Prospective evaluation of functional outcomes after laparoscopic sigmoidectomy with high tie of the inferior mesenteric artery for diverticular disease in consecutive male patients.

BACKGROUND: To date, there has been no consensus concerning the vascular approach during sigmoid colectomy for diverticular disease. The aim of this study was to determine the functional impact of elective laparoscopic sigmoidectomy performed with high ligation of the inferior mesenteric artery for diverticulitis in consecutive male patients. METHODS: Twenty-five consecutive patients of median age 53 years were enrolled in a prospective single-centre pilot study at a tertiary teaching hospital. Main outcome measures were functional results. Patients were asked to complete standardized, validated questionnaires to evaluate preoperative and 6 months postoperative bowel symptomatology (Jorge-Wexner Incontinence Score and KESS score), urinary function (IPSS), and sexual function (IIEF). Secondary outcomes were surgical data, morbidity, and quality of life (SF-36). RESULTS: There were no significant differences between preoperative and 6 months postoperative total scores for bowel symptomatology, urinary function, and sexual function. There were no perioperative deaths. The morbidity rate was 12% including three minor and no major events. Quality of life demonstrated statistically better general health (p < 0.01) and better medical status over the prior 4 weeks at 6 months after surgery, compared to baseline. This single-centre prospective study has a limited number of patients, relatively short follow-up time, and includes only male patients. CONCLUSION: Laparoscopic sigmoidectomy with high tie of the inferior mesenteric artery for diverticular disease does not induce functional disorders at 6 months after surgery. The benefit of the operation for quality of life is even greater for general health and medical status.

Strategies for improving production and purification of a recombinant protein: rP30 of Toxoplasma gondii expressed in the yeast Schizosaccharomyces pombe.

Many problems concerned with the production and the purification of recombinant proteins must be addressed prior to launching an industrial production process. Among these problems, attention is focused on low-level expression that complicates the purification step and can jeopardise the process. The expression of a membrane protein, rP30, of Toxoplasma gondii in the yeast Schizosaccharomyces pombe led to a secretion of only 0.5 microg ml(-1). In order to obtain a sufficient quantity for biochemical characterization and evaluation in vitro diagnostic test development, strategies for both production and purification had to be optimized. First, the influence of four nitrogen sources (three peptones and yeast extract) on the growth rate, but also on the separation between the protein and the components of the fermentation broth was assessed. Second, batch and fed-batch fermentations were compared in terms of final biomass and rP30 concentrations. Third, three different protocols that included fixed and expanded bed ion exchange chromatography were compared for processing a large volume of feedstock. By using the most appropriate strategies, i.e. fed-batch fermentation, capture on EBA cation exchanger and affinity chromatography polishing, a purification factor of 1778 and a yield of 49% were achieved. These performances allowed a 12.5-fold increase for the overall rP30 process productivity.

[Topical anesthesia for out-patient adult cataract surgery without an anesthesiologist]. / L'anesthésie topique en chirurgie de la cataracte ambulatoire de l'adulte sans anesthésiste présent.

To review the local anesthesia environment in France in adult out-patient cataract surgery. The author considers the presence or absence of an anesthesiologist in the operating room. The report shows that in many circumstances there is no need for an anesthesiologist if the following criteria are respected: the adult is fully informed, in rather good health, with no acute risk factors, and surgery is performed by a senior surgeon in a certified operating room. In France, a move toward more flexible regulations is expected, with a new cooperation between ophthalmologists and anesthesiologists that will lead to a new true risk/benefit/obligation ratio. Respect of the individual and safety remain crucial requirements making systematic generalizations impossible.

Autoantibodies recognizing citrullinated rat filaggrin in an ELISA using citrullinated and non-citrullinated recombinant proteins as antigens are highly diagnostic for rheumatoid arthritis.

The objective of the study was to determine the diagnostic value for rheumatoid arthritis (RA) of anti-filaggrin autoantibodies (autoAb) recognizing citrullinated recombinant rat filaggrin (ACRF) in community cases of very early arthritis. To evaluate the diagnostic value of ACRF, were studied sera from patients with different classified rheumatic diseases and healthy subjects (group 1, n= 422) and 314 community cases of very early arthritis (group 2) that were classified as RA (n = 176), non-RA (n = 63) and undifferentiated (n = 75) arthritides after 1 years of follow-up. ACRF were measured using a new ELISA, with results expressed as the difference between the OD value obtained on citrullinated minus that on noncitrullinated rat filaggrin (differential ACRF; dACRF). For both groups, rheumatoid factors (RF), anti-keratin autoAb (AKA) and anti-perinuclear factor (APF) were tested; for group 2, anti-CCP autoAb were also tested. Different reactivity patterns against citrullinated and noncitrullinated filaggrin were observed. Almost all sera reacting with citrullinated but not noncitrullinated filaggrin were from RA patients. Among RA and non-RA sera that recognized both forms of filaggrin, a positive result was obtained only with RA sera. For groups 1 and 2, dACRF sensitivity was 58.4% and 30.7%, and specificity for RA was 99.5% and 98.4%, respectively. In group 2, dACRF specificity for RA was better than that of RF (92.1%), APF (95.2%), AKA (96.8%) and anti-CCP (95.2%). dACRF positive predictive value was high (98.2) and close to that given by the concomitant positivity of RF and anti-CCP autoAb. Despite a high positive correlation between AKA, APF, anti-CCP and dACRF test results, they were complementary since some sera were positive for only one test. Thus, in a community setting, anti-citrullinated rat filaggrin reactivity detected by a new ELISA, whose originality is based on the difference between serum's reactivities on the citrullinated and native forms of filaggrin, had a higher diagnostic value for RA than other autoAb.

[The temporal periosteum: anatomical study and surgical implications]. / Le périoste temporal: étude anatomique et intérêt chirurgical.

The periosteum of the temporal area is mentioned at different places in the literature: either against the osseous plane like everywhere in the human body, or between the deep and the superficial temporal fascia. The subperiosteal subtemporal approach in craniofacial surgery in children is in favour of a juxta-osseous localization of the periosteum. Ten premature still-born neonates and two adults cadavers have been dissected for this study and, permit anatomical and histological (with HES coloration) studies. With every specimen, the authors concluded that the temporal periosteum is against the outer table of the calvarium. It became thinner in adults because of direct insertions of the temporalis muscle in the calvaria. An anatomical description of the layers of the temporal area is realised and discussed with an extensive review of the literature. The authors have proposed a subperiosteal subtemporal approach in craniostenosis surgery.

Highly specific anti-estradiol antibodies: structural characterisation and binding diversity.

Subtle modulation of antibody-binding properties by protein engineering often lies with an accurate structural and energetic description of how an antigen is recognised. Thus, with the intent to increase the affinity and add a bias in favour of natural estradiol compared with its chemically modified immunogen, we have determined the crystal structure of two anti-estradiol monoclonal antibodies, 10G6D6 and 17E12E5. Although generated against the same estradiol derivative, these antibodies share little sequence identity, which is reflected in dissimilar binding pockets and in different positioning of the steroid. In both antibodies the characteristic 17-hydroxyl group is buried deeply at the bottom of hydrophobic pockets and stabilised by hydrogen bonds. Apart from this similarity, the steroid is oriented differently in the respective binding pockets. The high specificity of both antibodies has been mapped out, and even closely related steroids show low cross-reactivity. The structural studies of the complex formed between 10G6D6 and 6-CMO-estradiol have identified contacts between the 6-CMO coupling linker and an arginine residue from the heavy chain CDR2 segment. This segment is now being targeted by random mutagenesis to select mutants with a preference for natural estradiol compared to the branched hapten.

An immunoreactive peptide of the FSH involved in autoimmune infertility.

The purpose of this study was to identify autoantigens contained in human ovary extracts. Serum samples from 36 infertile women with anti-ovary antibodies as detected with an ELISA technique were tested in Western blot against human ovary extracts. A reactive protein with a molecular mass matching that of the FSH was detected in 34 cases. These serum samples also reacted strongly in Western blot and ELISA with purified FSH and, in immunofluorescence, with pituitary cells. Using the Pepscan approach, with overlapping peptides matching the amino acid sequence of the human FSH beta-chain, several immunoreactive regions were evidenced. The 78-93 amino acid sequence of the human FSH beta-chain appeared as one of the major epitopes. Synthetic peptides of this region were prepared and demonstrated to react with human serum samples from women with anti-ovary antibodies. These data demonstrate that FSH can be an autoantigen, recognized by autoantibodies associated with infertility.

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies.

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.

Performance of two ELISAs for antifilaggrin autoantibodies, using either affinity purified or deiminated recombinant human filaggrin, in the diagnosis of rheumatoid arthritis.

OBJECTIVE: To develop a standardisable enzyme linked immunosorbent assay (ELISA), using human filaggrin, for detection of antifilaggrin autoantibodies in rheumatoid arthritis (RA). To compare the diagnostic performance of the ELISA with those of reference tests: "antikeratin antibodies" ("AKA"), and antibodies to human epidermis filaggrin detected by immunoblotting (AhFA-IB). METHODS: Two ELISAs were developed using either affinity purified neutral-acidic human epidermis filaggrin (AhFA-ELISA-pur) or a recombinant human filaggrin deiminated in vitro (AhFA-ELISA-rec) as immunosorbent. Antifilaggrin autoantibodies were assayed in 714 serum samples from patients with well characterised rheumatic diseases, including 241 RA and 473 other rheumatic diseases, using the two ELISAs. "AKA" and AhFA-IB tests were carried out in the same series of patients. The diagnostic performance of the four tests was compared and their relationships analysed. RESULTS: The titres of "AKA", AhFA-IB, and the AhFA-ELISAs correlated strongly with each other. The diagnostic sensitivity of the AhFA-ELISA-rec, which was better than that of AhFA-ELISA-pur, was 0.52 for a specificity of 0.95. This performance was similar to those of "AKA" or AhFA-IB. However, combining AhFA-ELISA-rec with AhFA-IB led to a diagnostic sensitivity of 0.55 for a specificity of 0.99. CONCLUSION: A simple and easily standardisable ELISA for detection of antifilaggrin autoantibodies was developed and validated on a large series of patients using a citrullinated recombinant human filaggrin. The diagnostic performance of the test was similar to that of the "AKA" and AhFA-IB. Nevertheless, combining the AhFA-ELISA-rec with one of the other tests clearly enhanced the performance.

Differential diagnosis of prostate cancer and benign prostate hyperplasia using two-dimensional electrophoresis.

Prostate specific antigen (PSA) is a protease which is characteristic of the prostate. It is widely used as a serum marker for the early diagnosis of prostate cancer (PCa). Nevertheless, for concentrations between 4 and 10 ng/mL, PSA does not enable PCa to be distinguished from benign diseases, such as benign prostate hyperplasia (BPH). In sera, the use of a ratio between free PSA (PSA uncomplexed with protease inhibitor) and total PSA (free PSA and PSA bound to alpha-1 anti-chymotrypsin) enables the "gray zone" to be reduced, but an important proportion of patients are still wrongly classed. Using two-dimensional electrophoresis, we demonstrated using 52 PCa and 40 BPH well-documented clinical cases that BPH sera show a significantly greater percentage of low-molecular-weight free PSA elements (IwPSA) than PCa sera. In our study, the use of a ratio between IwPSA and standard free PSA enables the correct diagnosis of 100% of PCa and 82.5% of BPH cases as against when 73.1% and 42.5% respectively were correctly diagnozed using the total PSA and the free/total PSA ratio. This important finding may be related to differences in the mechanism secreting PSA from the prostate into the bloodstream. We have shown how a tissue marker may be turned into a powerful tumor marker by events probably unrelated to its expression.

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation.

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M, 21,000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.

Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments.

Monoclonal antibodies are now widely used to measure the concentration of steroid hormones in human serum samples. The great development of molecular engineering techniques over the past 10 years has made possible the improvement of specificity and/or sensitivity of selected antibodies. We have obtained two monoclonal antibodies, 17E12E5 and 10G6D6, using estradiol-6-ethyl methoxy carbonyl (EMC)-bovine serum albumin (BSA) as immunogen. To tentatively improve their affinities for natural estradiol, we have initiated their structural and functional studies. For this purpose, we have cloned and sequenced the genes encoding the variable fragments of each antibody. Single chain variable fragments (scFv) were produced into the periplasmic space of E. coli using the pLIP6 expression vector. Mapping of the functional structures of both antibodies was obtained by combination of modelling and mutational analyses together with cross-reaction studies. The two binding pockets are described and models of estradiol complexed to 17E12E5 and 10G6D6 are proposed.

Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris: evidence of hyper O-glycosylation.

A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris. The truncated protein lacked the C-terminal residues which, in native SAG1, encompass a glycosylphosphatidylinositol anchorage site. The single potential N-glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C-terminal of the construction to aid purification by immobilized metal-chelate chromatography. Recombinant SAG1t was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAG1t heterogeneity was related to the presence of O-linked oligosaccharides containing alpha1-2-, alpha1-3- or alpha1-6-linked mannoses. The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human-serum-derived antibodies, specific for the native SAG1, which suggested that the O-glycosylations had no major effect on the protein conformation. However, ELISA and Western-blot analysis with human sera showed that the O-carbohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SAG1t species or deglycosylation is required in order to use recombinant SAG1t as a diagnostic reagent. Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P. pastoris is a potential drawback that should be considered when using this expression system.

Use of free radical chemistry in an immunometric assay for 17 beta-estradiol.

BACKGROUND: We wished to develop an enzyme immunometric assay for 17 beta-estradiol (E2) in human serum using solid-phase immobilized epitope immunoassay (SPIE-IA) technology and free radical chemistry. METHODS: We used an anti-estradiol monoclonal antibody as capture antibody and Fenton-like reagents to cross-link it to E2. The same antibody, labeled with acetylcholinesterase, was used for detection. Serum was diluted 10-fold before assay. RESULTS: After correction by the dilution factor, the detection limit was 5 ng/L for human serum and intra- and interassay CVs were <7% and 15%, respectively, at concentrations of 169-2845 ng/L. No cross-reactivity was seen with other natural steroids. In comparison with a competitive commercial RIA performed on 88 undiluted human sera, the slope (SD) of the regression line was 1.05 (+/- 0.02) and the intercept was 47 (+/-27) ng/L (S(y/x) = 186 ng/L) at concentrations of 20-5000 ng/L (r(2) = 0.97). CONCLUSIONS: The use of Fenton-like chemistry in SPIE-IA technology allows a sensitive measurement of E2 in human serum and could be a new approach for the development of sensitive immunoassays.

Analysis of prostate specific antigen and alpha1-antichymotrypsin interaction using antipeptide monoclonal antibodies.

PURPOSE: The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs). MATERIALS AND METHODS: Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane. RESULTS: Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA. CONCLUSIONS: The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.

Involvement of the C-terminal end of the prostrate-specific antigen in a conformational epitope: characterization by proteolytic degradation of monoclonal antibody-bound antigen and mass spectrometry.

Prostate-specific antigen (PSA), a 237-amino acid glycoprotein, encoded by the hKLK3 gene, is widely used as a serum marker for the diagnosis and management of prostate cancer. We report here the localization of a conformational epitope recognized by the anti-total PSA monoclonal antibody (mAb) 11E5C6, by proteolytic degradation of mAb-bound antigen followed by mass spectrometric analyses of the peptides generated. These two technologies, combined with molecular display, allowed the identification of amino acid residues contained within three different peptides distant on the PSA sequence, but close in the PSA three-dimensional structure, that may be part of the mAb 11E5C6 epitope. The last four C-terminal amino acid residues are included in this epitope, as well as certain other C-terminal residues between Y225 and T232. The involvement of the PSA C-terminal end in the mAb 11E5C6 epitope was confirmed by western blotting experiments with the recombinant protein proPSA-RP1, resulting from the cloning of an alternative transcript of the hKLK3 gene, in which the PSA C-terminal end was deleted and replaced by another sequence. Although the anti-total PSA mAb 5D5A5 used as a control bound proPSA-RP1, mAb 11E5C6 did not. The requirement of the C-terminal end for the recognition by mAb 11E5C6 may be useful for the discrimination of PSA-related forms.

[Homage to Leon Tripier. Description of the first musculocutaneous flap and current indications]. / Hommage à Léon Tripier. Description du premier lambeau musculocutané et indications actuelles.

The double pedicle flap taken from the upper lid was described for the first time in 1888 by Tripier. To our knowledge, it is the first case of reconstruction by a myocutaneous flap described in the literature. Other authors subsequently modified the first description. The same flap taken with a single inner pedicle is now usually employed. For the authors, the Tripier's flap, with a double and permanent pedicle, still has some indications in eyelid reconstruction.

Anti-free prostate-specific antigen monoclonal antibody epitopes defined by mimotopes and molecular modeling.

BACKGROUND: Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer, and the free PSA/total PSA ratio has been shown to be efficient for distinguishing prostate cancer from benign prostatic hyperplasia. We report here the characterization of seven mouse monoclonal antibodies (mAbs) and the partial localization of two conformational epitopes identified by anti-free PSA mAbs. METHODS: The mAbs were studied by competition and sandwich assays, and the epitope localization of the two anti-free PSA mAbs (6C8D8 and 5D3D11) was performed using phage displayed peptide libraries and molecular modeling. RESULTS: The seven mAbs were classified into three groups according to their recognition specificities and their ability to inhibit the enzymatic activity of PSA and the formation of PSA-alpha1-antichymotrypsin (ACT) complex. Among the anti-free PSA mAb group, 6C8D8 recognized the phage displayed peptide RKLRPHWLHFHPVAV, two parts of which presented similarities with two regions distant on the PSA sequence but joined in the tridimensional structure. mAb 5D3D11 recognized the peptide DTPYPWGWLLDEGYD, which is similar to a PSA region located on the board of the groove containing the PSA enzymatic site. Both epitopes were located in the theoretical ACT binding site described previously. Moreover, these mAbs were able to inhibit the enzymatic activity of PSA. CONCLUSIONS: These epitope localizations are in agreement with the ability of both mAbs to inhibit enzymatic activity and ACT fixation. The results presented here could bring information for the generation of clinically relevant PSA assays.

Two-dimensional electrophoresis of prostate-specific antigen in sera of men with prostate cancer or benign prostate hyperplasia.

Prostate-specific antigen (PSA), the main marker for prostate cancer (PCa), is released from the prostate into the blood stream at nanogram level and may increase in PCa and nonmalignant disease such as benign prostate hyperplasia (BPH). More recently, advantage was taken of PSA's ability to bind to protease inhibitors in serum in order to improve discrimination between PCa and BPH, using the free PSA to total PSA ratio. The understanding of this phenomenon at molecular level, which is still unknown, may promise new improvements in the field of diagnostics. For this purpose, we determined the pattern of PSA forms in PCa and BPH sera, using the high resolving power of two-dimensional electrophoresis (2-DE) in conjunction with the high sensitivity of chemiluminescence detection. Serum PSA differs drastically from seminal PSA: apart from complexed forms, serum PSA shows few cleaved forms. Moreover, 2-DE patterns from PCa are relatively homogeneous, whereas patterns from BPH may in some cases present a higher proportion of cleaved forms and in other cases present slightly more basic spots. We therefore demonstrated, for the first time, that an increase in the free to total PSA ratio in BPH cases may be due to cleaved PSA forms (which are enzymatically inactive and unable to bind inhibitors), or possibly related to basic free PSA, which may represent the zymogen forms.

Cross-linking of 17 beta-estradiol to monoclonal antibodies by direct UV irradiation: application to an enzyme immunometric assay.

Ultraviolet irradiation was used to cross-link 17 beta-estradiol directly to monoclonal anti-17 beta-estradiol antibodies coated on 96-well microtiter plates. Cross-linking efficiency was directly correlated with both irradiation energy and wave-length. The best results were obtained at 254 (10 J/cm2, 45-min irradiation) and 312 nm (40 J/cm2, 160-min irradiation). The irradiation fully denatured both individual molecules (i.e., 17 beta-estradiol and monoclonal anti-17 beta-estradiol antibody), but 17 beta-estradiol was at least partly protected when immunologically bound to the paratope of the antibody. Four different monoclonal anti-17 beta-estradiol antibodies yielded positive results, demonstrating that this photo-cross-linking has considerable potential. We used this original approach to develop a new enzyme immunometric assay of 17 beta-estradiol based on our previously described immunometric procedure, solid-phase immobilized epitope immunoassay, which uses chemical agents to cross-link haptens via amino groups to specific antibodies. The assay was specific (no cross-reactivity with other natural steroids), precise, and sensitive (detection limit of 38 pg/mL in human serum). It correlated well with two competitive commercial immunoassays when tested on 40 human sera.