Monoclonal Reagents Research Articles

Selection of monoclonal antibodies for the identification of D variants: ability to detect weak D and to split epD2, epD5 and epD6/7.

Red cells from known D variant donors were tested with 41 monoclonal anti-D reagents, 26 IgG and 15 IgM, with the view to selecting a panel to aid the identification of unusual D types. These antibodies gave reaction patterns which allowed the identification of most of the known D category cells, recognizing epD2, epD5, epD6/7, epD8 and epD9, but were unable to distinguish category III from normal D-positive cells. Reactivity with HMi, HMii, DFR, DBT and RoHar cells split epD2, epD5 and epD6/7 into two, three and eight groups, respectively. A panel comprising 15 monoclonal anti-D, 11 IgG and four IgM, was selected as representative of the antibodies tested. Reactivity of monoclonal anti-D was dependent on antibody concentration and antibody avidity. An antibody concentration of at least 12 micrograms/ml was required for optimum reactivity of the two monoclonal antibodies tested. A simple calculation of division of the titre by the antibody concentration provided a relatively simple means of establishing the reactivity performance of the antibody and correlated well with ability to detect weak D (Du) cells. A characteristic variable reduction in reaction strength with all the IgG anti-D was observed with weak D cells. The IgM antibodies, except the high avidity RUM-1, T3D2T6, D9A4 and BS226, performed poorly in detecting weak D. The majority of the IgM antibodies tested reacted with RoHarr cells, while only one IgG antibody was positive.

Molecular aspects and the pathological basis of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) has been considered to be a 'model auto-immune disease' for more than two decades. However, the underlying pathophysiology of PBC and the relationship with the associated serological abnormalities have been hitherto elusive. Beginning in 1987 with the cloning and subsequent identification of the mitochondrial autoantigens of PBC, progress has come rapidly and we can now sketch several potential pathogenic pathways through which disease occurs. More than 90% of patients with PBC produce autoantibodies to mitochondria, and the antoantigens involved have been identified as related components of the 2-oxo-acid dehydrogenase complexes (OADC), including the E2 subunits of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxo-acid dehdrogenase complex and 2-oxo-glutarate dehydrogenase complex, Protein X and E1 alpha. The cDNAs of each of the E2 subunits of OADC have been cloned and characterized. Moreover, the epitopes of the antimitochondrial antibodies (AMA) have been mapped at the highly conserved lipoyl domain E2 subunits. The use of recombinant peptides produced by these clones has greatly facilitated the detection of AMA. In addition, nucleotide sequence analysis of PDC-E2 specific human monoclonals and combinatorial Fabs strongly suggests that these autoantibodies are derived from clonal selection of a restricted set of somatically mutated immunoglobulin germline genes. Most interestingly, however, the use of monoclonal reagents to PDC-E2 has demonstrated that there is an increased expression of either PDC-E2, or a cross-reactive molecule, on the luminal surface of biliary epithelial cells in patients with PBC. These data provide a scenario to explain the tissue specific pathology associated with PBC and several interesting underlying pathophysiological mechanisms.

Selection of Monoclonal Antibodies for the Identification of D Variants: Ability to Detect Weak D and to Split epD2, epD5 and epD6/7

Red cells from known D variant donors were tested with 41 monoclonal anti-D reagents, 26 IgG and 15 IgM, with the view to selecting a panel to aid the identification of unusual D types. These antibodies gave reaction patterns which allowed the identification of most of the known D category cells, recognizing epD2, epD5, epD6/7, epD8 and epD9, but were unable to distinguish category III from normal D-positive cells. Reactivity with HMi, HMii, DFR, DBT and R(0)^Har cells split epD2, epD5 and epD6/7 into two, three and eight groups, respectively. A panel comprising 15 monoclonal anti-D, 11 IgG and four IgM, was selected as representative of the antibodies tested. Reactivity of monoclonal anti-D was dependent on antibody concentration and antibody avidity. An antibody concentration of at least 12 µg/ml was required for optimum reactivity of the two monoclonal antibodies tested. A simple calculation of division of the titre by the antibody concentration provided a relatively simple means of establishing the reactivity performance of the antibody and correlated well with ability to detect weak D (D^u) cells. A characteristic variable reduction in reaction strength with all the IgG anti-D was observed with weak D cells. The IgM antibodies, except the high avidity RUM-1, T3D2T6, D9A4 and BS226, performed poorly in detecting weak D. The majority of the IgM antibodies tested reacted with R(0)^Har cells, while only one IgG antibody was positive.

Evaluation of monoclonal anti-D reagents using D variant cells.

Monoclonal anti-D antibodies submitted to the Third Monoclonal International Workshop were evaluated against a number of D variant cells using standard serological techniques. The monoclonal antibodies were able to discriminate between the cells of Categories Va, VI and DFR but not Category III cells. Cells within each category did not give any aberrant results. The Rh:33 cells behaved as normal Rh(D) positive cells.

Flow cytometric analysis of 68 monoclonal anti-D reagents.

68 monoclonal IgG anti-D were assessed for quantitative and qualitative binding to D variant red cells using a flow cytometric indirect immunofluorescence test. One antibody failed to bind to normal RhD pos. cells, but reacted weakly with D cat VI and D Cat VII cells. Quantitatively, binding varied approximately twenty-fold between different MAbs and different D variants. D cat III cells gave the lowest level of binding when compared with the D pos. controls. Qualitatively, R1VIr, R2VIr, R1VIIr and DFR samples failed to react with various MAbs regardless of the levels of binding of these MAbs to the D pos. controls, thus supporting the idea of missing epitopes in such variants.

Development of a clinically applicable high-speed flow cytometer for the isolation of transplantable human hematopoietic stem cells.

A high-speed cell sorter capable of a throughput speed 4-5-fold greater than commercially available systems was developed and evaluated as a processing tool for isolating purified hematopoietic stem cell grafts. The clinical high-speed sorter (CHSS) serves as a single-pass, multiparameter processing tool that provides the means to isolate a highly purified population of cells from starting cell populations with extremely low frequencies. The sorter incorporates environmental barriers to create a sterile environment for cell processing. Monoclonal antibodies and reagents produced under good manufacturing practices (GMP) are used to isolate hematopoietic stem cells by means of the CHSS. Using this technology, the CD34+Thy-1+Lin- hematopoietic stem cell population has been isolated from normal adult bone marrow and mobilized peripheral blood. The sorted cells have been shown to be sterile and viable and to retain hematopoietic function.

Evaluation and validation of a monoclonal immunofluorescent reagent for direct detection of Bordetella pertussis

An outbreak of pertussis in Manitoba, Canada, provided an opportunity to evaluate the recently developed monoclonal antibody (MAb) BL-5 for the direct detection of Bordetella pertussis. The MAb recognizes a lipooligosaccharide epitope. A total of 1,507 consecutive nasopharyngeal swabs for culture and companion smears for direct fluorescent-antibody (DFA) detection were evaluated at Cadham Provincial Laboratory between September and November 1994. The cutoff for DFA positivity was four fluorescing organisms with morphology characteristic of B. pertussis. PCR analysis for B. pertussis DNA was performed on a subset of 100 smears by eluting material from the slides after DFA examination. In comparison with culture, the sensitivity, specificity, and positive and negative predictive values of BL-5 were 65.1% (41 of 63 samples), 99.6% (1,438 of 1,444 samples), 87.2% (41 of 47 samples), and 98.5% (1,438 of 1,460 samples), respectively. The sensitivity of culture compared with PCR was 45.5% (10 of 22 samples) for the subset of 100 specimens tested by both procedures. An expanded "gold standard" of positivity by culture or PCR for these 100 specimens resulted in DFA sensitivity, specificity, and positive and negative predictive values of 32.3, 97.1, 83.3, and 76.1%, respectively. The utility of MAb BL-5 for direct detection of B. pertussis in a clinical laboratory setting has been demonstrated by this investigation.

Identification of two new D variants, DHMi and DHMii using monoclonal anti-D.

Fifteen examples of red cells are described which showed discrepant reactivity on routine grouping with two monoclonal anti-D typing reagents, HAM-A being negative and MAD-2 strongly positive. The reaction profile of these cells with a number of monoclonal anti-D characterised the samples into two new variant groups, DHMi and DHMii. DHMi was characterised by a negative reaction with BRAD-8 and DHMii by a negative reaction with BRAD-5. DHMi cells were found to have a papain-sensitive site, identified by Mab 21 G6, which was not detectable in DHMii. The presence of allo-anti-D in one case of DHMi suggests a partial D in these individuals. The positive reaction of HAM-A with sialidase- and endo-F-treated DHMii cells suggests that sialic acid and/or N-glycans could possibly be involved in blocking the reaction with untreated cells. All samples typed as C-E+. One was e negative while the rest had variable depression of the e and f antigens. Marginal depression of E was seen with those DHMi cells tested, but not with DHMii cells. Data from family studies suggest that the variant D in both DHMi and DHMii is inherited as a cDE gene complex and is controlled by the RH locus.

Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes

In chickens, single functional immunoglobulin variable and joining gene segments at each of the heavy and light chain loci undergo V(D)J rearrangement. Diversity is subsequently introduced by conversions templated by upstream pseudo V region genes in such a way that practically all V regions in mature B cells have identical ends. This greatly simplifies the representative amplification of V region genes. Furthermore, the entire naive repertoire of the adult chicken is produced in the bursa of Fabricius of the young bird. These special properties of the generation of immunoglobulin diversity in chickens have been exploited in the development of procedures to produce large libraries of diverse antibody combining sites derived from chicken Ig genes and expressed on filamentous bacteriophage. The utility of this library was assessed by selection of specifically binding phage using three solid phase-bound protein antigens, hen egg white lysozyme, bovine thyroglobulin and bovine serum albumin. The sequences of the V region genes thus isolated demonstrated that selection was specific and that the library contained useful diversity of binding sites. This library provides access to a repertoire whose diversity is based on a mechanism different from that underlying previously available libraries. The demonstrated feasibility of generating chicken phage antibodies may lead to the production of monoclonal reagents from immunised chickens, and the derivation of reagents for studying immunoglobulin mediated selection in avian B cell development.

Monoclonal anti-D specificity and Rh D structure: criteria for selection of monoclonal anti-D reagents for routine typing of patients and donors.

The Rh blood group system is the next most important to the ABO system in terms of its clinical significance in blood transfusion. It is vital to the safe, efficient practice of transfusion medicine that Rh D phenotyping tests are selected, executed and interpreted correctly. However, the Rh D blood group antigen has been shown to be subject to many phenotypic variations, and different reagents and typing techniques vary in their ability to detect these variants. The range of D-positive phenotypes are reviewed in terms of their reactivity with monoclonal antibody reagents and their clinical significance. In view of the available evidence, it is suggested that patient typing can be safely achieved by the duplicate use of one high-avidity or two very similar IgM monoclonal anti-D reagents that detect most variants except category DVI in simple tube or microplate saline tests. Antiglobulin testing for weak D should not be carried out on patient samples. Donor typing can be safely achieved by the use of the same monoclonal, used in parallel with a polyclonal anti-D reagent that detects DVI on sensitive automated equipment.

Phospholipid binding specificities and idiotype expression of hybridoma derived monoclonal autoantibodies from splenic cells of patients with systemic lupus erythematosus.

To analyse the phospholipid binding specificity, functional characteristics and idiotype expression of human hybridoma derived monoclonal autoantibodies (MAb) derived from the spleens of two patients with active systemic lupus erythematosus (SLE). The IgM MAbs binding to phospholipids were generated from spleen cells of two patients (RSP and RT) with active SLE and their specificity of binding to neutral phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, platelet activating factor, sphingomyelin) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol and cardiolipin (CL)) analysed. Binding specificity of cross reactive antibodies (those binding to CL and DNA) was confirmed by fluid phase inhibition assays. Lupus anticoagulant activity and beta 2-glycoprotein-1 (beta 2 GP-1) requirement for the antigen binding of these MAbs were detected using the modified dilute Russell's viper venom test and modified anti-CL enzyme linked immunosorbent assay (ELISA), respectively. Expression of idiotypes (Id) Id RT-84 and Id H3 was analysed using rabbit polyclonal and murine monoclonal anti-idiotype reagents, respectively. Twelve clones from the patient RSP and eight clones from patient RT were reactive with phospholipids. Marked differences in phospholipid binding of these MAbs were noted, varying from truly polyreactive (RT-72 bound to most phospholipids tested) to monospecific (RT-84 bound only to CL). Furthermore, MAbs RT-84, RT-129, and RSP-57 had lupus anticoagulant activity and required beta 2 GP-1 for CL binding. It was found that 75% of phospholipid binding antibodies from RT clones expressed RT-84 Id, but none from RSP clones did so, and that Id H3 was expressed only by the RT-83 antibody. These results show that human anti-phospholipid MAbs are heterogeneous with respect to phospholipid binding, functional characteristics, and Id expression.

Humoral immune response in a marsupial Monodelphis domestica: Anti-isotypic and anti-idiotypic responses detected by species-specific monoclonal anti-immmunoglobulin reagentS

We examined the humoral immune response of the laboratory opossum ( Monodelphis domestica) as a model marsupial species. To evaluate antibody responses, IgM and IgG preparations were purified from the sera of naïve Monodelphis. These two immunoglobulin (Ig) preparations were used to generate specific murine monoclonal antibodies for use in ELISA-based serology. Individual Monodelphis were then immunized with a multidetenninant protein antigen, a murine monoclonal antibody (Mab) IgG preparation designated Pab 405. In contrast with the primary IgM response of eutherian mammals, the primary response of Monodelphis to mouse IgG involved both IgG and IgM. The specificity of this anti-mouse IgG response appeared isotypic in nature, specifically, the immune sera recognized Ig determinants common to both Pab 405 and a control IgG Mab. Further, to evaluate the antibody responses to mouse IgG, immune sera were adsorbed against a control IgG Mab to remove the anti-isotypic reactivity. The adsorbed Monodelphis sera recognized idiotope specificities expressed on Pab 405. Based on an inhibition ELISA, the anti-idiotype (anti-Id) response recognized an idiotope on Pab 405 associated with its antigen combining site. These results demonstrate that Monodelphis respond to a multidetenninant protein antigen such as murine IgG, similarly but not identically to eutherian mammals, and can serve as a useful marsupial model for additional comparative immunological studies.

Restriction fragment-length polymorphism analysis of the major histocompatibility complex in New Zealand white rabbits.

As a model system, rabbits are particularly useful in transplantation studies because of their size and accessibility. However, inbred rabbits of known MHC haplotype are not commercially available, and there are few monoclonal antibody reagents available to permit serological typing of experimental animals prior to transplant. Here we present a rapid and reliable method to distinguish rabbits that differ at their MHC class I and class II loci, and present 34 class I and 16 class II haplotypes determined by restriction fragment-length polymorphism analysis of outbred heterozygous rabbits. The applicability of this molecular typing system to transplantation experiments in the New Zealand White rabbit is discussed.

Hepatocellular codistribution of c100, c33, c22, and NS5 hepatitis c virus antigens detected by using immunopurified polyclonal spontaneous human antibodies

Hepatitis C virus (HCV) antigens in liver biopsy have been detected by immunohistochemistry using both spontaneous human IgG and murine monoclonal or rabbit polyclonal monospecific reagents. Conflicting results have been obtained in different studies. This was probably because of the incapacity of single experimental antibodies, raised against synthetic or recombinant peptides, to recognize native tissue antigens. To overcome this possibility, we immunopurified monospecific spontaneous polyclonal human Ig, therefore induced by native antigens, from the single antigen-containing bands of RIBA 3 strips. Antibodies to c100, c33, c22, and NS5 antigens were obtained from the serum of a patient affected by chronic hepatitis C. The IgG fraction of this serum had proved to stain tissue HCV antigens. Eight biopsies were selected on the basis of strong hepatocellular reactivity with the whole IgG fraction in a variable number (from 5% to 75%) of cells. The four antigens were detected in all biopsies; a clear cellular codistribution was observed on serial sections. These data demonstrate that the possibility to identify HCV antigens in liver biopsies is higher when using human antibodies induced by native antigens rather then experimental antibodies. The approach of immunopurification of human antibodies can be extended to other HCV-related epitopes to obtain reagents useful for the selection and optimization of monoclonal or polyclonal antibodies.

Hepatocellular codistribution of c100, c33, c22, and NS5 hepatitis C virus antigens detected by using immunopurified polyclonal spontaneous human antibodies

Hepatitis C virus (HCV) antigens in liver biopsy have been detected by immunohistochemistry using both spontaneous human IgG and murine monoclonal or rabbit polyclonal monospecific reagents. Conflicting results have been obtained in different studies. This was probably because of the incapacity of single experimental antibodies, raised against synthetic or recombinant peptides, to recognize native tissue antigens. To overcome this possibility, we immunopurified monospecific spontaneous polyclonal human Ig, therefore induced by native antigens, from the single antigen-containing bands of RIBA 3 strips. Antibodies to c100, c33, c22, and NS5 antigens were obtained from the serum of a patient affected by chronic hepatitis C. The IgG fraction of this serum had proved to stain tissue HCV antigens. Eight biopsies were selected on the basis of strong hepatocellular reactivity with the whole IgG fraction in a variable number (from 5% to 75%) of cells. The four antigens were detected in all biopsies; a clear cellular codistribution was observed on serial sections. These data demonstrate that the possibility to identify HCV antigens in liver biopsies is higher when using human antibodies induced by native antigens rather than experimental antibodies. The approach of immunopurification of human antibodies can be extended to other HCV-related epitopes to obtain reagents useful for the selection and optimization of monoclonal or polyclonal antibodies.

Construction of bacteriophage expressing mouse monoclonal Fab fragments directed against the human MN glycophorin blood group antigens.

The MN human blood group antigens are complex glycopeptide antigens at the amino terminus of glycophorin A. Many different mouse monoclonal antibodies to these antigens have been produced and characterized. The construction of combinatorial immunoglobulin libraries displaying antibody Fab fragments on the surface of bacteriophage (Fab-phage) represents a novel approach for developing monoclonal reagents, for exploring the diversity of the immune response to specific antigens, and for understanding the molecular basis of the interaction of an antibody with its antigen. However, it is necessary to determine whether Fab fragments displayed on bacteriophage surfaces retain immunologic characteristics similar to the intact antibodies. Fab-phage were constructed from three anti-N (AH7, N61, and N92) and two anti-M (425/2B and M2A1) murine hybridomas. The Fab-phage and parental hybridomas were compared by enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. In each case, the Fab-phage and its parental hybridoma antibody had similar immunologic characteristics. In particular, their dependence on the pH of the buffer and on sialylation of the target antigen was similar. These results suggest that Fab-phage may provide novel reagents with applications in immunohematology and may be useful in the study of the immune response to human blood group antigens.

ABO discrepancy with monoclonal ABO reagents caused by a pH-dependent autoantibody

Abstract ABO discrepancy was noted when a patient’s unwashed saline red cell suspension was tested with monoclonal ABO reagents. The discrepancy was resolved when a washed (× 3) saline red cell suspension was used in repeat testing. The patient’s serum contained an autoagglutinin that reacted optimally below pH 7.0. Tire discrepancy occurred when monoclonal ABO reagents, formulated at a low pH, lowered the pH of the reaction mixture, and autoagglutination was observed. Immunohematology 1995;11:71–73.

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes.

Microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of beta-tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated CD4+ and CD8+ T-cell subsets in Wegener's granulomatosis.

Several lines of evidence argue in favour of an involvement of T cells in the pathogenesis of Wegener's granulomatosis (WG). These include the presence of highly specific IgG autoantibodies to proteinase 3, perivascular T-cell infiltrates and elevated amounts of soluble interleukin-2 (IL-2) receptors in patient's serum. In order to further address this question we evaluated by double immunofluorescence and flow cytometry the expression of several cell surface molecules associated with T-cell activation. As compared to healthy controls (n = 15), the CD4+ subset was significantly diminished, while the percentage of CD8+ T cells was elevated in WG patients (n = 24). Within the CD4+ T-cell subset we found a highly significant increase in activation/memory markers (CD25, CD29, HLA-DR). Within the CD8+ T-cell subset the expression of CD11b, CD29 and CD57 was significantly elevated, while the expression of VD28 was reduced. The use of 10 V beta-, 1 V alpha- and 1 V gamma-specific monoclonal reagents failed to reveal any significant bias in the peripheral T-cell receptor V-gene repertoire of WG patients. There was also no correlation between T-cell activation markers and laboratory parameters [C-reactive protein (CRP), ESR], disease duration or therapy. A significant correlation was found only for the degree of organ involvement and the increase in CD4+ T cells coexpressing HLA-DR, as well as the increase in CD57 expression on CD8+ T cells. In conclusion, both CD4+ and CD8+ T-cell subsets were activated in WG. Cytotoxic CD8+CD57+CD11b+CD28- T cells may directly contribute to damage of vascular endothelium.

Development of a radioimmunoassay method for ethynylestradiol in plasma using a monoclonal antibody

An ethynylestradiol (EE 2) radioimmunoassay method (RIA) using a monoclonal antibody (MAB) reagent is described. 76 plasma and 67 buffer blanks spiked with known concentrations of EE 2 were used as positive samples. The steroid was extracted using dichloromethane method with an extraction efficiency of 78 ± 2.6%. The plasma extracts were not purified. Separation of the free from bound tritiated EE 2 was by centrifugation with dextran-coated charcoal suspension in phosphate-gelatin buffer. Within 95% confidence limits, the sensitivity of the assay was 10 pg/ml in buffer samples, and 43 pg/ml in unpurified plasma samples. The accuracy and precision of the method were lower for the plasma samples than for the buffer solutions but within accepted limits (i.e., 80–120% for accuracy, and up to 20% for precision). The mean recovery accuracy ( R) of the method for measuring 38 pg/ml EE 2 in buffer was 101 ± 0.9% ( n = 67), while for 2.39 pg/ml to 18.0 pg/ml EE 2 in plasma was 97 ± 12% ( n = 76). The buffer solutions and plasma samples containing 600 pg/ml EE 2 had a mean R of 102% ( n = 18) and 90% ( n = 16), respectively. The average interassay variation (%CV) between buffer samples was 14 ± 1.2%, and between plasma samples was 19 ± 8%. Plasma samples with 18 pg/ml and 2.39 pg/ml EE 2, together with the blank negative plasma samples showed high interassay variations (CV > 20%). Except for the above samples, however, the precision of the method in all sample groups was within accepted limits. The intra-assay variation of replicate assays of a single plasma sample containing 1.2 ng/ml EE 2 and 4 ng/ml levonorgestrel was 13% ( n = 7). In the presence of very high concentration of closely related levonorgestrel, the R of the method was 106%. The increase in average variability (%CV) between results for the plasma samples (CV 19 ± 8%) over that of the spiked buffer solutions (CV 14 ± 1.2%) suggest some possible interference by co-extracted materials from plasma, for example lipids, which interfered in the ligand-antibody reactions. Further investigation may prove that a pre-assay purification of the sample, e.g., HPLC, would eliminate this interference. Nevertheless, the immunoassay described above completely eliminates the problem of cross-reactivity caused by the natural estrogens in plasma with the conventional polyclonal antibodies. It possesses an acceptable intra- and interassay variation with very high specificity to EE 2. It promises to be clinically useful in the assessment of extremely low levels of EE 2 expected in patients having a daily intake of 30 μg of the estrogenic hormone contained in many oral contraceptives.