Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.     

Nonionic block polymer surfactants enhance the avidity of antibodies in polyclonal antisera against Streptococcus pneumoniae type 3 in normal and Xid mice

Avidities of antibody (sub)classes in polyclonal antisera against Streptococcus pneumoniae type 3 (S3) can be (semi) quantitatively determined with a specific inhibition ELISA. A hexasaccharide was isolated from the hydrolyzed S3 capsular polysaccharide and coupled to a protein-carrier. Mixtures containing these conjugates and nonionic block polymer (NBP) surfactants were used for immunization. After various immunizations of these conjugates without NBP the anti-S3 specific antibodies of IgM and IgG2a isotype decreased in both antibody level and avidity. The adjuvants NBP 1501 and L121 not only enhanced the hexasaccharide-protein induced IgM and IgG antibody levels but also clearly increased the avidity of the two antibody (sub)classes IgM and IgG2a. This effect was observed in normal (data not shown) and X-linked immunodefective mice. A maturation of the IgG antibody response was realized by the second immunization with hexasaccharide-protein conjugate whereas the third immunization showed no further increase in antibody level and avidity.     

The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C_H3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcγ receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcγ receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.     

Mab-ZAP: a tool for evaluating antibody efficacy for use in an immunotoxin

Immunotoxins, consisting of antibodies coupled to toxins, are extremely useful tools in the elimination of specific cell populations in vitro and in vivo for research and therapeutic applications. The antibody is used to target the toxin to a specific cell population, which is distinguished by its cell-surface antigen. Not all antibodies are suitable for creating an immunotoxin, and large numbers of antibodies may need to be screened. This is a time-consuming and expensive process if each potential candidate must be conjugated to the toxin and purified. A faster and more economical way to identify potential targeting antibodies is to use a second immunotoxin, an anti-IgG antibody that is coupled to the toxin. The second immunotoxin eliminates the need to couple every candidate antibody to the toxin because it can simply be added to cells in culture with the antibody of interest. Using this method, many antibodies can be screened quickly and efficiently for their ability to internalize.     

Antibodies against cortisol block suppressive effects of corticosteroids on lymphocytes in vitro.

Lymphocyte transformation assays were used to test the ability of antibodies against cortisol to reduce bioactivity of corticosteroids in vitro. Mononuclear cells were separated from whole bovine blood and cultured in the presence of PHA alone, PHA + steroid, PHA + steroid + anticortisol, or PHA + steroid + anti-bovine serum albumin. Tritiated thymidine uptake was determined for all groups during the last 24 hr of a 72-hr culture period by scintillation counting. Polyclonal anticortisol against cortisol-bovine serum albumin conjugated in the 21 position was more effective in blocking cortisol activity than monoclonal anticortisol built against conjugates in the 3 position. The steroids that suppressed PHA-induced lymphocyte proliferation in a concentration-dependent manner were: cortisol, corticosterone, dexamethasone, prednisolone, 11-deoxycortisol, and 11-deoxycorticosterone. Aldosterone, cortisone, cholesterol, estradiol, and progesterone did not exhibit concentration-dependent effects and, thus, were not considered suppressive. These concentration-independent steroids were also the least suppressive (with the exception of aldosterone). Anticortisol was able to reduce bioactivity of suppressive corticosteroids that had an 11-hydroxy group, suggesting the antibody was primarily made against this site. Anti-BSA was not effective in blocking corticosteroid activity, but it did enhance proliferation of lymphocytes if added in combination with weakly suppressive steroids. Anticortisol also had an enhancing effect when added with some weakly suppressive steroids. We conclude that antibodies against cortisol are capable of reducing bioactivity of steroids that strongly suppress lymphocyte proliferation. Additionally, the 11-hydroxy group may be an important antigenic determinant of steroid molecules.     

Interaction of steptolysin O with sterols.

A quantitative study of the specific inhibitory power of cholesterol and other sterols on the hemolytic properties of streptolysin O is reported. This streptococcal exocellular protein is a cytolytic toxin which disrupts cytoplasmic membranes of eukaryote cells. The structural characteristics, particularly the stereochemical ones required for a steroid molecule to inhibit the cytolytic activity of streptolysin O, have been investigated in detail. By immunodiffusion techniques, in agar gel plates or tubes containing sterols, the formation of hydrophobic complexes between streptolysin O and inhibitory steroids, but not non-inhibitory steroids except lanosterol, is shown. Upon interaction with inhibitory steroids streptolysin O loses its immunoreactive properties towards neutralizing and precipitating homologous antibodies. An interpretation of the mechanism of biomembrane disorganization by streptolysin O is discussed in the light of its steroid binding properties.     

Metrics for antibody therapeutics development

A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.     

I. Preparation of steroid-antigens through positions of the steroid not bearing functional groups

Dehydroepiandrosterone (DHA) and Dihydrotestosterone (dht) derivatives were prepared in order to obtain antibodies to these steroids. DHA and DHT were coupled to bovine serum albumin through positions which left the functional groups of the steroid free.An intercalated bond with a carboxylic function was linked to C-7 or C-15 of DHA and C-1 off DHT.     

Internal image properties of a monoclonal auto-anti-idiotypic antibody and its binding to aldosterone receptors

A monoclonal anti-idiotypic antibody (H10E4C9F) that interacts with the aldosterone receptors was generated using an auto-anti-idiotypic approach by immunizing a mouse with a 3-O-carboxymethyloxime of aldosterone coupled to bovine serum albumin. This antibody, an IgG1, displayed internal image properties of aldosterone and was considered as an Ab2 beta according to the following criteria. (i) H10E bound to Fab fragments of affinity-purified rabbit anti-aldosterone antibody that had high affinity for aldosterone (Kd = 5 x 10(-10) M). Binding was inhibited by aldosterone but not by estradiol. (ii) H10E inhibited [3H]aldosterone binding to rabbit polyclonal antibodies and also to murine monoclonal antibodies raised during the same fusion. Inhibition was concentration-dependent. These results are consistent with the antibody recognizing an interspecies cross-reacting epitope involved in the aldosterone combining site. (iii) The antibody could be affinity-purified on an immobilized monoclonal anti-aldosterone antibody. (iv) It inhibited [3H]aldosterone binding to rabbit kidney cytosolic aldosterone receptors but had no effect on glucocorticoid receptors. Additional evidence for the interaction of H10E with aldosterone receptors was provided by glycerol gradients analyses: the anti-idiotypic antibody displaced [3H]aldosterone and [3H]corticosterone from the native untransformed 9 S aldosterone receptor in the presence of RU 26988, a specific marker of glucocorticoid receptors. All of the above are consistent with the first successful production of a monoclonal antibody that mimics aldosterone and interacts specifically with the steroid binding domain of aldosterone receptors.     

Selection and characterization of cell binding and internalizing phage antibodies

Many therapeutic targets are cell surface receptors, which can be challenging antigens for antibody generation. For many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but also are internalized into the cell. This allows use of the antibody to deliver various payloads into the cell to achieve a therapeutic effect. Phage antibody technology has proven a powerful tool for the generation and optimization of human antibodies to any antigen. While applied to the generation of antibodies to purified proteins, it is possible to directly select cell binding and internalizing antibodies on cells. Potential advantages of this approach include: cell surface receptors are in native conformation on intact cells while this might not be so for recombinant proteins; antibodies can be selected for both cell binding and internalization properties; the antibodies can be used to identify their tumor associated antigens; and such antibodies can be used for human treatment directly since they are human in sequence. This review will discuss the factors that impact the successful selection of cell binding and internalizing antibodies. These factors include the cell types used for selection, the impact of different phage antibody library formats, and the specific selection protocols used.     

Isoluminol as a marker in direct chemiluminescence immunoassays for steroid hormones

We have used isoluminol steroid hormone conjugates in competitive heterogeneous chemiluminescence immunoassays (ClA) for estradiol, progesterone and estriol in body fluids. The assays did not require prior extraction of the steroid hormone with an organic solvent. Polyclonal or monoclonal antibodies, covalently coupled to polyacrylamide microspheres or adsorbed onto the plastic surface of microtritation plates via an intermediate second antibody, were used as solid-phase. The latter solid-phase system allows rapid processing of incubation mixtures. Sensitivie and reliable direct CIAs for estradiol in serum, progesterone in serum and in saliva and for estriol in saliva have been developed and their clinical utility has been assessed. Sensitivities ranged from 0.54 to 0.04nmol/l, and precision (% CV) ranged from 10% to 16%.     

Influence of the site of conjugation on the specificity of antibodies to progesterone

In order to determine how the site on the molecule used for conjugation influences the specificty of the resulting antiserum, progesterone was conjugated to bovine serum albumin (BSA) through substituents on the A(C3), B(C6), C(C11), and D(C20) rings for use as a hapten to elicit antibody formation in rabbits. Specificty of the antisera was determined by testing the ability of 24 representative steroids to displace radioactive progesterone in a radioimmunoassay procedure. Progesterone-tyrosine methyl ester (TME) conjugates were radioiodinated and used as the radioactive form of the hormone and radioactivity bound to antibody was separated from free radioactivity by a double antibody procedure. Immunization with progesterone conjugated at C20 resulted in the formation of antibodies which could not distinguish between progesterone and other Δ⁴-3-ketosteroids with structures similar in the A, B, and C ring (namely 17-hydroxyprogesterone, 20α and 20β-hydroxy-4-pregnen-20-one, deoxycorticosterne and testosterone). Immunization with progesterone-3-BSA resulted in the formation of antisera which were fairly specific for progesterone while immunization with progesterone conjugated at the 11 or 6 positions resulted in antisera which were very specific for progesterone. It was concluded that steroid hormones should be conjugated to protein at sites on the B or C ring of the molecule for the production of specific antisera.     

Isolation and affinity maturation of hapten-specific antibodies

More and more recombinant antibodies specific for haptens such as drugs of abuse, dyes and pesticides are being isolated from antibody libraries. Thereby isolated antibodies tend to possess lower affinity than their parental, full-size counterparts, and therefore the isolation techniques must be optimized or the antibody genes must be affinity-matured in order to reach high affinities and specificities required for practical applications. Several strategies have been explored to obtain high-affinity recombinant antibodies from antibody libraries: At the selection level, biopanning optimization can be performed through elution with free hapten, analogue pre-incubation and subtractive panning. At the mutagenesis level, techniques such as random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling (chain, DNA and staggered extension process) have been used with various degrees of success to affinity mature or modify hapten-specific antibodies. These techniques are reviewed, illustrated and compared.     

Further evidence that there is more than one adrenal 21-hydroxylase system

The 21-hydroxylase activity of microsomes isolated from bovine adrenal cortex have been assayed using [21-3H]17-hydroxypregnenolone and [1,2-3H]17-hydroxyprogesterone as substrates. When the assays are performed in the presence of an NADH regenerating system, to inhibit steroid 3 beta-hydroxy isomerase-dehydrogenase activity, the microsomes oxidize the 3 beta-hydroxy-5-ene steroid at a rate of 0.37 nmol/min.nmol cytochrome P-450 and the 3-keto-4-ene steroid at a rate of 6.4 nmol/min.nmol. When the microsomes are solubilized with Triton CF-54 they lose the ability to oxidize the 3-hydroxy-5-ene steroid, while the specific activity of the microsomes for the 3-keto-4-ene steroid is enhanced 3-fold. In contrast, when the microsomes are solubilized with sodium cholate, their specific activity towards the 4-ene steroid is decreased by 50% while the specific activity for a low concentration of the 5-ene steroid, 1 microM, is unchanged. In addition, when the oxidations of the labeled steroids (at 1 microM) by the microsomes, are examined in the presence of unlabeled 17-hydroxyprogesterone (at 20 microM) the oxidation of the 3-keto-4-ene steroid is inhibited by 92% while the oxidation of the 3 beta-hydroxy-5-ene steroid is only inhibited by 20%. These results all suggest that there are at least two 21-hydroxylases in bovine adrenal tissue, one of which can utilize the 3-keto-4-ene steroids only, the other of which, in addition, can utilize the 3 beta-hydroxy-5-ene steroids as substrates.     

A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free Reagents,Immunofluorescence, and Multiple Antibody Staining

Immunohistochemistry on mouse tissue utilizing mouse monoclonal antibodies presents a challenge. Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue. This can lead to significant spurious staining. Therefore, a “mouse-on-mouse” staining strategy is needed to yield credible data. This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system. The mouse primary antibody is first combined with an Fab fragment of an anti-mouse antibody in a tube and allowed sufficient time to form an antibody complex. Any non-complexed secondary antibody is bound up with mouse serum. The mixture is then applied to the tissue. The flexibility of this method is confirmed with the use of different anti-mouse antibodies followed by a variety of detection reagents. These techniques can be used for immunohistochemistry (IHC), immunofluorescence (IF), as well as staining with multiple primary antibodies. This method has also been adapted to other models, such as using human antibodies on human tissue and using multiple rabbit antibodies in dual immunofluorescence.     

Synthesis of 7 alpha- and 7 beta-carboxymethyl-9(11)-ene derivatives of estrone and estradiol

As part of a search for derivatives designed to conjugate to amino groups, either of a protein carrier for antibody production or of an immobilized side-chain on a polymer for affinity chromatography, functionalized estrone and estradiol analogues were prepared. These modified steroids were obtained via the introduction of a carboxymethyl side-chain at the C-7 alpha and C-7 beta position on an adrenosterone derivative and were then aromatized on the A ring. These new compounds are unsaturated at the C-9 (11) position, which could be useful for a second modification.     

Immunostaining with dissociable antibody microarrays

The availability of a large number of biological materials such as cDNA, antibodies, recombinant proteins, and tissues has promoted the development of microarray technologies that make use of these materials in high-throughput screening assays. However, because microarray technologies have been less successful in examining proteins than DNA and mRNA, there is a need for improved protein microarray systems. To address this need, we developed an antibody microarray-based immunostaining method that can analyze the properties of a large number of proteins simultaneously. In this method, antibodies are arrayed and immobilized on a solid support and cells bearing antigens of interest are attached to a second support. Apposition of the two supports allows the antibodies to dissociate from the array support and bind to the cellular antigens. After separation of the supports, antigen-bound antibodies can be detected by standard secondary antibody techniques. These "dissociable" antibody arrays were used to detect both the expression and subcellular localization of a large number of specific proteins in various cultured cell types.     

Fecal steroid research in the field and laboratory: improved methods for storage, transport, processing, and analysis

Since the pioneering paper "Measurement of Excreted Steroids in Macaca nemestrina" [Risler et al., American Journal of Primatology 12:91-100, 1987] was first published, field primatologists have been using fecal extraction techniques to examine adrenal and gonadal hormones. These techniques have allowed investigators to determine reproductive conditions in wild primates without causing any disruption to the populations. Over the years, many techniques have been developed to improve the ease of analysis, transportation, and purification. More of the processing can now be done in the field. This paper describes the methodology developed or adapted at the Wisconsin National Primate Research Center, and the factors involved in preparing fecal samples for steroid analysis. We provide information on the steps involved in extracting and purifying steroids from feces for measurement. The latest methods include field processing of samples, such as drying collected material or separating steroids from the fecal material by solid phase extraction (SPE). How samples are processed in the field determines the requirements for international transportation and the methods used in the laboratory. The pros and cons of the different processing methods are discussed. We also report on recent advances in laboratory quantification, with implications for steroid isolation prior to analysis. The different processes involved in isolating and measuring fecal steroids discussed here will enable investigators to understand the components necessary to ensure accurate and reliable results.