W. J. A. Boersma

Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy.

The human androgen receptor (hAR) is an important regulatory protein particularly in male sexual differentiation. The investigation of hAR functionality has been hampered by the lack of AR specific monoclonal antibodies recognizing the functional domains of the receptor. Therefore production of high affinity mono-specific polyclonal (PAbs) and monoclonal antibodies (MAbs) directed to the hAR was initiated following the synthetic peptide (SP) strategy. Five hAR specific peptides were selected on the basis of their predicted antigenic properties avoiding homology with other steroid hormone receptors. Peptide specific polyclonal antisera were obtained following selected immunization protocols. Mono-specific polyclonal antibody responses were elicited to all peptides in mice and rabbits. Crossreactivity of the peptide specific antisera with the native hAR in various biochemical assays was observed with two out of five peptides. Peptide SP61 (hAR residues 301-320) was used for the generation site-directed MAbs specific for the hAR. Specificity for the hAR was established by immunoprecipitation, immune-complex density gradient centrifugation and immunohistochemistry on human prostate tissue sections. The multi-assay performance of the selected high affinity antibodies proved the usefulness of the straight forward peptide approach and opens a wide field of possible biochemical and physiological investigations into questions related to androgen action.

Immunohistochemical detection of the androgen receptor with monoclonal antibody F39.4 in routinely processed, paraffin-embedded human tissues after microwave pre-treatment.

We describe the immunohistochemical detection of the human androgen receptor (AR) in routinely processed, paraffin-embedded tissue with the monoclonal antibody (MAb) F39.4. Deparaffinized sections were heated in a microwave oven for antigen retrieval. A panel of human male- and female-derived tissues was investigated. We observed a nuclear staining pattern consistent with previous results on frozen sections. Moreover, we studied the possibility of detecting AR in prolonged formalin-fixed tissue and in paraffin-embedded archival material. After prolonged fixation times or long-term storage of paraffin-embedded tissue, the staining intensity for the AR did not deteriorate. Blocking experiments with the specific synthetic peptides demonstrated the specificity of this technique. We conclude that this method is specific, allows retrospective AR studies, and offers optimally preserved morphology.

Developmental pattern and regulation by androgens of androgen receptor expression in the urogenital tract of the rat

Distribution and regulation of androgen receptor expression during fetal and neonatal virilization of the rat fetus was assessed by immunohistochemistry. In mesonephric duct derivatives the androgen receptor expression became evident first in the efferent ductules and epididymis (on fetal day 14), subsequently in the vas deferens and finally in the seminal vesicle. Mesenchymal cells of the urogenital tubercle were positive for androgen receptors from fetal day 14 onwards. In the mesenchymal cells of the prostate anlagen, androgen receptor positive cells were found first on fetal day 16. Administration of 5alpha-dihydrotestosterone to pregnant rats from day 11 to day 20 of gestation caused a stabilization of the wolffian duct in female fetuses. The androgen receptor expression pattern became similar as found in mail fetuses, and showed an increase in density and in frequency of androgen receptor positive cells. Administration of the androgen antagonist flutamide during the same interval caused a reduction in density and frequency of androgen receptor positive cells in male fetuses. These findings indicate that androgens enhance the expression of androgen receptors in the developing rat genital tract by induction of androgen receptor positive cells, and by increasing the frequency. The developmental pattern of androgen receptor expression in the rat mesonephric duct system reflects the androgen-responsiveness of the ducts, and is consistent with induction of the androgen receptor along the ducts by testosterone reaching these structures in an exocrine fashion.

Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor.

Antibodies against the N-terminal domain of the human androgen receptor (hAR) were prepared by two different approaches. Firstly, rabbits were immunized with a beta-galactosidase-hAR (amino acids (aa) 174-353) fusion protein. Secondly, two synthetic peptides corresponding to potentially antigenic sites located within this fragment (aa 201-222 and 301-320) were used as immunogens. The obtained antisera contained high titer anti-hAR antibodies as was established with several independent methods (e.g. sucrose gradient centrifugation, immunoprecipitation, Western blotting). The two anti-peptide antisera specifically stained nuclei of glandular epithelial cells in frozen sections of human prostate tissue. Progesterone, estradiol and glucocorticoid receptors were not immunoprecipitated with these antisera. The specific hAR antibodies provide new tools for the characterization of this steroid receptor as well as for diagnostic purposes in pathology of the human prostate and androgen resistance.

A novel carbodiimide coupling method for synthetic peptides: Enhanced anti-peptide antibody responses

Coupling of peptides to immunogenic protein carriers is required for the generation of anti-peptide antibody responses. Carbodiimides are hetero bi-functional coupling reagents that are utilized for coupling reactions through carboxyl and amino groups. The procedures generally used for carbodiimide coupling of peptides and proteins result in conjugates which generate immunodominant antibody responses directed against the neodeterminants on the carrier protein. These determinants are induced by the reaction of carrier and/or peptide with the coupling agent. We have investigated the potential inhibiting effect of an imidazole intermediary on the formation of unwanted neodeterminants during carbodiimide coupling. The serum antibody responses elicited with the peptide-protein conjugates produced were evaluated in ELISA. We have modified and improved the coupling with a watersoluble carbodiimide (EDC) in such a way that a high response to the coupled peptide is obtained in association with negligible levels of anti-neodeterminant antibodies.

An improved conjugation method for controlled covalent coupling of synthetic peptides to proteins using glutaraldehyde in a dialysis method

Controlled and efficient conjugation of synthetic peptides to proteins, for use in immunization or in assay procedures, is a prerequisite for the immunological applications of synthetic peptides. This study describes a new method of conjugating synthetic peptides to proteins in such a way that no homopolymers of synthetic peptides or proteins occur. To achieve this, the protein is first activated with glutaraldehyde and subsequently excess glutaraldehyde is removed. Then coupling of the synthetic peptide to the activated protein occurs while subsequently the surplus reactive glutaraldehyde groups on the protein are blocked with lysine. Excess free peptide and lysine is then removed by dialysis. This improvement not only results in better defined conjugates when compared to classical glutaraldehyde coupling, but also in the consumption of smaller amounts of synthetic peptide during conjugate formation. When used for immunization we obtained similar and sometimes even better responses with the glutaraldehyde based conjugates than with succinimidyl (MBS) conjugates of the same peptides. The performance of the modified conjugates in ELISA procedures, immunization and immunocytochemistry suggests that they are superior to conjugates formed by classical glutaraldehyde coupling.

Immunohistochemical detection of co-localizing cytokine and antibody producing cells in the extrafollicular area of human palatine tonsils.

In vitro experiments have documented the role of cytokines in the regulation of the human humoral immune response. Which cytokines arc operative in vivo and in which lymphoid compartment interactions between cytokine‐producing T cells and antibody‐forming B cells occur is still unclear. For that reason we studied human tonsils using immunohistochemical techniques. In tissue sections from tonsils in a resting stage after recurrent tonsillitis we observed cells producing IL‐1 α and tumour necrosis factor‐alpha (TNF‐α) which were exclusively localized in the mantle zone of the follicle and in the extrafollicular area. Furthermore, a high frequency of interferon‐gamma (IFN‐γ)‐producing cells was detected in the extrafollicular area, but not inside the follicles. Occasional IL‐2‐ and IL‐4‐producing cells were found in the extrafollicular area. Immunohistochemical detection of antibody isotypes revealed that B cells, IgM‐membrane‐positive, were localized inside the follicles and mantle zones, whereas IgD‐membrane‐positive cells were mainly found in the mantle zones of secondary follicles. In contrast, plasma cells producing IgGl‐4 and IgAl‐2 were found in the extrafollicular area. No IgD and IgE antibody‐forming cells were detected in tonsils, whereas IgM antibody‐forming cells were detected in the xtrafollicular area. The co‐localization of cytokine‐producing cells and antibody‐forming cells in human tonsil suggests that T‐B cell interactions, required for B cell differentiation and isotype switching, take place in the extrafollicular area.

Synthetic peptide conjugates with horseradish peroxidase and β-galactosidase for use in epitope-specific immunocytochemistry and ELISA

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-beta-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to beta-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.

Immunocytochemical determination of antigen and epitope specificity of HIV-1-specific B cells in lymph-node biopsies from HIV-1-infected individuals

Knowledge about B-cell dysfunction and HIV-specific antibody production is necessary for the understanding of both HIV-1-related immunopathology and the (vaccine-induced) humoral immunity involved in protection against AIDS. This paper describes the application of recently developed methods to detect epitope specificity of B cells in lymph-node biopsies with antigen-enzyme conjugates. Cryosections of five lymph-node biopsies from HIV-1-infected individuals and four control tissues were stained with a panel of HIV-1 antigen-enzyme conjugates: recombinant HIV-1 proteins (gp 160, gp 120 and p24), labelled with peroxidase, and synthetic peptides representing neutralizing epitopes from gp120 and gp41, labelled with alkaline phosphatase. Antibody-forming cells (AFCs) were detected in all the HIV-1-infected biopsies with gp160, gp120 and/or p24, in numbers up to 350 per section. AFCs producing specific antibodies against peptide 101 (SP 101), representing the neutralizing epitope 586-608 of gp41, were detected in one patient. These techniques allow correlation of in vivo function of B cells with lymph-node pathology, clinical stage of the disease and serological data. Their potential for the elucidation of HIV-related immunopathogenesis and the development of vaccines is discussed.

Type-specific immunodetection of human heart fatty acid-binding protein with polyclonal anti-peptide antibodies

SummaryIn order to develop specific antibodies against human heart cytoplasmic fatty acid-binding protein (HFABPc), four oligo-peptides of 15–20 amino-acids each and corresponding with different antigenic parts of the human H-FABPc molecule, were synthesized. Polyclonal antibodies against these synthetic peptides were raised in mice (Balb/C) and rabbits (Flemish giant). When tested in enzyme linked immunosorbent assays (ELISA, antibody-capture assay), antisera against three of the four peptides showed a high immunoreactivity with the synthetic peptide selected for immunization as well as with the native human H-FABPc. Some cross-reactivity with the other synthetic peptides was observed for the rabbit antisera but not for those from mice. Polyclonal antibodies against synthetic peptides can be applied for the specific detection of the native protein in biological preparations containing proteins that show a high degree of homology with the protein to be assayed.