The impact of symbolism on immunogenetics: an application to HLA

The genes coding for the class I human lymphocyte antigens (HLA) are located on chromosome 6. These antigens are involved with the immunological interaction between cells. In some immunogenetic systems, such as HLA in humans, genes are defined by antibody/antigen reaction and are denoted by single symbolic identifiers. This symbolization assumes a one-to-one correspondence between antibodies, antigens and genes. Recent molecular studies, however, suggest that HLA antibody/antigen reaction is complex and most HLA class I specific antibodies may not uniquely identify a single allelic product. Where cross-reactivity is present in an immunogenetic system it is important to label each reagent with symbols corresponding to all genes coding for antigens with which the reagent will react. The problems of cross-reactive groups and unexplained linkage relations may be elucidated by the redefinition and clarification of certain HLA antigens. A computer program can suggest such labelling schemes using input given by phenotype reaction patterns with a panel of reagents. When this program was applied to data on the class I HLA antigens a genetic model was suggested that differs somewhat from the currently accepted model. The new model is able to predict what would appear as linkage relations in the accepted model. Our methodology can provide alternate models to guide in typing cloned genes in terms of the HLA locus and alleles.     

Analyzing antibody specificity with whole proteome microarrays

Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins. Furthermore, new applications such as antibody arrays and monoclonal antibody therapeutics have increased the demand for more specific antibodies to reduce cross-reactivity and side effects. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity, because it allows the simultaneous screening of thousands of proteins for possible cross-reactivity. As an initial test of this approach, we screened 11 polyclonal and monoclonal antibodies to approximately 5,000 different yeast proteins deposited on a glass slide and found that, in addition to recognizing their cognate proteins, the antibodies cross-reacted with other yeast proteins to varying degrees. Some of the interactions of the antibodies with noncognate proteins could be deduced by alignment of the primary amino acid sequences of the antigens and cross-reactive proteins; however, these interactions could not be predicted a priori. Our findings show that proteome array technology has potential to improve antibody design and selection for applications in both medicine and research.     

Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.     

Immunocytochemistry of brain-reactive monoclonal antibodies in peripheral tissues

Among a total of 135 tissue-reactive monoclonal antibodies previously prepared, 81 were brain-selective and were classified into neuronal and non-neuronal categories. The neuronal antibodies were again subdivided into antineurofibrillar, antiperikaryonal-neurofibrillar, and antisynapse-associated groups. On the basis of morphologic, developmental, biochemical, and pathologic criteria, the antibodies in at least two of these groups were found to detect heterogeneous antigens (called "neurotypes") rather than different antigenic determinants in single antigens. On examining the distribution in peripheral organs of staining patterns of 11 antineuronal brain-reactive antibodies, we now confirm that these antibodies are, indeed, largely brain-specific. In general, non-neuronal elements in liver, lung, heart, thymus, intestine, adrenal, and spleen remained unstained. However, most of the antibodies stained peripheral neural elements. Occasional antibodies did stain selected, non-neuronal structures. Four out of five antineurofibrillar antibodies stained nerve fibers in adrenal medulla, intestine and thymus. All of three antiperikaryonal-neurofibrillar antibodies also stained nerve fibers in the adrenal medulla, but not in other organs. Two out of three antisynapse-associated antibodies stained what appear to be nerve contacts on adrenal medullary cells, but not on any other peripheral cells examined. The non-neuronal peripheral staining patterns were restricted to selective nuclear staining exhibited by two out of five antineurofibrillar antibodies and the staining of macrophage and selected cardiac muscle nuclei by two of three antisynapse-associated antibodies. However, one antineurofibrillar antibody also stained the cytoplasm of selected liver cells. Among non-neuronally reacting antibodies, two antibodies stained nuclei of all cells except neurons in brain as well as peripheral organs. An antibody staining the ciliary epithelium of choroid plexus also stained basal bodies of ciliated bronchial epithelium. The overall data suggest that the specificity of brain-reactive antibodies is high and that their cross-reactivity with epitopes in non-nervous tissue is rare. In these cases, the antibodies seem to provide specific reagents for these additional structures as well as for their specific brain antigens.     

Parallel sets and the internal image of antigen within the idiotypic network

Antibodies carrying the internal image of antigens and antibodies with different specificities sharing idiotypes, which were called parallel sets, have been looked on as mere curiosities or odd by-products of antibody diversity. New findings supporting the existence of these antibodies in various experimental systems were presented. In addition, molecular studies of parallel sets in an arsonate system suggested that the genes encoding for these antibodies originate from germ line genes but use a different JH segment. The functions of these antibodies within the idiotype network were discussed.     

Comparing genomes with duplications: a computational complexity point of view

In this paper, we are interested in the computational complexity of computing (dis)similarity measures between two genomes when they contain duplicated genes or genomic markers, a problem that happens frequently when comparing whole nuclear genomes. Recently, several methods ( [1], [2]) have been proposed that are based on two steps to compute a given (dis)similarity measure M between two genomes G_1 and G_2: first, one establishes a oneto- one correspondence between genes of G_1 and genes of G_2 ; second, once this correspondence is established, it defines explicitly a permutation and it is then possible to quantify their similarity using classical measures defined for permutations, like the number of breakpoints. Hence these methods rely on two elements: a way to establish a one-to-one correspondence between genes of a pair of genomes, and a (dis)similarity measure for permutations. The problem is then, given a (dis)similarity measure for permutations, to compute a correspondence that defines an optimal permutation for this measure. We are interested here in two models to compute a one-to-one correspondence: the exemplar model, where all but one copy are deleted in both genomes for each gene family, and the matching model, that computes a maximal correspondence for each gene family. We show that for these two models, and for three (dis)similarity measures on permutations, namely the number of common intervals, the maximum adjacency disruption (MAD) number and the summed adjacency disruption (SAD) number, the problem of computing an optimal correspondence is NP-complete, and even APXhard for the MAD number and SAD number.     

Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays

In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems.     

Isotype-specific rabbit antibodies against chinchilla immunoglobulins G, M, and A.

Chinchillas have become a preferred animal model for studying otitis media, and are also useful in studying insulin release, gastrin physiology, intestinal infection, and hepatocellular pathophysiology. Immunopathologic studies in the model, however, have been limited by absence of specific antibody reagents against chinchilla immunoglobulins. We describe a method for preparing isotype-specific rabbit antibodies against the heavy-chain components of chinchilla immunoglobulins G, M, and A. Chromatographic techniques were used to isolate chinchilla immunoglobulins from serum and breast milk; heavy-chain fractions were isolated and used as antigens to produce isotype-specific antibodies in New Zealand White rabbits. Enzyme-linked immunosorbent assay of these antisera disclosed anti-light chain cross-reactivity, which was removed by affinity chromatography. The isolation and affinity purification techniques were highly reproducible. The availability of these reagents should greatly enhance the utility of the chinchilla in modeling human disease.     

A restricted human antitetanus clonotype shares idiotypic cross-reactivity with tetanus antibodies from most human donors and rabbits: reactivity with antibodies of widely differing electrophoretic mobility

Antitetanus antibodies from each of 20 hyperimmunized human donors were isolated on a tetanus immunoadsorbent, eluted with acidic buffer, and examined by isoelectric focusing (IEF). There was electrophoretic restriction, as determined by IEF, in the IgG of only 20% of the purified antibodies studied. The remaining 80% showed a more diffuse polyclonal spectrotype. Several IEF bands from the most electrophoretically restricted sample were isolated and used to immunize rabbits. Virtually every IgG-IEF band in the antitetanus antibodies of the original donor shared idiotypic cross-reactivity as detected by one or more of the three rabbit anti-Id reagents, even though major qualitative differences in binding from one rabbit anti-Id reagent to another were noted. Antitetanus antibodies of each of the 20 donors were separated by IEF and transferred to a nitrocellulose membrane. By using a sensitive and specific ELISA detection method, cross-reactivity was detected with the rabbit anti-Id reagents in 1 to 50% of the antitetanus antibodies of individual donors. This cross-reactivity was greater than 10% in 15 of the 19 antisera studied. In addition, these cross-reactive antibodies had very different electrophoretic mobility. Binding of the rabbit anti-Id reagents to the tetanus antibodies was almost completely blocked by pretreatment with soluble tetanus toxoid antigen. This idiotypic cross-reactivity with antibodies of different electrophoretic mobility from the same and unrelated donors suggests sharing among these antibodies of one or more of the germ-line DNA-encoded hypervariable regions present in the antibody-combining site.     

Antibody-induced association between discrete regions of HLA class I and II antigens

The anti-HLA-DR + DP monoclonal antibody (MoAb) CR11-462 was unexpectedly found to cross-inhibit the binding to B lymphoid cells of the anti-HLA Class I MoAb CR10-215 and CR11-115. The latter two antibodies recognized the same or spatially close antigenic determinant. The cross-blocking of anti-HLA Class I MoAb CR10-215 and CR11-115 by MoAb CR11-462 reflects neither its contamination by anti-HLA Class I antibodies nor its cross-reactivity with HLA Class I antigens. On the other hand, the cross-blocking appears to reflect redistribution of HLA Class II antigens by the MoAb CR11-462, since the MoAb CR10-215 and CR11-115 are not susceptible to blocking when lymphoid cells are treated with 0.025% glutaraldehyde or are coated with Fab' fragments of the MoAb CR11-462. Furthermore, immunoprecipitates from B lymphoid cells preincubated with the MoAb CR11-462 before solubilization contain HLA Class I antigens. Therefore, these results have shown for the first time an antibody-induced association between discrete regions of HLA Class I and Class II antigens on the membrane of B lymphoid cells.     

The epitope space of the human proteome

In the post-genome era, there is a great need for protein-specific affinity reagents to explore the human proteome. Antibodies are suitable as reagents, but generation of antibodies with low cross-reactivity to other human proteins requires careful selection of antigens. Here we show the results from a proteome-wide effort to map linear epitopes based on uniqueness relative to the entire human proteome. The analysis was based on a sliding window sequence similarity search using short windows (8, 10, and 12 amino acid residues). A comparison of exact string matching (Hamming distance) and a heuristic method (BLAST) was performed, showing that the heuristic method combined with a grid strategy allows for whole proteome analysis with high accuracy and feasible run times. The analysis shows that it is possible to find unique antigens for a majority of the human proteins, with relatively strict rules involving low sequence identity of the possible linear epitopes. The implications for human antibody-based proteomics efforts are discussed.     

Identification of a 75 kDa highly immunodominant antigen from Mycobacterium smegmatis and cross-reactivity with other species

Three monospecific antibodies MSAb 1, MSAb 2 and MSAb 3 were raised in BALB/C mice against respective antigens. M. smegmatis whole cell lysate was first separated on SDS-PAGE and randomly chosen bands were cut and then used for immunization. Antibodies were collected as ascites by injecting mice with myeloma cell line P3X63 Ag 658.4. All the three antibodies showed high reactivity with denatured antigens compared to native. Different extent of cross-reactivity was observed as evident from ELISA. MSAb1 recognized a 75 kDa immunodominant antigen from M. smegmatis and 66 kDa from M. tuberculosis (H37Ra), respectively. An apparently similar molecular weight antigen shown to be present in M. tuberculosis (H37Ra) an avirulent strain and BCG, but not recognized by MSAb1. The 75 kDa antigen has a stimulatory effect on T-cell proliferation.     

Heterogeneity of primary and metastatic human malignant melanoma as detected with monoclonal mntibodies in cryostat sections of biopsies

Summary Monoclonal antibodies were generated against established melanoma cell lines and characterized by their reactivity with various sublines. The antibodies selected for their reaction with melanoma-associated antigens were tested on cryostat sections of melanoma tissue from various stages and on other tumors. The reactivity with normal tissues was also determined. Of 30 antibodies reacting with melanoma cell lines 11 did not react with melanoma biopsies. Of the remaining 19 antibodies nine displayed broad cross-reactivity with normal cells and structures and other benign or malignant tumor cells. Among the remaining antibodies five types were defined that detected antigens (nevocellular I, nevocellular II, neural, endothelial, basal cell) found on certain normal tissues and structures and on certain tumor phenotypes. Even though there seems to be a tendency for some antigens to be preferentially associated with certain stages of melanoma, it has not yet been possible to establish any clear-cut correlation between the expression of one of the differentiation antigens and a particular stage or malignancy potential of melanoma.     

Antigenic Mimicry Between Helicobacter pylori and Gastric Mucosa: Failure to Implicate Heat-Shock Protein Hsp60 Using Immunoelectron Microscopy

Background. Helicobacter pylori infection induces autoantibodies that cross-react with human gastric mucosa from infected individuals. Candidates for the antigens responsible for molecular mimicry causing autoreactivity include the heat-shock protein HspB (Hsp60, sometimes called Hsp54) or Lewis x and Lewis y carbohydrate antigens.
Objective. Our goal was to investigate the involvement of HspB (Hsp60) in autoreactivity between H. pylori and gastric biopsy tissue.
Materials and Methods. Immunoelectron microscopy was used to study cross-reactivity among biopsy tissues from a patient with gastritis, gastric ulcer, and duodenal ulcer and his own serum as well as reactivity with serum raised against HspB from H. pylori and monoclonal antibodies against Lewis antigens.
Results. The patient serum reacted with gastric mucosa, and the antibodies involved were predominantly IgG. Antibody raised to H. pylori HspB (Hsp60) reacted only with H. pylori cells but not with gastric mucosal tissue. In contrast, monoclonal antibodies specific for Lewis x and Lewis y antigens reacted with both H. pylori and human gastric epithelial tissue.
Conclusions. Hsp60 (Hsp54) is unlikely to be involved in autoreactivity seen in individuals infected with H. pylori. In contrast, we could not rule out the role of Lewis x and Lewis y carbohydrate antigens, expressed as a component of H. pylori lipopolysaccharides, in molecular mimicry and autoantibody production.     

Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever

Abstract Sera from patients suffering from Mediterranean spotted fever (i.e. an infection due to Rickettsia conorii ) were studied by immunoblot to investigate cross-reactivity. A prevalence of IgM antibodies to Proteus OX 19, Proteus 0X 2, to the Rickettsia typhus group, to Legionella pneumophila serovars 4 and 5, to L. bozemanii Wiga and to L. micdadei Tatlock was found. Western blot confirmed that the antibodies were directed against the lipopolysaccharide as demonstrated by proteinase K digestion of the antigens. Cross-adsorptions showed that there is a common cross-reacting epitope among L. bozemanii Wiga, R. typhi and Proteus OX 19 but cross-reacting antibodies to L. micdadei and OX 2 were distinct and independent. This IgM cross-reaction could lead to a misdiagnosis.     

Phenotypic variations in blood and milk of the Somali camel

132 blood samples and 54 milk samples obtained from Somali camel were analysed for red blood cell antigens with the cattle reagents and for Hb, Ca, X proteins, Tf, Alb, Am, SOD, alpha-La, beta-Lg and casein systems respectively. Positive lytic reactions were obtained with the anti-B, -Q, -Q', -W, -F1 and -J reagents. No biochemical polymorphism was observed except for Hb, X protein and beta-Lg systems.     

Phenotypic variations in blood and milk of the Somali camel*

132 blood samples and 54 milk samples obtained from Somali camel were analysed for red blood cell antigens with the cattle reagents and for Hb, Ca, X proteins, Tf, Alb, Am, SOD, α-La, β-Lg and casein systems respectively. Positive lytic reactions were obtained with the anti-B, -Q, -Q, -W, -F₁ and -J reagents. No biochemical polymorphism was observed except for Hb, X protein and β-Lg systems.     

Do X and Y spermatozoa differ in proteins?

This article reviews the current knowledge about X- and Y-chromosomal gene expression during spermatogenesis and possible differences between X- and Y-chromosome-bearing spermatozoa (X and Y sperm) in relation to whether an immunological method of separation of X and Y spermatozoa might some day be feasible. Recent studies demonstrated that X- and Y-chromosome-bearing spermatids do express X- and Y-chromosomal genes that might theoretically result in protein differences between X and Y sperm. Most, if not all, of these gene products, however, are expected to be shared among X and Y spermatids via intercellular bridges. Studies on aberrant mouse strains indicate that complete sharing might not occur for all gene products. This keeps open the possibility that X and Y sperm may differ in proteins, but until now, this has not been confirmed by comparative studies between flow-cytometrically sorted X and Y sperm for H-Y antigen or other membrane proteins.     

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.