Antigenic variation and the murine immune response to Giardia lamblia

The protozoan parasite Giardia lamblia is an important causative agent of acute or chronic diarrhoea in humans and various animals. During infection, the parasite survives the host's reactions by undergoing continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). The VSPs form a unique family of cysteine-rich proteins that are extremely heterogeneous in size. The relevance of antigenic variation for the survival in the host has been most successfully studied by performing experimental infections in a combined mother/offspring mouse system and by using the G. lamblia clone GS/M-83-H7 (human isolate) as model parasite. In-vivo antigenic variation of G. lamblia clone GS/M-83-H7 is characterised by a diversification of the intestinal parasite population into a complex mixture of different variant antigen types. It could be shown that maternally transferred lactogenic anti-VSP IgA antibodies exhibit cytotoxic activity on the Giardia variant-specific trophozoites in suckling mice, and thus express a modulatory function on the proliferative parasite population characteristics. Complementarily, in-vitro as well as in-vivo experiments in adult animals indicated that non-immunological factors such as intestinal proteases may interfere into the process of antigen variation in that they favour proliferation of those variant antigen-type populations which resist the hostile physiological conditions within the intestine. These observations suggest that an interplay between immunological and physiological factors, rather than one of these two factor alone, modulates antigenic diversification of a G. lamblia population within an experimental murine host and thus influences the survival rate and strategy of the parasite.     

In vitro synthesized immunoglobulin A from nu/+ and reconstituted nu/nu mice against a dominant surface antigen of Giardia lamblia

Nu/+ mice (ZU.ICR-strain) experimentally infected with Giardia lamblia (clone GS/M-83-H7) cleared the infection by day 45 postinfection (p.i.). Athymic nu/nu mice were reconstituted with immune Peyer's patch lymphocytes obtained from self-healed nu/+ littermates and thus acquired the potential to decrease their intestinal parasite mass. Intestinal B-cells from self-healed nu/+ mice as well as from immune-reconstituted athymic nude mice synthesized in vitro parasite-specific immunoglobulin A (IgA). This IgA was subsequently analyzed by immunoblotting, showing a predominant reaction with the major surface antigen (a 72,000-Da polypeptide) characterizing the Giardia clone in question. The hypothesis on the causative role of intestinal IgA and immune lymphocytes in the control of G. lamblia infection thus deserves further attention.     

Antigenic variation in Giardia lamblia and the host's immune response

Giardia lamblia, a protozoan parasite of the small intestine of humans and other animals, undergoes surface antigenic variation. The antigens involved belong to a family of variant-specific surface proteins (VSPs), which are unique, cysteine-rich zinc finger proteins. The patterns of infection in humans and animals fail to show the expected cyclical waves of increasing and decreasing numbers of parasites expressing unique VSPs. Nevertheless, changes in VSP expression occur within the population in vivo owing to selection of VSPs by both immune and non-immune mechanisms. After inoculation of a single G. lamblia clone (able to persist in the absence of immune pressure) expressing one VSP (> or = 90%) into mice or humans, the original VSP continues to be expressed until 2 weeks post inoculation (p.i.), when many other VSPs gradually replace it. Selection by immune-mediated processes is suggested because switching occurs at the same time that humoral responses are first detected. In most mouse strains, switching also occurs at about two weeks. Almost all trophozoites are eliminated at three weeks (p.i.), but a barely detectable infection persists over months. In neonatal mice, apparent self-cure is delayed until the sixth or seventh week. Antigenic switching does not occur in adult or neonatal severe combined immunodeficiency disease (SCID) mice, but does occur in neonatal nude mice, thus implicating B-cell-mediated mechanisms in immune switching. Not all VSPs are expressed to the same degree in vivo. Some VSPs appear to be preferentially selected whereas others are eliminated on a non-immune basis. In infections in which immunity does not play a role, such as in SCID mice, and during the first week of infection in immunocompetent mice or gerbils, persisting VSPs are preferentially expressed and maintained whereas non-persisting VSPs are replaced within the first week of infection. The purpose of antigenic variation may be presentation of a wide assortment of VSPs to hosts, increasing the chance of a successful initial infection or reinfection. Immune selection of variants comes into play following biological selection.     

An immobilised peptide array identifies antibodies to a discontinuous epitope in the extracellular domain of the bovine growth hormone receptor

Using an array of overlapping decapeptides representing the extracellular domain of the bovine (b) growth-hormone receptor (GHR) we have mapped the continuous, dominant epitopes defined by five rabbit and one guinea pig polyclonal antisera to recombinant bovine growth-hormone-binding protein (rbGHBP). We report that six major epitopes are identified by these antisera and that these largely occur in areas of non-ordered secondary structure, although there is some contribution from the extensive beta-sheet structure of GHBP. Similar to our previously described studies for growth hormone (GH), we have again found slight differences between animals in the exact location of these epitopes. Using peptide-affinity chromatography we have isolated a population of antibodies reactive with epitope 1 (the N-terminal epitope:GHBP residues 21-38). Analysis of these antibodies by further peptide affinity chromatography and competitive radioimmunoassay experiments indicated cross-reactivity of epitope-1-specific antibodies with epitope 4 (in the interdomain hinge region of the GHBP:residues 111-126). We suggest that, although separate in the primary structure of the molecule, the tertiary fold exhibited by GHBP may bring into close proximity areas of sequence representing epitope 1 and epitope 4 such that they represent a conformational epitope. Under these conditions our experiments indicate that peptides 1 and 4 may represent partial functional epitopes for this antibody population and consequently demonstrate that this approach may be useful in describing discontinuous epitopes.     

Characterization of epitopes in native and unfolded cobrotoxin: evidence of an immunodominant C-terminal region related to the production of precipitating and non-precipitating antibodies against cobrotoxin

Rabbits hyperimmunized with cobrotoxin from Taiwan cobra venom produced non-precipitating as well as precipitating antibodies. Both antibody preparations exhibited higher affinity for native cobrotoxin than for reduced and S-carboxymethylated (RCM) cobrotoxin. This indicated that the epitope structures in cobrotoxin are mostly conformation-dependent. In order to identify the conformational epitopes, native cobrotoxin was hydrolyzed with acid protease A, and 12 peptides were obtained on HPLC. Three peptide fragments, AP-10, AP-11, and AP-12, showed pronounced antigenicities toward precipitating as well as non-precipitating antibodies. AP-10, AP-11, and AP-12 contained a common segment in the C-terminal region of cobrotoxin, residues 43 to 62, with intact disulfide linkages. Complete removal of the C-terminal antibodies from antisera and precipitating antibodies on a C-terminal segment-Sepharose affinity column resulted in the loss of their precipitability with cobrotoxin, whilst restoration of precipitability was observed on the addition of the C-terminal antibodies to the C-terminal antibody-depleted antisera and precipitating antibodies. Studies on the antigenic structures of RCM-cobrotoxin revealed that RCM-cobrotoxin contains an immunodominant epitope at positions 22-38. The N-terminal and C-terminal regions of RCM-cobrotoxin encompass other epitopes which exhibit low reactivities toward anti-RCM-cobrotoxin antibodies. However, no precipitated antigen-antibody complexes were observed with the mixture of anti-RCM-cobrotoxin antibodies and RCM-cobrotoxin. These results suggest that the inherently different immunogenicities with different segments might affect the precipitabilities of the resulting antibodies, and that the notable immunogenecity of the C-terminal region is related to the production of precipitating and non-precipitating antibodies against cobrotoxin.     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (II). Epitope mapping by monoclonal antibodies

PSP94 has shown potential to be a serum biomarker for evaluating prostate cancer. Studies of the epitope structure is crucial for this endeavour. In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with the results obtained from our previous work using polyclonal antibody and recombinant PSP94. Firstly, we determined the relative activities of the 15 MAb population by direct and competitive ELISA. The two predominant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further studies of the epitope structure. By comparing the binding activities of recombinant GST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 was shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody. Using recombinant GST fusion protein with PSP94 and with each half of the N- and C-terminal 47 amino acids (GST-PSP-N47/C47) in E. coli cells, the different epitopes recognized by 15 monoclonal antibodies were delineated and the polar distribution of the epitope structure of PSP94 was characterized. Results of direct ELISA of recombinant N47 and C47 and their competitive binding against natural PSP94 (competitive ELISA) showed that the N- and C-termini represent the immuno-dominant and immuno-recessive area separately. A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein. Using GST-PSP-N47 as a standard protein, an epitope map of the 15 monoclonal antibodies was obtained. The results of this study will help to define the clinical utility of PSP94.     

Monomer arrangement in HSP90 dimer as determined by decoration with N and C-terminal region specific antibodies

Electron microscopy using the low-angle rotary shadowing replica method showed that the HSP90 dimer consists of four globular domains aligning in a tandem fashion. When decorated with two monoclonal antibodies against epitopes mapped on the N-terminal region of HSP90, these antibodies bound to both ends of the HSP90 dimer. A C-terminal region specific antibody was shown to bind to the side of HSP90. These results support a model for HSP90 dimer whereby two HSP90 monomers are arranged in an antiparallel fashion and dimerize through the C-terminal domain. Treatment of HSP90 at elevated temperatures or with ATP at room temperature, though not with ADP, induces molecular transformation of the linear HSP90 dimer into an O-ring-shaped structure.     

Induction of differential T-helper-cell responses in mice infected with variants of the parasitic nematode Trichuris muris

The resistance or susceptibility of mice to infection with the intestinal nematode parasite Trichuris muris is closely correlated with polarization of T helper (Th) cell responses to the type 2 (Th2) or type 1 (Th1) subset. Comparison of infections with three isolates of T. muris (E/K, E/N, and S) in three inbred strains of mice (CBA, C57BL/10, and B10.BR) has shown that host Th response phenotype can be parasite determined. Although the mouse strains used show genetically determined variation in ability to respond to T. muris (CBA > C57BL/10 > B10.BR), the speed of worm expulsion in a given strain depended upon the isolate used for infection (E/K > E/N > S). The two isolates that induced the most effective resistance (E/K and E/N) elicited parasite-specific host antibody responses that were dominated by immunoglobulin G1 (IgG1), and antigen-stimulated T cells from infected mice released interleukin-5 in vitro. With the isolate that induced the least host resistance (S), the dominant antibody response was IgG2a, and T cells released gamma interferon in vitro. These data show clearly that parasite variant-specific factors play a major role in Th subset polarization during infection.     

Passive immunization with antibodies against three distinct epitopes on Plasmodium yoelii merozoite surface protein 1 suppresses parasitemia

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an approximately 42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142 is further cleaved to an N-terminal approximately 33-kDa polypeptide (MSP-133) and a C-terminal approximately 19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142 but not with either of the constituents MSP-133 and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, and B6, B10, F5, and G3 bound to MSP-119. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.     

An investigation of the high-avidity antibody response to glycoprotein 120 of human immunodeficiency virus type 1

The avidity of antibodies for antigens can be measured by determining what remains bound after exposing the antibody-antigen complex to a chaotropic agent such as urea. This method has been gaining popularity for assessing the immune response to the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120 (or its counterpart from simian immunodeficiency virus), during natural infection or after subunit vaccination. High-avidity antibodies have been considered to be a possible correlate of protection. We have examined the avidity assay to determine what it, in fact, measures. First, we studied the development of the anti-gp120 response in seroconverting individuals. Urea elution reduced the polyclonal anti-gp120 titers by 3- to 10-fold. After allowing for the consequent reduction in assay sensitivity, there was no obvious change in the rate of development of the high-avidity and unfractionated antibody responses. Furthermore, in the one individual who developed a strong autologous, virus-neutralizing response, the appearance of neutralizing antibodies and high-avidity antibodies did not coincide. Antibodies to the V3 loop, when present, comprised a major fraction of the polyclonal response that survives urea elution. We next examined the effect of urea elution on the binding to gp120 of a panel of monoclonal antibodies (MAbs). Urea treatment preferentially eluted MAbs to discontinuous rather than continuous epitopes, independent of their affinities. Furthermore, these patterns of epitope stability were unaltered by the presence of polyclonal anti-gp120 antibodies. As most broadly neutralizing anti-gp120 antibodies recognize discontinuous epitopes, this skewing effect must be taken into account when interpreting studies using polyclonal sera.     

Perceived importance of clinical teaching characteristics for nurse anesthesia clinical faculty

Cancer-related, mucin-type carbohydrate epitopes, principally mannose and sialo-syl residues, are expressed on the envelope protein gp 160 of the human immunodeficiency virus (HIV). Anticarbohydrate antibodies directed toward these and other carbohydrate epitopes are known to neutralize HIV-1 infection by cell-free virus. Carbohydrates, however, being T cell-independent antigens, typically elicit diminished immune responses. To overcome this potential draw back, we have examined the ability of peptides that mimic such epitopes to elicit immune responses that cross-react with carbohydrate structures. We report that mouse polyclonal antisera generated against peptides that mimic mucin-related carbohydrate epitopes have anti-HIV-1 activity. Generation of antibodies was not lr-gene restricted, as at least two different strains of mice. Balb/c (H-2d) and C57Bl/6 (H-2b), responded equally to the peptides. The antipeptide sera displayed neutralizing activity against HIV-I/MN and HIV-I/3B viral strains. This neutralization was as good as human anti-HIV sera. These results indicate that peptide mimics of carbohydrates provide a novel strategy for the further development of reagents that elicit immune responses to carbohydrate epitopes associated with many infectious organisms and tumor cells.     

The platelet-stimulating effect of adrenaline through alpha 2-adrenergic receptors requires simultaneous activation by a true stimulatory platelet agonist. Evidence that adrenaline per se does not induce human platelet activation in vitro

In the murine model for EAMG we investigated the relation between disease susceptibility and fine specificity of anti-AChR antibodies obtained from high susceptible C57Bl/6 and low susceptible BALB/c mice after immunization with Torpedo acetylcholine receptor (tAChR). Anti-AChR MoAbs with fine specificity for the main immunogenic region (MIR), the alpha-bungarotoxin (alpha-BT)/acetylcholine binding sites and other extra- and intracellular epitopes were isolated from both mouse strains. In total, nine out of 38 MoAbs obtained from C57Bl/6 mice were directed against extracellular epitopes on mouse AChR in contrast to only one out of 27 MoAbs from BALB/c mice. A difference in antibody repertoire may underlie the difference in pathogenic response observed between these mouse strains. These results indicate that strain-specific differences in disease susceptibility in murine EAMG may be related to differences in the available repertoire of potential pathogenic antibodies.     

Role of magnetic resonance in hemodialysis patients with amyloid shoulder arthropathy]

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.     

Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys

We have shown that conserved B epitopes were immunodominant in animals hyperimmunized with parasite-purified or recombinant merozoite surface protein MSP1 of Plasmodium falciparum. Cross-priming studies also suggested that a conserved T-helper epitope(s) is efficient in inducing the anti-MSP1 antibody response. In this study, we determined whether a similar profile of immune responses was induced during live P. falciparum infections. Naive Aotus monkeys were infected by blood-stage challenge with either one of the two dimorphic MSP1 alleles represented by the FUP and FVO parasites. Sera collected after parasite clearance were analyzed by enzyme-linked immunosorbent assays (ELISAs). Monkeys infected with parasites carrying one allelic form of MSP1 had antibodies that were equally reactive with homologous or heterologous MSP1s. This preferential recognition of conserved epitopes of MSP1 was confirmed by competitive binding ELISAs. Studies with Plasmodium yoelii and P. falciparum show that the C-terminal 19-kDa fragment of MSP1, MSP1(19), is the target of protective immunity. Thus, monkey sera were assayed for recognition with recombinant MSP1(19)s expressing variant and conserved B epitopes. Results of direct and competitive binding ELISAs showed that the anti-MSP1(19) antibodies were also directed primarily against conserved determinants. The similarities between vaccine- or infection-induced antibody responses suggest a possible reciprocal enhancement of the two populations of anti-MSP1 antibodies when a subunit MSP1 vaccine is introduced into populations living in areas where malaria is endemic. This together with previous observations that conserved determinants are important in MSP1-mediated immunity provides an optimistic outlook that a subunit MSP1 vaccine may be effective and practical for field applications in malaria-exposed populations.     

A method for rapid and complete substitution of the circulating erythrocytes in SCID mice with bovine erythrocytes and use of the substituted mice for bovine hemoprotozoa infections

We have previously developed an in vivo experimental system for a bovine hemoprotozoan parasite, in which SCID mice were periodically transfused with bovine red blood cells (Bo-RBCs), followed by infection with the parasite. The SCID mice prepared by the original method, however, had both mouse and bovine RBCs in the circulation, and their proportion always fluctuated significantly. In the present study, we aimed to deplete the mouse RBCs circulating in SCID mice and, thereby, to create SCID mice having complete Bo-RBC substitution. An anti-erythropoietin rabbit serum, an anti-mouse RBC rabbit serum and 23 monoclonal anti-mouse RBC rat antibodies were prepared for this purpose. They were examined, after administration into SCID mice, for their ability to decrease hematocrit value and also for any other adverse effect. A monoclonal antibody, clone 2E11, was found to have potent ability to induce clearance of the mouse RBCs in SCID mice without causing toxic effects. SCID mice receiving this antibody together with periodic transfusion of Bo-RBCs had their circulating RBCs completely substituted with Bo-RBCs. Infection of Bo-RBC-SCID mice with bovine hemoprotozoan parasites demonstrated that elimination of the mouse RBCs from Bo-RBC-SCID mice resulted in augmentation of parasite growth.     

Adsorption-induced antigenic changes and their significance in ELISA and immunological disorders

The functional properties of 125I-labeled antibodies and antigens adsorbed on polystyrene and silicone were compared to their counterparts immobilized by non-adsorptive methods. Less than 20% of polyclonal (pAb) and 1-2% of monoclonal (mAb) capture antibody equivalents remained functional after adsorption as a monolayer. Survivability circa doubled or was totally rescued, when the same antibodies were immobilized via a streptavidin bridge or by using a first stage polyclonal antiglobulin capture antibody, respectively. Similarly, the antigenicity of bovine IgGs for pAb and mAb anti-IgGs was highest when the IgGs were immobilized via a streptavidin bridge or when secondarily adsorbed to an albumin monolayer. IgGs in these configurations were significantly more antigenic than when directly adsorbed on polystyrene or a silicone elastomer. Similar activity was seen after adsorption on polystyrene or silicone. Interestingly, these IgGs were equally antigenic when denatured and subsequently adsorbed in 6M guanidine-HCl versus adsorption in PBS without prior denaturation. Although many of the above finding on antibodies and antigens could be explained by the greater accessibility of antigenic epitopes or antibody binding sites when molecules are immobilized by some type of underlying molecular layer, we also show that certain mAb and pAbs preferentially recognized allotopes on IgG2a when IgG2a was adsorbed. Furthermore, such antigenicity was highest when IgG2a was adsorbed at low, sub-monolayer concentrations. Finally, we show that differences in antigenicity need not be related to the method of immobilization, but can also result from differences in the microenvironment of the epitope. This was demonstrated using a filamentous phage clone specific for fluorescein (FLU). This clone recognizes the fluorescein hapten differently depending on the carrier protein used and the method of conjugation. Data presented in this report indicate that antibodies and antigens adsorbed on hydrophobic polymers undergo changes in their functional properties. Data suggest that both changes in conformation and the accessibility of antigen epitopes or antibody binding sites, most likely occur. Especially in the case of the latter, the functional concentration may be 1-2 orders of magnitude lower than the antibody protein concentration. These observations have implications for immunodiagnostics and emphasize the need to determine the specificity of an antibody in the assay in which it is employed and to make no assumptions about the behavior of solid-phase antigens and antibodies from their behavior in solution. Our studies are also relevant to the use of silicone medical prostheses. The antigenicity of IgGs adsorbed on silicone as a multilayer (secondary layer) is much higher than when directly adsorbed. Since such surfaces would be exposed to very high protein concentrations in vivo, multilayers not a monolayer, would be expected. Thus it would seem from these studies that host protein adsorbed on silicone would be expressed to the immune system at the surface of multilayers. This being the case, it seems unlikely that the adsorption of host protein in vivo would generate new epitopes against which the host's immune system could respond and subsequently initiate an autoimmune syndrome.     

Characterisation of Australian ovine adenovirus isolates

The influenza virus haemagglutinin has an important role in the infectious cycle of the virus and carries multiple B and T cell epitopes. It is synthesized as a single polypeptide chain but viral infectivity depends on its post-translational enzymatic cleavage. The cleavage site of a trypsin-like enzyme responsible for this modification is found in the most conserved intersubunit region of the molecule. In this study the role of this region in antibody recognition was investigated. Synthetic peptides comprising the intact and cleaved forms of the intersubunit segment were used to examine the specificity of virus- or peptide-induced antibodies. The immune response elicited by viral infection resulted in the appearance of antibodies capable of neutralizing the virus without interfering with its binding to the receptor. A monoclonal antibody (MoAb) of such functional properties was shown to recognize the intact intersubunit region both in the uncleaved haemagglutinin molecule and in a 25-mer synthetic peptide comprising the intact intersubunit region. Specificity and functional studies revealed the conformation-dependent recognition of the C-terminal segment of the haemagglutinin 1 subunit by this MoAb. The binding of the antibody was shown to inhibit the trypsin-mediated cleavage of the haemagglutinin molecule and the membrane fusion event. The enzymatic cleavage of the haemagglutinin was demonstrated to abolish antibody recognition of the infective virus suggesting an escape mechanism mediated by the functional destruction of this highly conserved region. The synthetic peptide corresponding to the intact intersubunit region is characterized by an ordered structure and is able to elicit an antibody response in BALB/c mice while its subfragments are nonimmunogenic. Furthermore, this peptide elicited a protective immune response demonstrated by in vivo experiments.     

p53 immunolabeling in archival paraffin-embedded tissues: optimal protocol based on microwave heating for eight antibodies on lung carcinomas

The prognostic value of p53 gene mutations is dealt with by several recent reports. However, retrospective assessment of p53 tumor status on archived samples has been prevented by p53 epitope alteration during routine fixation and embedding procedures. This study aimed at establishing a reproducible low-cost protocol to retrieve not only N-terminal, but also midregion and C-terminal, epitopes, with special attention to possible artifacts induced by epitope retrieval procedures. Using microwave heating, we compared the epitope retrieval efficiency of five solutions with eight commercial antibodies on 21 lung carcinomas for which frozen tissue and samples fixed with formalin and Bouin's liquid were available. All eight epitopes were retrieved, citrate buffer proving efficient for seven. PAb 240 epitope was restored by target unmasking fluid only. No false positivity was observed. Fixation-induced loss of p53 immunoreactivity was minimal for formalin (two of 10 tumors for one antibody each), more significant for Bouin (six of 10 tumors for one to five antibodies). On the other hand, staining intensity was maintained or even improved, and nonspecific staining reduced, through fixation. We conclude that p53 stabilization can be detected on routinely processed archival tumor samples with a reliability similar to that of frozen tissue by means of a microwave-based procedure and a panel of at least three antibodies, with epitopes on the N-terminal, C-terminal, and midpart of the molecule.     

Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies

A majority of monoclonal antibodies (mAbs) raised against soluble oligomeric human immunodeficiency virus type 1 isolate IIIB (HIV-1IIIB) envelope (env) glycoprotein reacted with conformational epitopes within the gp120 or gp41 subunits. Of 35 mAbs directed against gp41, 21 preferentially reacted with oligomeric env. A subset of these mAbs reacted only with env oligomers (oligomer-specific mAbs). In contrast, only 1 of 27 mAbs directed against the gp120 subunit reacted more strongly with env oligomers than with monomers, and none were oligomer-specific. However, 50% of anti-gp120 mAbs preferentially recognized monomeric env, suggesting that some epitopes in gp120 are partially masked or altered by intersubunit contacts in the native env oligomer. Two mAbs to oligomer-dependent epitopes in gp41 neutralized HIV-1IIIB and HIV-1SF2, and binding of these mAbs to env was blocked by preincubation with HIV-1-positive human serum. Thus, immunization with soluble, oligomeric env elicits antibodies to conserved, conformational epitopes including a newly defined class of neutralizing antibodies that bind to oligomer-specific epitopes in gp41, and may also minimize the production of antibodies that preferentially react with monomeric env protein.