence of positive cooperativity between the binding sites of this MAb.     

Measurement of the association constants of the complexes formed between intact C1q or pepsin-treated C1q stalks and the unactivated or activated C1r2C1s2 tetramers

The association constants between C1q and C1r2C1s2 and between C1q and C1r2C1s2 were measured in solution using a new technique which employs sucrose gradient ultracentrifugation to estimate thermodynamic association constants. In this technique, zones of dilute, radioiodine-labeled C1q were sedimented through uniform concentrations of either C1r2C1s2 or C1r2C1s2. The zones remained intact, indicating that the dynamic equilibrium was rapid compared with the time of centrifugation. The observed increases in the sedimentation coefficients of the C1q zones were assumed to be directly proportional to the fraction of C1q bound in the dynamic equilibrium. Binding curves were constructed by performing the measurements at many C1r2C1s2 and C1r2C1s2 concentrations. The association constants were estimated from the midpoints of the binding curves and found to be 6.7 X 10(7)M-1 for C1r2C1s2 binding to 125I-C1q. After activation of the C1r2C1s2 the association constant decreased 10-fold to 7.1 X 10(6)M-1. These association constants refer to solvent conditions of pH 7.35, 1 mM Tris, 5 mM Ca2+ and 150 mM NaC1, pH 7.35. Similar measurements were performed with the collagenous peptic fragment of C1q and both 125I-C1r2C1s2 and 125I-C1r2C1s2. The association constants were independent of the state of activation and both found to be about 2 X 10(7) M-1, suggesting that most if not all of the interactions between C1q and C1r2C1s2 were confined to the collagenous portion of C1q.     

Complex signatures of locus-specific selective pressures and gene conversion on Human Growth Hormone/Chorionic Somatomammotropin genes

Reduced birth weight and slow neonatal growth are risks correlated with the development of common diseases in adulthood. The Human Growth Hormone/Chorionic Somatomammotropin (hGH/CSH) gene cluster (48 kb) at 17q22-24, consisting of one pituitary-expressed postnatal (GH1) and four placental genes (GH2, CSH1, CSH2, and CSHL1) may contribute to common variation in intrauterine and infant growth, and also to the regulation of feto-maternal and adult glucose metabolism. In contrast to GH1, there are limited genetic data on the hGH/CSH genes expressed in utero. We report the first survey of sequence variation encompassing all five hGH/CSH genes. Resequencing identified 113 SNPs/indels (ss86217675-ss86217787 in dbSNP) including 66 novel variants, and revealed remarkable differences in diversity patterns among the homologous duplicated genes as well as between the study populations of European (Estonians), Asian (Han Chinese), and African (Mandenkalu) ancestries. A dominant feature of the hGH/CSH region is hyperactive gene conversion, with the rate exceeding tens to hundreds of times the rate of reciprocal crossing-over and resulting in near absence of linkage disequilibrium. The initiation of gene conversion seems to be uniformly distributed because the data do not predict any recombination hotspots. Signatures of different selective constraints acting on each gene indicate functional specification of the hGH/CSH genes. Most strikingly, the GH2 coding for placental growth hormone shows strong intercontinental diversification (F(ST)=0.41-0.91; p<10(-6)) indicative of balancing selection, whereas the flanking CSH1 exhibits low population differentiation (F(ST)=0.03-0.09), low diversity (non-Africans, pi=8-9 x 10(-5); Africans, pi=8.2 x 10(-4)), and one dominant haplotype worldwide, consistent with purifying selection. The results imply that the success of an association study targeted to duplicated genes may be enhanced by prior resequencing of the study population in order to determine polymorphism distribution and relevant tag-SNPs.