Epitope-specific Monoclonal Antibodies Research Articles

Addition of a c-myc epitope tag within the VEGF protein does not affect in vitro biological activity

The overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in diseases involving neovascularization. VEGF exists in as many as six different isoforms, each showing a unique pattern of tissue distribution and activity. To investigate the effect of individual VEGF isoform overexpression in neovascular disease models, we inserted c-myc epitope tags into the three VEGF isoforms expressed in retinal pigment epithelial cells, VEGF121, VEGF165, and VEGF189. We found that the 12-amino acid insertion between the receptor binding and heparin binding domains did not affect VEGF transcription, translation, or secretion. In addition, VEGF isoforms containing the c-myc epitope tag were able to stimulate endothelial cell proliferation as efficiently as non-tagged VEGF isoforms and they could be individually identified by Western blotting and immunocytochemistry using the c-myc epitope specific monoclonal antibody 9E10.Key words: Vascular endothelial growth factor, VEGF, c-myc epitope tag, immunocytochemistry.

Addition of a c-myc epitope tag within the VEGF protein does not affect in vitro biological activity

The overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in diseases involving neovascularization. VEGF exists in as many as six different isoforms, each showing a unique pattern of tissue distribution and activity. To investigate the effect of individual VEGF isoform overexpression in neovascular disease models, we inserted c-myc epitope tags into the three VEGF isoforms expressed in retinal pigment epithelial cells, VEGF121, VEGF165, and VEGF189. We found that the 12-amino acid insertion between the receptor binding and heparin binding domains did not affect VEGF transcription, translation, or secretion. In addition, VEGF isoforms containing the c-myc epitope tag were able to stimulate endothelial cell proliferation as efficiently as non-tagged VEGF isoforms and they could be individually identified by Western blotting and immunocytochemistry using the c-myc epitope specific monoclonal antibody 9E10.

Phosphocholine-containing, zwitterionic glycosphingolipids of adult Onchocerca volvulus as highly conserved antigenic structures of parasitic nematodes

Human Onchocerca volvulus infection sera were found to recognize zwitterionic glycolipids of O. volvulus and to cross-react with those of other parasitic nematodes (Ascaris suum, Setaria digitata and Litomosoides sigmodontis). By the use of an epitope-specific monoclonal antibody, zwitterionic glycolipids of all these nematode species were observed to contain the antigenic determinant phosphocholine. A hyperimmune serum specific for arthro-series glycolipid structures reacted with the various neutral glycolipids of all these nematodes, which demonstrated that their oligosaccharide moieties belonged to the arthro-series of protostomial glycolipids. These results indicated that arthro-series glycosphingolipids carrying, in part, phosphocholine substituents, represent highly conserved, antigenic glycolipid markers of parasitic nematodes. Three glycolipid components of the O. volvulus zwitterionic fraction were structurally characterized by matrix-assisted laser-desorption/ionization time-of-flight MS, methylation analysis and exoglycosidase treatment. Their chemical structures were elucidated to be phosphocholine-6GlcNAc(beta1-3)Man(beta1-4)Glc(1-1)ceramide, GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta1-4)Glc(1-1) ceramide and Gal(alpha1-3)GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta 1-4)Glc(1-1)ceramide for the zwitterionic ceramide tri-, tetra- and penta-hexosides respectively. The ceramide composition was found to be dominated by 2-hydroxylated docosanoic (C(22h:0)), tricosanoic (C(23h:0)) and tetracosanoic (C(24h:0)) acids, and C(17) sphingosine (C(d17:1)) (where (h) is hydroxylated and (d) is dihydroxylated).

Characterization and formulation of multiple epitope-specific neutralizing monoclonal antibodies for passive immunization against cryptosporidiosis.

The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [alphaCSL]), 3H2 (alphaGP25-200), and 1E10 (alphaP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.

Nucleocapsid Formation and/or Subsequent Conformational Change of Rabies Virus Nucleoprotein (N) Is a Prerequisite Step for Acquiring the Phosphatase-Sensitive Epitope of Monoclonal Antibody 5-2-26

We investigated the antigenic maturation of rabies virus N protein, for which we used some conformational epitope-specific monoclonal antibodies (MAbs) and an MAb (5-2-26) against a phosphorylation-dependent linear epitope. Infected cells were lysed with a deoxycholate-free lysis buffer and separated by ultracentrifugation into the soluble top and the nucleocapsid fractions. None of the study MAbs recognized N proteins in the top fraction, whereas nucleocapsid-associated N proteins were recognized by all of the MAbs. Immunoprecipitation with polyclonal anti-N antibodies coprecipitated the P proteins from the top fraction, indicating that soluble N proteins are mostly associated with the P protein. The N proteins dissociated from both the N–P complex and nucleocapsids were recognized by none of the study MAbs, whereas the MAb 5-2-6 recognized the SDS-denatured N proteins of the nucleocapsid but not of the top fraction. In addition, the phosphorylation-deficient mutant N proteins were shown to be similarly accumulated as the wild-type N proteins into the viral inclusion bodies, defined as the virus-specific structures composed of viral nucleocapsids, that are produced in the cytoplasm of the infected cells. Based on these results, we believe that newly synthesized N proteins are not immediately phosphorylated at serine-389 (a common phosphorylation site) but are first associated with the P protein. After being used for encapsidation of the viral RNA, the N proteins undergo conformational changes, whereby epitopes for the conformation-specific MAbs are formed and become phosphorylated at serine-389.

Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection.

The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, E1, E2/NS1, NS2, NS3, NS4, and NS5 regions. The U. S. Food and Drug Administration-licensed 2.0G immunoassay for the detection of anti-HCV uses proteins from the core, NS3, and NS4 regions (McHutchinson et al., Hepatology 15:19-25, 1992). The 3.0G enzyme-linked immunosorbent assay includes the protein from the NS5 region (Uyttendaele et al., Vox Sang. 66:122-129, 1994). The necessity of detecting antibodies to viral envelope proteins (E1 and E2) and to different genotype samples has been demonstrated previously (Chien et al., Lancet 342:933, 1993; Lok et al., Hepatology 18:497-502, 1993). In this study we have attempted to improve the sensitivity of the anti-HCV assay by developing a single multiple-epitope fusion antigen (MEFA; MEFA-6) which incorporates all of the major immunodominant epitopes from the seven functional regions of the HCV genome. A nucleic acid sequence consisting of proteins from the viral core, E1, E2, NS3, NS4, and NS5 regions and different subtype-specific regions of the NS4 region was constructed, cloned, and expressed in yeast. The epitopes present on this antigen can be detected by epitope-specific monoclonal and polyclonal antibodies. In a competition assay, the MEFA-6 protein competed with 83 to 96% of genotype-specific antibodies from HCV genotype-specific peptides. This recombinant antigen was subsequently used to design an anti-HCV chemiluminescent immunoassay. We designed our assay using a monoclonal anti-human immunoglobulin G antibody bound to the solid phase. Because MEFA-6 is fused with human superoxide dismutase (h-SOD), we used an anti-human superoxide dismutase, dimethyl acridinium ester-labeled monoclonal antibody for detection. Our results indicate that MEFA-6 exposes all of the major immunogenic epitopes. Its excellent sensitivity and specificity for the detection of clinical seroconversion are demonstrated by this assay.

Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis.

Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.

Studies on the structures and antigenic properties of rabies virus glycoprotein analogues produced in yeast cells

We investigated two forms (designated as yGI and yGII) of rabies virus glycoprotein (G) analogues produced in the G cDNA-transfected yeast cells. Molecular weights of yGI and yGII were estimated as 66 and 56 kDa, respectively, according to their relative mobility in SDS-PAGE. Although being produced in large amounts, yGI was present mostly in insoluble forms and hardly extractable with non-ionic detergents. The yGI reacted with polyclonal anti-G antibodies, but did not react with our conformational epitope-specific anti-G monoclonal antibodies (G-MAbs). No protective immunity was induced by yGI in guinea pigs nor in mice. On the other hand, yGII was Triton-soluble, but was only small in amount (at most 1% of total G proteins) and was shown to lack the cytoplasmic domain. The yGII, however, reacted with the G-MAbs and induced protective immunity in guinea pigs as well. When the G-cDNA was expressed in animal cells in culture, a single form (about 66 kDa) of G protein was produced, which displayed similar behaviors as seen in its reactivity with the MAbs and intracellular distribution as seen in the virus-infected cells. These results suggest that most G protein molecules were not processed normally in yeast cells, resulting in abnormal folding and multimer formation, while only a small fraction were occasionally folded normally to have conformational epitopes but were mostly deprived of the C-terminal portion.

The αEC Domains of Human Fibrinogen420Contain Calcium Binding Sites But Lack Polymerization Pockets

AbstractThe extended α (αE) isoform unique to Fibrinogen420 (Fib420) is distinguished from the conventional α chain of Fibrinogen340 by the presence of an additional 236-residue carboxyl terminus globular domain (αEC). A recombinant form of αEC (rαEC), having a predicted mass of 27,653 Daltons, was expressed in yeast (Pichia pastoris) and purified by anion exchange column chromatography. Purified rαEC appears to be predominantly intact, as judged by N-terminal sequence analysis, mass spectral analysis of the C-terminal cyanogen bromide (CNBr) fragment, and comparison of recognition by epitope-specific monoclonal antibodies. Carbohydrate determination, coupled with analysis of CNBr digestion fragments, confirms N-linked glycosylation at Asn667, the site at which sugar is attached in αE. Analysis of CNBr digestion fragments confirms that two disulfide bridges exist at cysteine pairs αE613/644 and αE780/793. In the presence of 5 mmol/L EDTA, rαEC is highly susceptible to plasmic degradation, but Ca2+ (5 mmol/L) renders rαEC resistant. No protective effect from plasmic degradation was conferred to rαEC by the peptides GPRPamide or GHRP, nor did rαEC bind to a GPR peptide column. These results suggest that the αEC domain contains a calcium-binding site, but lacks a polymerization pocket. By analogy with the site elucidated in the γC domain, we predict that the αEC calcium binding site involves residues αE772-778: DADQWEE.

The EC Domains of Human Fibrinogen420Contain Calcium Binding Sites But Lack Polymerization Pockets

The extended (E) isoform unique to Fibrinogen420 (Fib420) is distinguished from the conventional chain of Fibrinogen340 by the presence of an additional 236-residue carboxyl terminus globular domain (EC). A recombinant form of EC (rEC), having a predicted mass of 27,653 Daltons, was expressed in yeast (Pichia pastoris) and purified by anion exchange column chromatography. Purified rEC appears to be predominantly intact, as judged by N-terminal sequence analysis, mass spectral analysis of the C-terminal cyanogen bromide (CNBr) fragment, and comparison of recognition by epitope-specific monoclonal antibodies. Carbohydrate determination, coupled with analysis of CNBr digestion fragments, confirms N-linked glycosylation at Asn667, the site at which sugar is attached in E. Analysis of CNBr digestion fragments confirms that two disulfide bridges exist at cysteine pairs E613/644 and E780/793. In the presence of 5 mmol/L EDTA, rEC is highly susceptible to plasmic degradation, but Ca2+ (5 mmol/L) renders rEC resistant. No protective effect from plasmic degradation was conferred to rEC by the peptides GPRPamide or GHRP, nor did rEC bind to a GPR peptide column. These results suggest that the EC domain contains a calcium-binding site, but lacks a polymerization pocket. By analogy with the site elucidated in the gammaC domain, we predict that the EC calcium binding site involves residues E772-778: DADQWEE.

Recombinant antibodies with conformationally constrained HIV type 1 epitope inserts elicit glycoprotein 160-specific antibody responses in vivo.

Although neutralizing epitopes have been identified on the HIV-1 gp120/gp41 envelope complex, efforts to exploit this information through the construction of synthetic peptide vaccines have been largely unsuccessful. Unfortunately, synthetic peptides tend to be poorly immunogenic, and most often lack the conformational characteristics of the corresponding epitope in the native protein. In an effort to circumvent these difficulties, we have utilized an anti-class II MHC antibody as a molecular scaffold for the construction of two conformationally constrained neutralizing HIV-1 epitopes. Previously we demonstrated that anti-class II MHC antibodies can function as vectors for the induction of adjuvant-independent antibody responses to incorporated epitopes. In this instance, one epitope, IHIGPGRAFYT, is the crown of the V3 loop from gp120, and the other, ELDKWAS, is a neutralizing epitope from gp41. The insertion of these epitopes into a specific loop region of the immunoglobulin heavy chain FR3 was found to preserve the anti-class II MHC-binding activity of these recombinant antibodies, and the inserts were recognized by epitope specific monoclonal antibodies. When utilized as immunogens, each of these epitope insertion antibodies was able to induce high-titer anti-HIV-1 gp160 responses in guinea pigs. These responses were conformation specific in that the anti-gp160 binding was not inhibited by the synthetic peptide corresponding to the epitope in question. These data demonstrate the potential to construct conformationally constrained HIV-1 epitope immunogens, and thus establish an alternative approach to the design of an effective HIV-1 subunit vaccine.

An assessment of the effects of cadaver donor bone marrow on kidney allograft recipient blood cell chimerism by a novel technique combining PCR and flow cytometry.

A new technique, the PCR-flow assay is described that has allowed for the serial identification and quantitation of discrete mononuclear cell subsets of donor (or recipient) bone marrow derived cells in cadaver kidney transplant recipients infused postoperatively with donor vertebral body bone marrow cells. With fixed permeabilized cells in flow cytometry the amplification power of the polymerase chain reaction (PCR), using fluorescent-labeled primers to identify single copy HLA class II DRbeta1 genes of either donor or recipient origin, is combined with multi-color fluorochrome-labeled CD epitope-specific monoclonal antibodies. The details of the methodology are described; these support the utility of the assay. Initial observations were made on the chimeric makeup of the peripheral blood as well as iliac crest bone marrow between six months and one year posttransplantation in recipients serially followed weekly and then monthly, concomitantly compared with a control group of stable kidney transplant recipients using similar therapeutic protocols, who did not receive cadaver bone marrow. Several findings are of note. In 14 recipients of two bone marrow infusions totalling a mean of 6.29+/-2.18x10(10) cells, donor CD34 positive (+) (immature) cells were fourteen times as numerous in peripheral blood six months postoperatively as in six recipients given half as many bone marrow cells in one infusion (averaging 3.02+/-0.5x10(10)). These donor CD34+ cells unexpectedly averaged 36+/-7% of the total (donor plus recipient) CD34+ subset counted. Moreover, iliac crest bone marrow aspirates contained an average of thirteen times this number of CD34+ cells than in the peripheral blood, supporting the notion of engraftment. Of additional interest, between six months and one year posttransplant although no donor cells could be detected in peripheral blood of the controls there was an identifiable presence of donor CD34+ cells in their iliac crest bone marrow, albeit 10-fold less than the marrow-infused patients. In the clinical follow-up, although there were three unrelated mortalities, there were no additional kidney losses with current serum creatinine concentrations averaging 1.3+/-0.06 mg/dl. In conclusion, the PCR-flow assay presents the possibility of identifying discrete subsets of donor or recipient cells that may have an immunoregulatory function.

A simple solid-phase competition assay with labeled antigen.

The determination of epitope specificities of monoclonal antibodies (MAbs) has usually been performed using the competitive solid-phase assay in which the antigen is immobilized, and a radiolabeled antibody and competing unlabeled antibodies are mixed in solution (Fig. 1A) (,). Although this method facilitates separation of free from bound antibody, it possesses the problem of labeling all antibodies to be tested. Since the number of MAbs to be screened is usually large, this method is time consuming and tedious, and the instability of radiolabels represents a significant drawback. In addition, radioactive hazards have to be taken into account. Recently, nonisotopic tracers, such as biotin (,) and fluorescein isothiocyanate (), have been introduced for determination of epitope specificities of MAbs, but these methods still have the problem of labeling all antibodies to be tested. Open image in new window Fig. 1. Diagrammatic representation of a conventional competition immunoassay with labeled antibody (A) and a new type of competition immunoassay with labeled antigen (B) used in epitope mapping. In the competition assay with labeled antibody, the antigen is immobilized, and a radiolabeled antibody as well as competing unlabeled antibodies are mixed in solution. Finally, the radiolabeled antibody bound to the immobilized antigen is detected in a γ-counter. Recently, biotinylated antibody is often used instead of radiolabels. On the other hand, in the competition assay with labeled antigen, a constant amount of biotinylated antigen is incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated antigen bound to the immobilized antibody are then reacted with avidin-peroxidase conjugate and the activity of the bound peroxidase is determined.

Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP.

The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutageneses. Nine of the 13 linker mutant genes expressed protein at levels comparable to those obtained with the wild-type gene. These mutant proteins were shown, by indirect immunofluorescence with an OprP-specific antiserum, to be properly exposed at the cell surface. Four of the linker mutant genes expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutant genes. Seven of the resultant epitope insertion mutant genes expressed surface-exposed protein. Two of these mutant genes presented the foreign epitope at surface-accessible regions as assessed by indirect immunofluorescence with a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer.

Apolipoprotein B and E basic amino acid clusters influence low-density lipoprotein association with lipoprotein lipase anchored to the subendothelial matrix.

Lipoprotein accumulation in the subendothelial matrix is an important step in atherogenesis. We have previously shown that addition of lipoprotein lipase (LPL) markedly increased binding of apolipoprotein B (apoB)-containing lipoproteins to an endothelial cell-derived matrix, and this enhanced lipoprotein binding was inhibited by apoE. In the present studies we examined the role of various regions of apoB in the binding of LDL to LPL-containing endothelial cell matrix and the ability of various apoE domains to decrease lipoprotein retention. We studied three apoB epitope-specific monoclonal antibodies for their ability to block the binding of 125I-LDL to LPL-containing matrix. Of these, monoclonal antibody 4G3, which recognizes an arginine-containing epitope in apoB, was the most effective in reducing LDL binding. Chemical modification of LDL apoB lysines or arginines markedly reduced the ability of the lipoprotein to block the binding of 125I-LDL to LPL-containing matrix, suggesting that apoB positively charged amino acids are involved in the interaction. Furthermore, polyarginine or polylysine markedly decreased 125I-LDL binding to LPL-containing matrix, whereas polyleucine was ineffective. These data suggest that apoB positively charged regions are important in LDL binding. To explore the role of charge modifications on apoE by single arginine-cysteine interchanges, we examined the effects of the three major human apoE isoforms (apoE2, apoE3, and apoE4). ApoE3 was the most effective in decreasing 125I-LDL retention, followed by apoE4; apoE2 was the least effective. Similarly, apoE2-containing HDL was much less effective than apoE3-containing HDL in decreasing 125I-LDL retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective allergic reactions to clavulanic acid

Clavulanic acid (CL) is a [Mactam antibiotic with weak antibacterial activity, but it is a potent inhibitor of [3-1actamases. In association with amoxycillin (AX) it has great antimicrobial activity and is increasingly prescribed in daily medical practice. We present two cases of selective adverse reactions to CL with good tolerance to other [3-1actam antibiotics in which a type I immunologic mechanism is probably implicated. To the best of our knowledge, these are the first reported cases of selective allergy to CL.

Tau phosphorylation in human, primate, and rat brain: evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro.

The extent of tau phosphorylation is thought to regulate the binding of tau to microtubules: Highly phosphorylated tau does not bind to tubules, whereas dephosphorylated tau can bind to microtubules. It is interesting that the extent of tau phosphorylation in vivo has not been accurately determined. Tau was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that tau isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated tau was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of tau were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of tau in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive tau was present as slow-mobility (66- and 68-kDa) forms of tau. The percentage of immunoreactive tau in faster-mobility pools (42-54 kDa) increased in proportion to the decrease in content of 66-68-kDa tau as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on tau is rapid and depends on neuronal phosphatases. Furthermore, tau is highly phosphorylated in normal-appearing human, primate, and rodent brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Rh phenotype on the antigen density of C, c, and D: flow cytometric study using a frozen standard red cell.

Flow cytometry is increasingly being used for the comparison of antigen density. Indirect immune fluorescence is more sensitive than direct immune fluorescence and thus allows the study of red cells (RBCs) with a weak D antigen. In indirect immune fluorescence, when the fluorescence is standardized by the use of aliquoted frozen standard RBCs, the coefficient of variation in fluorescence intensity was less than 5 percent, which allows accurate determination of minor variations of Rh antigen density. For D antigen, the well-known suppressive effect of C, and the low antigen density of the weak D phenotype, was demonstrated. Use of epitope-specific monoclonal antibodies yielded similar results and allowed the identification of a D category IV heterozygote; the relative antigen density measured with a monoclonal antibody that reacted with D(IV) was twice that measured with a monoclonal antibody that did not react with D(IV). RBCs from C and c homozygotes had significantly more antigen than those from heterozygotes. There was significant variation in antigen density, depending on Rh phenotype: for example, D+ RBCs had less C antigen than D- RBCs, and Rh:1,2,-3,4,5 (CcDee) RBCs had more c antigen than Rh:1,2,3,4,5 (CcDEe) RBCs. There was no difference in D, C, and c antigen density in neonatal and adult RBCs. Flow cytometry is an excellent tool for the demonstration of minor differences in antigen density.

European collaborative study of luteinizing hormone assay: 1. Epitope specificity of luteinizing hormone monoclonal antibodies and surface mapping of pituitary and urinary luteinizing hormone

This report describes the results of the first part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The whole study was designed to understand the causes of discrepancy among LH immunoassay methods. In the parent work, we studied the characteristics of 55 monoclonal antibodies to LH which allowed us to establish a detailed map of the antigenic surface of the hormone. In the present report we used this information to interpret the discrepancy in LH concentrations assayed with 12 different methods in 300 sera from subjects with various clinical conditions. The 55 monoclonal antibodies provided by 11 commercial companies were tested in various experiments: the apparent affinity of the antibodies was, generally but not always, higher for LH presented by a second antibody coated to plastic than for LH directly coated to plastic; 26% of the antibodies recognized the alpha subunit, 26% the beta subunit and 48% reacted only with the holomolecule (anti-alpha beta); only 28% of the antibodies were strictly specific for LH. Criss-cross experiments allowed us to distinguish 13 antigenic regions on the surface of LH: 6 were located on the alpha subunit, 3 on the beta subunit and 4 on the holomolecule. The monoclonal antibodies to the alpha beta regions further separated into 12 clusters of reactivity. Accordingly, LH appeared to exhibit at least 21 epitopes. Comparison of the immunoreactivity of various LH preparations indicated that highly purified pituitary LH and immunoaffinity purified urinary LH reacted similarly with the monoclonal antibodies and strongly differed from crude urinary LH. These data indicated that the immunoreactivity of an LH preparation depends mainly upon the degree of purification and not that much upon the origin of the preparation. The epitope specificity of the monoclonal antibodies used in 11 commercially available LH assay kits was also determined: 10 kits used at least one anti-alpha beta monoclonal antibody associated with an anti-beta monoclonal antibody in 7 cases or another anti-alpha beta monoclonal antibody in 3 cases; one kit used an anti-alpha monoclonal antibody associated with an anti-beta monoclonal antibody. None of the kits were strictly identical with regards to the epitope specificity of the monoclonal antibodies used.

Differentiation of pathogenic Entamoeba histolytica infections from nonpathogenic infections by detection of galactose-inhibitable adherence protein antigen in sera and feces.

We determined whether epitope-specific monoclonal antibodies to the galactose-inhibitable adherence protein (GIAP) of Entamoeba histolytica could be used in an enzyme-linked immunosorbent assay (ELISA) to detect antigen in serum and feces and differentiate between nonpathogenic zymodemes and the potentially invasive pathogenic organisms that require treatment. Overall, 57% of subjects from Cairo, Egypt, with symptomatic intestinal amebiasis and 42% with asymptomatic infection possessed GIAP antigen in their sera, whereas 4% of uninfected controls or subjects with other parasitic infections possessed GIAP antigen in their sera (P < 0.001). In subjects from Durban, South Africa, only 6% of uninfected controls or those with nonpathogenic E. histolytica infection were positive for GIAP in serum, whereas 3 of 4 with asymptomatic pathogenic intestinal infection and 75% with amebic liver abscess were positive for GIAP in serum. Fifteen stool samples from patients with intestinal amebiasis were available for study; all had a positive ELISA result for fecal GIAP antigen. Epitope-specific monoclonal antibodies identified 8 of 15 subjects with fecal antigen from pathogenic strains. Seven of those eight subjects had adherence protein antigen in their sera, whereas none of seven with apparent nonpathogenic E. histolytica infection had adherence protein antigen in their sera. In summary, we were able to detect E. histolytica adherence protein antigen directly in serum and fecal samples by ELISA. The presence of amebic antigen in serum demonstrated 94% specificity for pathogenic E. histolytica infection, and amebic antigen is present during asymptomatic intestinal infection. In conjunction with antibody detection, this method should be very useful in the diagnosis and management of intestinal amebiasis.