Interspecies Cross-reactivity Research Articles

RAPID PURIFICATION OF ISLETS USING MAGNETIC MICROSPHERES COATED WITH ANTI-ACINAR CELL MONOCLONAL ANTIBODIES

A simple, rapid method of islet purification is important in large-scale human islet isolation. We have previously identified monoclonal antibodies specific for acinar cells, but not islets, and described an immunologic method of purification by selective lysis of the acinar cells. An attractive alternative to lysis of the acinar cell is depletion by a magnetic immunomicrosphere technique. We report in this study a rapid, reproducible method of rat islet purification utilizing magnetic microspheres coated with acinar-cell-specific monoclonal antibodies. Pancreatic digestion with collagenase followed by depletion of acinar cells with the magnetic immunomicrospheres (MIMS) yields large numbers of intact islets. We compared the islets thus obtained with hand-picked (HP) islets (control) for yield, purity, in vitro insulin secretory capacities, and in vivo functional viability. The islet yield with the MIMS method (n = 35) was 72.7% that obtained with the HP method (n = 6) (378 +/- 8 vs. 519 +/- 31 islets per pancreas). The purity of the MIMS-isolated islets was 84 +/- 1.9%, ranging from 75-95%. Static glucose stimulation showed excellent function (2-3-fold increase of insulin release over basal levels) with no statistical difference in insulin secretion between MIMS and HP islets. Under microscopic examination, both groups revealed a well-preserved structure with healthy endocrine cells. When 1321 +/- 59 MIMS islets were transplanted into streptozotocin-induced diabetic rats (n = 10), normoglycemia (less than 200 mg/dl) was restored in all recipients following transplantation, and 100% of them remained normoglycemic on day 120 postgrafting. In summary, a rapid, consistent, and simple method of isolating viable, purified rat islets is described. The broad interspecies crossreactivity of the McAb suggests that this technique may be generally useful for islet purification in large mammalia, including man.

Studies on catecholamine-synthesizing enzymes

The purification and characterization of three enzymes involved in the biosynthesis of catecholamines is reviewed. Polyclonal antibodies against native tyrosine hydroxylase (TH) were raised in rabbits, and these antibodies inhibit TH activity. Polyclonal antibodies were also raised in rabbits against a segment of TH (TH-16) and these antibodies stimulate enzymatic activity. In addition, mouse monoclonal antibodies were raised but exerted no significant effect on enzymatic activity. The three types of antibodies were used for immunohistochemical localization of dopamine neurons in the CNS. Studies on activation of TH revealed that phosphorylation of the enzyme by protein kinases plays an important role in the short-term regulation of the enzyme activity. Dopamine ?-hydroxylase (DBH) was purified from human and rat pheochromocytoma tumors, and polyclonal antibodies against human and rat DBH were raised in rabbits. Enzyme inhibition studies with the corresponding antibodies revealed poor interspecies crossreactivity. Phenylethanolamine N-methyl-transferase (PNMT) was purified from bovine and rat adrenal glands. Polyclonal antibodies raised against rat adrenal PNMT were used for immunohistochemical localization of PNMT-containing neurons. The presence of PNMT-containing cell bodies in two clusters, termed C1 and C2, were found; ascending and descending projections were demonstrated.

Serological studies of peptostreptococci using an indirect fluorescent antibody test.

Members of the genus Peptostreptococcus are frequent isolates from periodontal lesions. A study was undertaken for determination of the serological relationships of oral and non-oral strains of several species of this genus. Eighty-nine strains of peptostreptococci representing seven species were tested by means of an indirect fluorescent antibody technique (IFA), with rabbit antisera to Peptostreptococcus anaerobius ATCC 27337, Peptostreptococcus micros VPI 2618-A, and Peptostreptococcus productus ATCC 27340. Each antiserum showed positive fluorescence when reacted with homologous cells. When anti-P. micros VPI 2618-A serum was added to suspensions of P. micros ATCC 33270 and clinical isolates of P. micros in the IFA, positive fluorescence was observed to a titer of 1:64 with 26 out of 29 strains. Positive fluorescence was also seen when P. anaerobius VPI 5737 and clinical isolates of P. anaerobius were tested with anti-P. anaerobius ATCC 27337 serum; a titer of at least 1:64 was observed in 10 out of 14 strains. These results support the presence of two serological groups of P. anaerobius, rabbit anti-P. anaerobius ATCC 27337 serum reacting with Group II organisms. No interspecies cross-reactivity was observed except with four strains of Peptostreptococcus magnus, which reacted with several antisera as well as with normal rabbit serum and the saline controls. These results indicate that rabbit antisera and the use of an IFA are useful in the identification of P. anaerobius and P. micros from oral lesions.

Inter-species cross-reactivity of human sperm antibodies demonstrated by immunoenzyme method

Inter-species cross-reactivity of human sperm antibodies demonstrated by immunoenzyme method

An Immunohistochemical Study of Canine Tissues with Vimentin, Desmin, Glial Fibrillary Acidic Protein, and Neurofilament Antisera

In a wide range of canine tissues the immunoreactivity with commercially available antisera against intermediate filament antigens viz. vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilament proteins, was studied. In addition, the results of formalin and Carnoy fixation were compared. Carnoy fixation appeared to result in optimal reactivity for all antisera. Epithelial cells did not react with any of the antisera, with exception of ovarian surface epithelium, which showed staining with the vimentin and desmin antisera. The vimentin antiserum induced staining of several cell types viz. fibroblasts, endothelial cells, chondrocytes, Schwann cells, ependymal cells, astrocytes, Leydig cells, synovial cells, podocytes and some parietal cells of Bowman's capsule. Sertoli cells showed a faint staining reaction. Muscle cells in various tissues reacted with the desmin antiserum. In the kidney a varying number of parietal cells appeared to react as did a restricted number of epithelial cells of proximal tubules and loops of Henle. GFAP reactivity was confined to glial cells, predominantly fibrous astrocytes, Schwann cells and axons. Additionally, some neuronal cell bodies in peripheral ganglia showed staining of varying intensity. Neurofilament staining was restricted to axons and some neurons. The immunoreactivity of canine tissues with these antisera is compared to findings in other species. The results confirm a broad interspecies cross-reactivity of these antisera. They can be used in studying the nature of canine tissues.

Characterization and binding properties of fatty acid-binding proteins from human, pig, and rat heart

Fatty acid-binding proteins (FABPs) were isolated from the cytosols of hearts of man, pig, and rat by gel filtration and anion-exchange chromatography. The heart FABPs had a M rmr of about 15,000 (pig, rat) and 15,500 (man); p I values were 5.2, 4.9, and 5.0 for human, pig, and rat heart, respectively. In contrast to liver FABPs, tryptophan was present in the heart FABPs. Binding characteristics for long-chain fatty acids determined with the radiochemical Lipidex assay were comparable for all three proteins. Heart FABPs also bind palmitoyl-CoA and -carnitine with an affinity comparable to that for palmitic acid. Other ligands investigated, heme, bilirubin, cholesterol, retinoids, and prostaglandins, could not compete with oleic acid for binding by human heart FABP. Binding parameters of FABP for oleic acid from multilamellar liposomes were comparable to those from the Lipidex binding assay. Immunological interspecies crossreactivity with antisera against the heart FABPs was much higher between man and pig than between rat and man or pig. None of the antisera reacted with liver FABPs. The IgG fraction of anti-human heart FABP serum inhibited fatty acid binding to human heart FABP.

Identification of a human glial fibrillary acidic protein cDNA: a tool for the molecular analysis of reactive gliosis in the mammalian central nervous system.

Two clones encoding human glial fibrillary acidic protein (GFAP) were isolated from a human astrocytoma cDNA library. The clones pHGFAP1 and pHGFAP2 were selected by the combined use of differential colony hybridization and hybridization-selection technique with polyclonal anti GFAP antiserum. The longer one, pHGFAP1, encompasses 3.0 kb and includes the 1.8 kb long 3' untranslated region specific to the human mRNA. Sequence data disclosed an extensive homology within the coding region of human and mouse GFAP cDNAs even in the end domains. Blot hybridization analysis of RNAs from human, rat and mouse brain revealed a single GFAP mRNA species of 3.1, 2.8 and 2.7 kb respectively and Southern blot experiments indicated that this mRNA is most probably transcribed from a unique gene. In situ hybridization performed with biotinylated probes on cultured mouse brain cells suggests both the sorting and the transport of GFAP mRNA throughout the cytoplasm and processes of the astrocytes. As a model of reactive gliosis secondary to degenerative disorders, 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra in the rat was performed. GFAP mRNA increased 1.4 fold in the ipsilateral striatum on day 10 after the lesion. It then declined to the control level 4 months later contrasting with the lower and more sustained increase in preproenkephalin (PPE) mRNA. The interspecies cross-reactivity of the HGFAP probes make them useful as a tool for the molecular analysis of reactive gliosis in various experimental models.

Probing the Receptor-Interaction of Glycoprotein Hormones with Monoclonal Antibodies

Having recently analyzed with monoclonal antibodies (MCA) the immunologic surface of human chorionic gonadotropin (hCG) as consisting of 9 distinct epitopes exposed on the molecule in a characteristic topographical manner (Schwarz, S., Berger, P., and Wick, G., Endocrinology 118, 189-197, 1986) we now attempted to confirm this result on a more general basis, e.g. by incorporating MCA that were just recently obtained and previously not included. What we were, however, interested most was the question whether the tentative model of the epitope map of hCG could represent a "target" with which hCG-related hormones such as LH, FSH, and TSH (the family of glycoprotein hormones, GPH) would (partially) match. Indeed, repeating various immunizations with GPH of human as well as animal origin revealed a remarkable reproducibility in terms of several anticipated epitope specificities of MCA. This indicates that MCA can be regarded as reliable probes for mapping epitopes and, as we have presumed, of receptor interaction domains of GPH as well. Extending the originally used 2-site MCA binding exclusion approach by an interspecies crossreactivity (Xr) analysis we now are able to refine our epitope model of hCG such that 2 additional epitopes were found which were not previously resolvable. Most surprisingly, two of 5 epitopes on the alpha subunit were now also detected on various non-human GPH, which is in striking contrast to a seemingly well established dogma. Yet all five alpha epitopes of hCG are present on hLH, hFSH, and hTSH as well and arranged in the same spatial relationship to each other as on hCG. Even the 2 conformational epitopes and their close topographical relationship to the alpha-epitopes appear to be remarkably conserved on all human GPH. Among the beta-epitopes we have found one that is not shared by hLH and that - surprisingly - is not the C-terminal peptide (CTP) by which hLH differs from hCG. On the basis of this refined epitope map a way was paved along which it should be feasible to elucidate the sterical relationship of the epitopes to the receptor interaction domain(s) of hCG. To this end the MCA were tested in principally two ways: first, as to which of the 11 MCA with different epitope specificities would be able (or not) to inhibit by preincubation the binding of radiolabeled hCG (or hLH, respectively) to rat testis LH/hCG receptors? Secondly (and inversely) which of the 11 epitopes of hCG would still be accessible to binding by radiolabeled MCA when the (unlabeled) hormone is bound to the receptor?(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibodies against human, bovine and rat prolactin: epitope mapping of human prolactin and development of a two-site immunoradiometric assay

Nine mouse hybridoma cell lines producing monoclonal antibodies (MCA) against human prolactin (hPRL), 19 cell lines against bovine prolactin (bPRL) and one MCA against rat prolactin (rPRL) were established. The MCA were characterized by one- and two-site radioimmunoassays (RIA) as well as indirect immunofluorescence (IIF) and used for epitope mapping of hPRL and immunoradiometric assays (IRMA). Interspecies cross-reactivity studies by RIA revealed two groups of anti-hPRL MCA: seven which reacted only with hPRL and two additionally recognizing bPRL and ovine prolactin (oPRL). The anti-bPRL MCA, which were tested on pituitary sections by IIF could be divided into 17 MCA cross-reacting with the closely related oPRL, and two MCA which showed additional cross-reactions with equine prolactin. The anti-rPRL antibody reacted exclusively with rPRL in direct binding RIA studies. No intraspecies cross-reactions with the closely related protein hormones placental lactogen and GH were detected. To elucidate the antigenic surface of hPRL all MCA directed against hPRL were then used for two-site epitope mapping studies in which pairs of MCA were assessed for simultaneous reaction with the same antigen. The native hormone was incubated with the first, solid-phase bound, so called 'capture MCA', and this complex treated with a second, 125I-labelled 'detection MCA'. Based on the results of these combinations, at least three sterically non-overlapping and (taking RIA cross-reaction studies into consideration) two additional epitopes could be defined.(ABSTRACT TRUNCATED AT 250 WORDS)

The human immune response to reconstituted bovine collagen.

The human immune response to bovine dermal collagen was characterized through histologic, serologic, and immunoblotting methods. Collagen-sensitive patients were identified by hypersensitivity to intradermal exposure to ZYDERM Collagen Implant--a pepsin-solubilized, reconstituted, bovine dermal collagen. Biopsies of test sites in the forearm were obtained from several collagen-sensitive patients. Histologic examination revealed an implant-associated palisading foreign body granuloma. The lesion also contained a mixed cell infiltrate of histiocytes, lymphocytes, and eosinophils. Sera were collected from patients who developed erythema or induration at intradermal test or treatment sites, and were evaluated for antibodies to bovine dermal collagen by an enzyme-linked immunosorbent assay (ELISA). Sera with anti-collagen antibodies were further characterized in this study. The circulating antibodies were reactive with both native and heat-denatured bovine dermal collagen. By using purified alpha 1(I) and alpha 2(I) polypeptides, these sera were found to have antibodies reactive with both alpha-chains. Each alpha-chain was fragmented by using cyanogen bromide (CB). The CB peptides were electrophoretically separated, and these sera were evaluated for antibodies to the major fragments by using an immunoblotting technique. Of the sera evaluated by this method, 89% (23/26) had antibodies to alpha 1-CB6; 77% (20/26) had antibodies to alpha 2-CB4; and 65% (17/26) had antibodies reactive with both CB fragments. In addition, most sera (77%) contained antibodies reactive with two or more (up to five) of the major CB peptides. The least antigenic fragment was alpha 2-CB3,5 (8%). In addition, these sera had antibody activity against both native and heat-denaturated bovine types III and II collagens. Little or no interspecies (rat or guinea pig) cross-reactivity (types I and II) was detected. Furthermore, these sera did not have antibodies against human types I, II, and III collagens.

Inter-species cross-reactivity of the prothoracicotropic hormone of Manduca sexta and egg-development neurosecretory hormone of Aedes aegypti

The cross-reactivities of the big and small forms of prothoracicotropic hormone (PTTH) from pupal brains of Manduca sexta and egg-development neurosecretory hormone (EDNH) from heads of adult Aedes aegypti were examined for PTTH by the in vitro Manduca prothoracic gland assay and for EDNH by the in vitro and in vivo Aedes ovary assays. The synthesis of ecdysone by both larval and pupal prothoracic glands of Manduca was increased in a dose-dependent manner by crude extracts of Aedes aegypti heads, reaching a maximum of approx. 3- and 2-fold, respectively. Gel filtration chromatography of the Aedes head extract revealed a peak of EDNH activity with an apparent mol. wt of 11 kD. This partially purified EDNH did not possess prothoracicotropic activity in the in vitro prothoracic gland assay, nor did any other fractions from the gel filtration column. Similarly, partially purified big and small PTTH did not activate Aedes atropalpus ovaries to synthesize ecdysone in vitro, nor did they cause ovarian maturation in vivo. Thus, it appears that the structural differences between PTTH and EDNH are sufficient enough to prevent functional cross-reactivity. The apparent discrepancy in the results obtained with the crude and partially purified EDNH and PTTHs raises questions about the reliability of bioassays for screening the presence and cross-reactivity of peptide neurohormones in crude extracts.

Localization of sodium/potassium adenosine triphosphatase in multiple cell types of the murine nervous system with antibodies raised against the enzyme from kidney

This report describes the development and characterization of a battery of highly specific antibodies to sodium/potassium (Na+ + K+)-ATPase and their use in localizing this enzyme in nervous tissue. The immunolabeling characteristics of polyclonal antibodies and monoclonal antibodies (Schenk, D. B., and H. L. Leffert (1983) Proc. Natl. Acad. Sci. U. S. A. 80: 5281-5285) raised against rat renal (Na+ + K+)-ATPase were compared. The interspecies cross-reactivity of the polyclonal anti-rat antibodies was examined by determining their binding to purified rat, eel, or dog enzyme. The immunostaining characteristics of the IgG fraction of the polyclonal antibody preparations, their affinity-purified derivatives, and the monoclonal antibodies were compared. The results obtained with each of these were similar, providing information about where focal concentrations of the enzyme exist within nervous tissue. The IgG fraction of the polyclonal antibody preparations provided the most sensitive probe, facilitating localization of the (Na+ + K+)-ATPase in the tissue sections from various regions of the nervous system. (Na+ + K+)-ATPase-like immunoreactivity was observed along the plasmalemma of alpha-motor neurons and at the nodal axolemma of myelinated axons from the central or peripheral nervous system. It was determined that the absence of labeling for the enzyme along the paranodal or internodal regions of the axolemma was not an artifact due to a limited accessibility of antibody to these regions. Some central nervous system glial cells demonstrated abundant amounts of plasmalemmal and intracellular (Na+ + K+)-ATPase-like immunoreactivity. These cells were identified as astroglia by positive labeling of cells in serial sections for glial fibrillary acid protein immunoreactivity in the soma and radial processes in optic nerve, or velous processes in the cerebellum. Astrocyte processes overlying the nodal axolemma also stained positively for the enzyme. (Na+ + K+)-ATPase-like immunoreactivity was not observed in association with oligodendroglia cell bodies or their processes forming myelin sheaths. In contrast, the plasmalemma of myelinating Schwann cells showed greatest immunoreactivity in the region of the node of Ranvier. Although a focal concentration of immunoreactivity was observed along node- and paranode-associated regions of Schwann cells, a lower level of uniform staining was noted along the entire Schwann cell surface membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Syngeneic monoclonal antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma.

It has previously been reported that a mouse (C57BL/6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to react only with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). In the present study, the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAc alpha 2-3Gal beta 1-4Glc (or -GlcNAc)) of gangliosides by three different assay systems including enzyme immunostaining on thin layer plates, sandwich radioimmunoassay, and enzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosides having NeuGc terminus, including NeuGc alpha 2-3Gal beta 1-4Glc-ceramide and NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide, nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was isolated from B16 melanoma cells, and its structure was determined to be NeuAc alpha 2-3Gal beta 1-4Glc-ceramide by fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:1 as its long-chain base and C16:0, C24:1, and C24:0 as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.

Monoclonal antibodies to Escherichia coli F1-ATPase. Correlation of binding site location with interspecies cross-reactivity and effects on enzyme activity.

Twenty-one hybridoma cell lines which secret antibodies to the subunits of the Escherichia coli F1-ATPase were produced. Included within the set are four antibodies which are specific for alpha, six for beta, three for gamma, four for delta and four for epsilon. The antibodies were divided into binding competition subgroups. Two such competition subgroups are represented for the alpha, beta, and epsilon subunits, one for delta and three for gamma. The ability to bind intact F1-ATPase was demonstrated for some of the antibodies to alpha and beta, and for all of those to delta, while the antibodies to gamma and epsilon gave unclear results. All of the antibodies to alpha and beta which bound ATPase were found to have effects on the ATPase activity of purified E. coli F1-ATPase. One of those to alpha inhibited activity by about 30%. Another anti-alpha was mildly stimulatory. The four antibodies to beta which bound ATPase inhibited activity by 90%. In contrast, membrane-bound ATPase was hardly affected by the antibodies to alpha, but was inhibited by 40-60% by the antibodies to beta. The other antibodies to alpha and beta bound only free subunits, or partially dissociated ATPase, suggesting that their epitopes are buried between subunits in ATPase. These antibodies had no effects on activity. The ability of the antibodies to recognize ATPase subunits present in crude extracts from mitochondria, chloroplasts, and a variety of bacteria was tested using nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. One anti-beta specifically recognized proteins in the range of 50,000-60,000 daltons in each of the extracts, although the reaction with mitochondrial beta was weak. Some of the other antibodies had limited cross-reaction, but most were specific for the E. coli protein. In some species, those proteins which were recognized by the anti-beta ran with a higher apparent molecular weight than proteins which were recognized by an anti-alpha. All antibodies which exhibited cross-reactivity were found to recognize sites which were not exposed in intact ATPase, implying that the surfaces which lie between subunits are most highly conserved.

Antigenic determinant and interspecies cross-reactivity of a monoclonal antibody to poly(ADP-ribose) synthetase

A monoclonal antibody (1F4) was prepared against calf thymus poly(ADP-ribose) synthetase. It was classified as IgG 1/κ and its antigenic determinant was localized on the 46 kDa portion of the enzyme molecule which contains the site for the binding of DNA. When calf thymus DNA-binding proteins were subjected to immunostaining after electrophoresis and transblotting to a nitrocellulose filter, the native enzyme (120 kDa) and its endogenous degradation products (80, 64 and 32 kDa) were detected. When the interspecies cross-reactivity was examined using DNA-binding proteins from 6 different sources, 1F4 reacted with the 120- and 32-kDa protein bands in HeLa cells, mouse testis and chicken liver as in the case of calf thymus. These results indicate that the antigenic structures ofpoly(ADP-ribose) synthetase and its degradation products are highly conserved in various animal cells.

Interspecies Cross-reactivity of Class II Antigen of MHC Determined by Syngeneic, Allogeneic and Xenogeneic B and T Cells

Although this chapter ought to summarize the role of MHC antigens in T cell activation, the immunobiological meaning of the polymorphism of Class II antigens, as well as that of Class I antigens, is still unresolved. The antigen-presenting ability of human APC is dominant over that of murine APC in the stimulation of antigen-specific xenogeneic T cells. In addition, xenoreactive murine T cells specific for human PBL failed to recognize the polymorphic determinant of Class II antigens of human MHC. On the basis of the data, Class II antigens may be seen to have some role as antigen-presenting molecules rather than as restricting molecules, at least, in the xenogeneic APC-T cell interaction or the xenogeneic MLR responses. These data together with the fact that the linkage disequilibrium found among the various groups of alleles encoding Class I and II antigens making up an MHC haplotypes suggest that the MHC may play a key role during evolution. These studies using xenogeneic cell interaction may shed some light on the immunobiological function of polymorphism of MHC antigens in the mechanisms of T cell activation, and the evolutional history of the polymorphism of the NHC in self or not-self recognition by T cells.

Identification of sperm antigenic determinants with phylogenetically diverse and limited distribution using monoclonal antibodies

Mouse monoclonal antibodies capable of recognizing mouse sperm antigens were produced and the inter-species cross-reactivity of the antigenic determinants was examined. Of the seven clones independently established, it was found by indirect immunofluorescence staining that one was bound to the tail, one could recognize the head and five other antibodies reacted with the crescent-shaped anterior acrosome. Ultrastructural studies showed that the anti-head antibody recognized the sperm surface, and the anti-tail antibody reacted with the fibrous sheath. Five acrosomal antigenic determinants were found in the acrosome. These antibodies reacted with sperm from the male reproductive tract (testis, cauda epididymis, vas deferens), but not with somatic tissues. The anti-tail antibody reacted with sperm from four other mammalian species (musk shrew, rat, boar and human). The anti-head antibody and one of the anti-acrosome antibodies were bound only to mouse sperm. The other four acrosomal antigenic determinants showed various degrees of inter-species cross-reactivity. None of these antibodies reacted with chicken sperm.

Interspecies cross-reactivity of monoclonal antibodies to various epitopes of human plasminogen

The immunological cross-reactivities of three conformationally specific monoclonal antibodies to distinct epitopes on human plasminogen toward plasminogens purified from 14 additional species have been examined. Antibody 10-F-1, which is produced against an epitope on the kringle 4 region of human plasminogen, shows a high degree (>80%) of cross-reactivity against baboon, goat, monkey, ovine, and rabbit plasminogens; more limited (20–50%) cross-reactivity against bovine, equine, goose, guinea pig, mouse, rat, and porcine plasminogens; and little comparable cross-reactivity against canine and chicken plasminogens. Antibody 10-H-2, generated to an epitope of the kringles 1–3 region of human plasminogen, shows extensive cross-reactivity (72%) only toward monkey plasminogen, more limited (22–35%) cross-reactivity toward equine and rabbit plasminogens, and much less cross-reactivity toward any other of the above plasminogens. Antibody 10-V-1, also produced against an epitope on the kringle 1–3 region of human plasminogen, which is distinct from the 10-H-2 epitope, shows extensive cross-reactivity (72–100%) with baboon, monkey, and rabbit plasminogens; more limited cross-reactivity with equine (48%) and mouse (28%) plasminogens; and a low level of such reactivity with the remaining plasminogens. These studies show that the extent of interspecies cross-reactivity of various plasminogens greatly depends upon the epitope in question. The K4 region of these molecules appears more extensively conserved than the K1-3 region, at least in regard to the particular epitopes examined in this study.

Sharing of Ia antigens between species. IV. Interspecies cross-reactivity of monoclonal antibodies directed against polymorphic mouse Ia determinants.

The specificity of interspecies Ia cross-reactions has been analyzed by testing a panel of monoclonal antibodies (mAb) to mouse I-E and I-A antigens for reactivity with pig Ia antigens. Our earlier studies showed that mouse anti-I-E alloantisera recognized common determinants on Ia antigens of other species, whereas anti-I-A alloantisera showed much more limited cross-reactivity. These results were confirmed using a panel of 17 anti-I-E mAb, 10 of which were cytotoxic to pig cells. 2D gel electrophoretic analyses of precipitates with these mAb of 35S-labeled, NP40 solubilized pig cells revealed a limited set of protein spots that appeared to be identical to the subset of pig Ia antigens precipitated by A.TH anti-A.TL alloantiserum. Because the cross-reactive mouse sera were produced in mouse strains that do not express an I-E molecule (H-2b and H-2s), it was anticipated that the cross-reacting antibodies would be reactive with the monomorphic determinant of the I-E molecule, Ia.7. However, comparison of the reactivity of these mAb with pig cells and mouse cells revealed that the cross-reactivity on pig cells correlated not with Ia.7 but rather with detection of epitope(s) of the I-E molecule associated with inter-strain polymorphism. Anti-I-A cross-reactions were also detected, but were weaker and more limited. These findings may have implications for the evolution of Ia antigens in mammalian species.

A cross-reactive mouse anti-I-E k monoclonal antibody detects an HLA-DR polymorphism linked to HLA-DR1

Two mouse monoclonal anti-I-E k antibodies (H7-8.26 and H40-242.3) also reacted with human B-cells. Cellular radioimmunoassays performed on lymphoblastoid B-cell lines suggest that these two antibodies recognized broad antigenic determinants on human B-cells. In order to determine whether the monoclonal antibody-HLA-DR antigen interaction had the same affinity on cells of different haplotypes, we measured the dissociation rate constants ( k ds) of radioiodinated Fab fragments from several human lymphoblastoid cell lines. Antibody H7-8.26 bound with the same avidity to all these B-cell lines regardless of their origin suggesting that its target antigenic determinant on HLA-DR molecules was non-polymorphic. By contrast antibody H40-242.3 displayed two patterns of binding avidity with either high or low k d values depending on the cell tested. This strongly suggested that this antibody cross-reacted with different determinants on human B-cell lines. High-avidity binding patterns correlated with the classical HLA-DR1 allospecificity and segregated with this antigen in a family study. The amount of HLA-DR antigen immunoprecipitated by H40-242.3 from 125I-radiolabeled NP-40 solubilized membrane extracts of cells from five members of the same family depended also on the presence or absence of serologically defined HLA-DR1 on the cells. These results indicated that: (a) avidity measurements can detect some degree of polymorphism of membrane antigens in cases where the cell binding reactions appear to be monomorphic; (b) the immunoprecipitation efficiency of a monoclonal antibody may depend on its avidity and can not be predicted from its cell binding reactivity alone; and (c) an interspecies cross-reactivity can also be related to an allospecificity in the species studied.