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Background and Objectives Paper biodiagnostics for blood typing are novel, cheap, fast and easy to use. Agglutinated red blood cells cannot travel through the porous structure of paper, indicating a positive antibody-antigen interaction has occurred. Conversely, non-agglutinated blood can disperse and wick through the paper structure with the ease to indicate a negative result. This principle has been demonstrated to detect blood group phenotypes: ABO and RhD. However, typing for red blood cell antigens such as Rh, Kell, Duffy and Kidd has not yet been explored on paper. Materials and Methods Two paper testing methods - an elution and a direct flow-through method - were investigated to detect red blood cell antigens excluding the ABO system and RhD. Antigens explored include the following: C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S and s, P1, Lea and Leb. The variables tested include the following: reaction time and reagent concentration. The importance of antibody type/structure for successful agglutination on paper was confirmed. Results Some blood group phenotypes showed less agglutination due to weaker antibody-antigen interactions. Most blood groups with antibodies available as IgM, such as C, c, E, e, K and k, and Jka and Jkb, and P1, were successful using both methods. However, other blood groups, especially those with antibodies only available as polyclonal antibodies, were unsuccessful and require further scrutiny. Conclusion Paper can be used as an alternative blood grouping diagnostic tool for selected blood group phenotypes. [ABSTRACT FROM AUTHOR]

The suitability of blood collected on filter papers in comparison with corresponding conventional serum samples in the diagnosis of bovine anaplasmosis was studied using the Complement Fixation Test (CFT), DOT-ELISA, Western immunoblot and Rapid Card Agglutination Test (RCAT). Dried blood on Whatman filter paper no. 1 was eluted in 1.8 ml of PBS 0.05% Tween 20 given an initial dilution of 1:100. The reactivity in both DOT-ELISA and Western immunoblotting was similar to that obtained with the sera diluted 1:100. Filter paper samples gave lower reactivity in all the tests as compared with corresponding serum samples. There was no significant difference in the reactivity between the eluates from filter papers stored at room temperature and those stored at 4°C. Storage at room temperature did not significantly affect reactivity for up to 6 months. Eluates from filter papers stored for 6 months at room temperature continued to give similar reactivity to those from freshly prepared filter papers in both DOT-ELISA and Western blot, and in the Rapid Card Agglutination Test. It is concluded that collecting blood on filter papers is a suitable technique for large-scale screening and for seroepidemiological studies on anaplasmosis, and offers many advantages especially in developing countries where transport and cold chain facilities are a major constraint. [ABSTRACT FROM AUTHOR]

Background: Lewis serologic reagents frequently give inaccurate phenotyping results. Furthermore these serologic reagents are often used in nonserologic assays such as inhibition and immunohistochemistry. In both scenarios knowledge of the fine specificity and cross-reactivity of these reagents will improve the quality of results obtained.Study Design and Methods: A range of contemporary and historical workshop and developmental Lewis reagents including mouse monoclonal (MoAb) and human and goat polyclonal (PoAb) reagents were evaluated. All were evaluated both against Lewis kodecytes expressing only single Le(a) , Le(b) , ALe(b) , BLe(b) , Le(x) , Le(y) , ALe(y) , or BLe(y) antigens and against the same antigens inkjet printed on a paper-based microplate and analyzed by enzyme immunoassay. Nine clinical samples were also evaluated. A kodecyte antigen dilution sensitivity assay was used to establish the ratio of Le(b) antigen between group A1 /A2 and O RBCs.Results: A continuum of cross-reactivity from Le(x) through to H was observed with MoAbs. All PoAb and few MoAb anti-Le(a) samples and reagents cross-reacted to some degree with Le(b) antigen. Some PoAb and MoAb anti-Le(b) did not cross-react with Le(a) . All polyclonal goat anti-Le(b) reagents showed substantial activity against ALe(b) and BLe(b) , while no MoAb reagent had this activity. A1 RBCs had less than half the Le(b) antigen of A2 /O RBCs.Conclusions: Substantial cross-reactivity of both MoAbs and PoAbs with related antigens highlights the risks of using serologic reagents in nonserologic assays or against synthetic antigens. The lack of ALe(b) activity in anti-Le(b) MoAbs explains their poor performance against blood group A1 Le(a-b+) phenotypes. [ABSTRACT FROM AUTHOR]

Human leucocyte lysosomal polypeptides of mol. wt 4000-5000, which constitute the neutrophil-immobilizing factor (NIF), were isolated from the 22,000 g supernate of sonicates of human neutrophils by filtration on Sephadex G-75. The larger (NIF-1) and smaller (NIF-2) of the polypeptides were resolved by filtration on Bio-Gel P6 and purified to homogeneity by sequential reverse-phase high performance liquid chromatography and paper electrophoresis. The results of analyses of amino acid composition indicated that NIF-1 and NIF-2 are distinct polypeptides composed of an apparent total of 41 and 38 amino acids, respectively. Both NIF polypeptides contain one cysteine and one methionine, lack isoleucine, tyrosine and phenylalanine, and are rich in histidine and praline. The sequence of 20 of the amino-terminal amino acids of both NIF polypeptides is identical, but NIF-2 possesses an additional alanine at the amino-terminus. Highly purified NIF-1 and NIF-2 inhibited human neutrophil random migration and chemotaxis to diverse stimuli in a concentration-dependent manner, with 50% inhibition of chemotaxis by 0.3-1 &times 10-8 M NIF-1 and 1-3 × 10-7 M NIF-2. Neither NIF polypeptide was cytotoxic for neutrophils, altered neutrophil phagocytosis or release of lysosomal enzymes, or inhibited mononuclear leucocyte chemotaxis. The leucocyte and functional specificity of the NIF polypeptides and the quantities released upon stimulation of human leucocytes suggest that the transition to a mononuclear leucocyte population in chronic inflammation may be attributable in part to the NIF thrived from the leucocyte infiltrates of acute responses. [ABSTRACT FROM AUTHOR]

Maternally inherited bacteria of the genus Wolbachia are known to affect the physiology and reproduction of several arthropod and nematode hosts. This paper presents the effects of using tetracycline to eliminate Wolbachia on the oogenesis of its host uzifly Exorista sorbillans (Diptera: Tachinidae), a parasitoid of silkworm Bombyx mori L. Earlier we reported cytoplasmic incompatibility, nucleo-cytoplasmic conflict, male biased sex ratio and reduction in fecundity upon curing of Wolbachia infection using an antibiotic. This paper presents the abnormal growth and proliferation of oocytes in the uninfected/cured uziflies. The anomalies include abnormal/arrested oocyte production with inhibition of normal functioning of nurse cell nuclei, malformation and agglutination in egg chambers, empty egg chambers due to arrested vitellogenesis and undeveloped ovarian tubules. These anomalies further lead to reduction in growth of the ovarian tubules, which resulted in a 20% reduction of fecundity. It is hypothesized that normal oocyte production in uzifly requires the infection of Wolbachia bacteria. Further, in the uzifly, infection is at transition stage from a facultative to an obligatory mode of symbiosis. In light of this, this paper discusses the relationship between Wolbachia and oocyte production. [ABSTRACT FROM AUTHOR]

The G1 cyclins, D1, D3 and E, were investigated in 38 lesions of oral epithelial dysplasia from the floor of the mouth or the lateral border of the tongue. Their immunohistochemical expression was observed and compared with that of Ki-67 and with the degree of dysplasia assessed by the semiobjective technique of Smith & Pindborg. Antibody labelled cells were quantified and expressed as a percentage (LI%) of the total nucleated cell population and per mm basement membrane length (LI/mm). The labelling indices of all of the antibodies were high and quantitatively similar. There were no significant correlations with the degree of dysplasia assessed by the atypia scores. There was a correlation between labelling indices for the various antibodies expressed as LI/mm but little correlation between the indices expressed as LI%. The distribution of the D cyclins was similar to that of Ki-67 with relatively few of the basal cells demonstrating immunoreactivity. The reasons for this are discussed in the paper. Some cross-reactivity was observed with the cyclin antibodies. We conclude that the antibodies against the cyclins used in the present study are not a useful adjunct in the study of the cell kinetics of oral epithelial dysplasia. [ABSTRACT FROM AUTHOR]

Six new analogs of the phagocytosis-stimulating peptide tuftsin were synthesized with the eventual aim of characterizing and isolating the tuftsin receptor. These analogs can be classified as follows; (a) photoaffinity labelling analogs for the specific covalent attachment to the tuftsin receptor; (b) fluorescent analogs containing either rhodamine or dansyl fluorescent probes for microscopic visualization of the tuftsin receptor; (c) biotin analog for separation and purification of the receptor by affinity methods. In this paper we describe the various synthetic pathways employed to introduce sensitive prosthetic groups into the tuftsin molecule while preserving its biological activity. Activities of the various analogs synthesized as compared to tuftsin in biological and receptor-binding assays are described. All analogs are able to stimulate phagocytosis of the macrophage cell as well as compete specifically for tuftsin binding sites on these cells. [ABSTRACT FROM AUTHOR]

The structure of the proteomannans from the cell-wall mutant Candida sp. and its parent strain was further studied by immunochemical methods. Alkaline titration showed that the phosphate is present in the form of diester and mild acid treatment released mannose from both of the mannans. In the wild-type mannan, the phosphorylated side-chain oligosaccharides gave 85 % inhibition and a mannopentaose side chain having α1 -3 linkage in the nonreducing terminal also gave 40 % inhibition in the homologous precipitin reaction. This indicates that the immunodominant side chain of the Candida sp. wild type mannan is the phosphorylated mannopentaose which has an (α1- 3)-Linked nonreducing terminal. In the mutant system, a significant reactivity in the cross reaction between the mutant antiserum and Kloeckera brevis mannan indicates that the immunodeterminant of the mutant mannan may be a structural analogue of the side-chain oligosaccharide from K. brevis, which has phosphorylated (α1-2)-linked mannotriose. However, no definite results were obtained in the inhibition studies. On the basis of the results from chemical, enzymatical and immunological experiments, the side-chain structure and macromolecular models of Candida sp. wild-type and mutant mannans are proposed. [ABSTRACT FROM AUTHOR]

Heat and pH stability studies and experiments with organic solvents show that the A-antigens discussed in the preceding paper (Augustin, 1959c) are much more labile than the I-(‘inner ring’) antigens. Breakdown products and/or aggregates are produced which no longer precipitate with antisera to the original extracts, but act as inhibitors. Solutions of pollen allergens, on the other hand, are found to withstand even autoclaving for 15 min. at 20 atm. and vigorous boiling over the naked flame of a bunsen burner. None of the carbohydrates tested has a demonstrable effect on skin reactivity which is, however, destroyed by crystalline pepsin, crystalline trypsin, a crystalline mould protease and a tissue protease (a partially purified extract from rabbit spleen). It follows that the bulk of the allergens—if not all—are proteins. The relation of skin reactivity, immuno-electrophoretic patterns, carbohydrate and protein reactions to the selective destruction of the pollen antigens is investigated. Pollen components prove to have a somewhat wider range of electrophoretic mobilities than serum proteins and are probably as complicated a mixture. The most and least highly negatively charged components are without skin reactivity in allergic subjects. The skin reactive allergens appear to have the mobilities of α- and β-globulins. Not all the hay fever subjects react equally to all the components, and Cocksfoot and Timothy activity patterns vary in different subjects. [ABSTRACT FROM AUTHOR]

Since the publication of the mouse genome, attention has focused on the strains that were selected for sequencing. In this paper we report the results of experiments that characterized the response to infection with the murine gastrointestinal nematode Heligmosomoides polygyrus of eight new strains (A/J, C57BL/6, C3H, DBA/2, BALB/c, NIH, SJL and 129/J), in addition to the well-characterized CBA (poor responder) and SWR (strong responder) as our controls. We employed the repeated infection protocol (consisting of 7 superimposed doses of 125L3 each administered at weekly intervals, faecal egg counts in weeks 2, 4 and 6 and assessment of worm burdens in week 6) that was used successfully to identify quantitative trait loci for genes involved in resistance to H. polygyrus . SWR, SJL and NIH mice performed indistinguishably and are confirmed as strong responder strains to H. polygyrus . CBA, C3H and A/J mice all tolerated heavy infections and are assessed as poor responders. In contrast, DBA/2, 129/J and BALB/c mice performed variably between experiments, some tolerating heavy worm burdens comparable to those in poor responders, and some showing evidence of resistance, although only in one experiment with female 129/J females and one with female BALB/c was the pattern and extent of worm loss much like that in SWR mice. Because the genetic relationships between six of the strains exploited in this study are now well-understood, our results should enable analysis through single nucleotide polymorphisms and thereby provide more insight into the role of the genes that control resistance to H. polygyrus. [ABSTRACT FROM AUTHOR]

Discusses the 'Immunology' paper by Smith et al. and its contribution to understanding at which point in the immune response to antigen the critical decision making between an active versus a tolerogenic response may take place. Induction of oral tolerance initially to provoke the activation of T cells; Possible role of the antigen-presenting cells present in the intestine in determining the T-cell response to antigen.

The function of MHC class-I molecules is to sample peptides from the intracellular environment and present them to CD8+ cytotoxic T lymphocytes. To understand the molecular details of the assembly (and disassembly) of peptide-β2m-class-I complexes a biochemical peptide-class-I binding assay has been generated recently and this paper reports on a similar assay for the interaction between β2m and class I. As a model system human β2m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined by size separation. It is specific and highly sensitive. The observed affinity of the interaction, KD, is close to 0.4 nM. The rate of association at 37°C is very fast (the ka is around 5 x 104/M/s) whereas the dissociation is slow (the kd is around 8 x 10-6/s); the ratio of dissociation to association yields a calculated KD close to the observed value. At 37°C almost all of the purified class I participates in binding of the exogenously offered β2m showing that a considerable exchange of the endogenous β2m occurs. Finally, it was demonstrated that exogenous β2m enhances binding to MHC class-I of short perfectly-matching peptides as well as longer peptides. [ABSTRACT FROM AUTHOR]

This paper presents a method for the isolation of highly enriched antigen-specific B lymphocytes from peripheral blood mononuclear cells (PBMC) by means of an immunomagnetic procedure. The method is illustrated by the isolation of B cells positive for rheumatoid factors (RF) from patients with rheumatoid arthritis and healthy blood donors. After Epstein-Barr virus transformation of isolated cells, more than 90% of the generated immunoglobulin-secreting cells produced RF in several experiments. Compared to the low frequency of B cells positive for RF in PBMC, this represents a 10³- to 104-fold enrichment of antigen-specific B cells. [ABSTRACT FROM AUTHOR]

The routine availability of nucleated human cells for experimental use is limited in the absenee of venipuncture. In this paper we have demonstrated that macrophages may be harvested routinely from the waste dialysis bags of patients undergoing continuous ambulatory peritoneal dialysis. These cells were identified as macrophages by morphology, adherence, phagocytosis, chemotaxis, non-specific esterase staining and peroxidase staining. Macrophages from patients with end-stage renal disease produced arachidonate cyclo-oxygenase products in a pattern similar to that of ascites macrophages obtained from patients with normal kidney function. Arachidonate metabolism was shown to be manipulatable. Thus, indomethacin blocked synthesis of cyclooxygenase products, and OKY-t58l. a specific thromboxane synthase inhibitor, increased the release of prostaglandin E2 and prostacyclin, measured as its stable breakdown product 6-keto-prostaglandin F1, whereas the thromboxane B2 synthesis was effectively inhibited. [ABSTRACT FROM AUTHOR]

The leaf apoplast is a site of preferential accumulation of heavy metals in the shoot when barley seedlings are grown in the presence of cadmium, nickel or zinc in hydroponic medium. It was shown recently that apoplastic protein content increased concomitantly in plants grown In the presence of high Zn (Brune et al. 1994, Journal Experimental Botany 45, 1189). Here it is demonstrated that apoplastic proteins increase to an even greater extent in Ni-treated plants, and that the response is intermediate for Cd-treated plants. The paper focuses on possible causes for the increase in apoplastic proteins. (1) Synthesis of cell wall proteins was studied by in vivo pulse labelling of leaf proteins With 35S methionine followed by extraction of extracellular polypeptides. Depending on growth conditions and leaf age, 1-6% of the protein synthesized de novo in the leaf was allocated to the extractable protein fraction of the leaf apoplast. Under control conditions, labelling reached its maximum after 2 h; labelling continued to rise in Ni-treated plants, where the maximum level of final labelling was reached only after several hours. Labelling decreased with leaf age and increased proportionally with Ni concentration in the nutrient solution. (2) Autolysis experiments on extracted apoplastic proteins and in situ digestion of infiltrated ovalbumin revealed a low proteolytic activity in the apoplast which was not altered in heavy metal-treated leaves. (3) Following a sudden application of Ni to the growth medium, protein content and peroxidase activity increased with a 48 h delay and approached the level obtained for continuously Ni-treated plants, even in previously fully expanded leaf tissue. It is concluded that altered cell wall structure is probably not involved in increasing the extraction yield of apoplastic proteins. (4) The increase in extractable apoplastic proteins was also not due to inhibited ionical or covalent immobilization of proteins in the cell w... [ABSTRACT FROM AUTHOR]

All ideas implicit in the papers since 1953 involved in applying mass-action thermodynamics to antibody-antigen reactions are unified by the use of: (a) the intermediary concept of extent of reaction; (b) the concept of intrinsic association constant; (c) a statistical analysis for probable complexes; and (d) identification of the complex or complexes that contribute to the bioassay. Several general theoretical examples are given that show the limitations of linear interpretations of equilibrium data. Two practical examples from the literature illustrate foot-and-mouth disease virus and influenza virus neutralization. [ABSTRACT FROM AUTHOR]

Immune responsiveness to the streptotoccal group polysaccharides at the clonal level can conveniently be monitored by analytical isoelectric focusing combined witb autoradiography. The parameters investigated in this paper can be summarized as follows: [l] Within the first week after injection antistreptococcal polysaccbaride responses art characterized by IgM antibodies, followed by subclass restriction in Balb/c mice to IgG2a antihodies. [2] The clonal pattern of specific IgG2a antibodies established in the second week after inimunization does not change within the following 6 weeks. [3] Persistence of a specific response pattern established within a primary immunization course holds for Balb/c mice and rabbits for as long as 12 to 13 months. [4] Repeated immunization courses in mice and rabbits maintain established clonal antibody patterns in toto. Temporal variations of clonal expression are generally quantitative rather than qualitative. B-cell memory within this system is therefore stable and long-lived. [ABSTRACT FROM AUTHOR]

Thirty-six guinea pigs were injected with a suspension of pooled guinea-pig adrenals incorporated into Freund adjuvants; twenty-two rabbits were immunized in a similar manner with rabbit adrenal suspensions. Sera of twenty-six guinea pigs and twelve rabbits reacted in complement fixation and tanned cell haemagglutination tests with adrenal extracts but not with extracts of other organs. Potent antisera also produced one line reaction in double diffusion gel precipitation test with adrenal extracts. The adrenal-specific antigen was thermolabile, possibly of protein character. Reactions of guinea-pig antisera were limited to guinea-pig adrenal; rabbit antisera reacted with adrenal of all tested mammalian species. The serological reactions exhibited by antigens prepared from the antibody producer's own adrenal were as strong as those produced by adrenals of other animals of the same species. Additional evidence for auto-specificity of the antibody under investigation was obtained from the experiment in which a guinea pig injected with its own gland removed by left adrenalectomy formed adrenal antibodies. Some but not all animals developed histological lesions in the adrenal gland. The interpretation of this observation was complicated by occasional positive histological findings in the adrenals of control animals. [ABSTRACT FROM AUTHOR]

Whilst the ability of isoantibodies to agglutinate mouse red cells in saline medium does depend in part upon the properties of the antibody molecules, the concentration of antigen upon the red cell and certain genetically determined properties of the cell surface are of even greater importance. The red cells of mice of any strain will give positive results in the human serum:dextran system whilst reactions in a saline medium are unusual. In this paper the term ‘incomplete antibody’ is used to denote antibodies that can only be detected in conjunction with other antibodies in the human serum:dextran system. The time of appearance of antibodies to homografts of an ascites sarcoma, an ascites leukosis and a solid mammary carcinoma has been studied. Incomplete antibody as defined above has been detected by two methods. In the blocking test incomplete antibody is allowed to react with red cells before contact with complete antibody, a positive result being observed as a fall in titre when the treated red cells are subsequently exposed to complete antibody. In the second type of test, the synergic test, incomplete antibody is mixed with suitably diluted complete antibody and fitrated against suitable red cells, a positive result being a significant rise in titre as compared with complete antibody mixed with normal serum. Antibodies produced by the antigen donors did not give significant blocking or synergic effects. Incomplete antibodies are sometimes detectible on the third day and with regularity on the fourth day. Antibodies ‘complete’ for A strain red cells may appear at any time from the fifth day onwards. There is sometimes a drop in fitre of varying duration on the sixth day. This corresponds with the commencement of marked inflammatory changes on the graft bed. The maximum titre is attained at about two weeks. Antibodies may disappear in under three months or persist as long as a year. The function of antibodies in homograft reactions varies with the target tissue. However, they appear before the onset of anatomical signs of homograff response regardless of the type of target cell. [ABSTRACT FROM AUTHOR]

When lymphoid cells from hypersensitive rabbits are incubated with antigen, biologically active substances are formed and released which are capable of inhibiting the migration of normal non-sensitized mesenchymal cells. In the present paper some basic parameters of their production were determined. These substances were regularly obtained after 6 and 18 hours incubation, but not after 2 hours. Under more favourable cultivation conditions (lower density of lymphocyte suspension) an increased activity in the cell extracts as compared with the supernatants was observed. Another critical factor in the production of these substances is the quantity of antigen added. Ten micrograms of PPD leads to the production and liberation of a highly effective substance. A lower dose of antigen results in the liberation of a substance into the supernatant which by itself is almost inactive, but becomes more active when more antigen is added. The efficiency of the released substances was determined by serial dilution. The inhibiting activity was maintained at 1:5 and 1:20 and sometimes at 1:100 dilutions. [ABSTRACT FROM AUTHOR]

This paper describes a form of red cell lysis differentiated from classical complement haemolysis by its occurrence in the absence of antibody on the cells and in the presence of EDTA. This type of haemolysis has been called reactive haemolysis. It is the result of the interaction, in the presence of red cells, of at least two serum factors called ‘reactor’ and ‘indicator’, respectively. Reactor only becomes active after incubation at 37° of serum with certain agents, notably antibody-coated bacteria, zymosan and agarose, in conditions similar to those required for complement activation. The potential for the formation of activated reactor could be demonstrated infrequently in healthy subjects but more frequently in sera from hospital patients. Activated reactor behaved as a protein sedimenting between 7S and 19S, and of α2-β1, electrophoretic mobility in agar. Indicator factors were present in all human sera studied, as well as in the sera of a number of mammalian species. They were demonstrable at high dilutions of the serum and required no prior activation for their action. They occurred maximally in the 7S fractions of serum proteins and migrated in the β2 position on electrophoresis. Reactive haemolysis was first observed and can most conveniently lie demonstrated in a red cell-agarose gel. It can also be demonstrated in the test tube following partial purification of activated reactor and indicator factors. Studies in the test tube indicated that a .soluble labile lytic factor was responsible for this type of haemolysis, [ABSTRACT FROM AUTHOR]

The studies reported here indicate that tanned red cells coated with a mixture of chromatographically purified ovalbumin and conalbumin give agglutination titres which are directly related to the amount of antibody (as meas- ured by quantitative precipitation) present against either of these antigens in mixed sera. With whole (unabsorbed) mixed sera, these coated cells record the titre of the major antibody present, whether this is anti-ovalbumin or anti-conalbumin. There was no evidence of greater sensitivity to anti-conalbumin. A trace of ovalbumin in a preparation of conalbumin used to coat cells is fully sufficient to enable them to react with any anti-ovalbumin in a serum. Twice recrystallized ovalbumin still contains some conalbumin. Sera raised against it may contain unexpectedly high levels of anti-conalbumin and this could cause confusion in tanned red cell tests. [ABSTRACT FROM AUTHOR]

Leptospirosis is a common disease in dogs, despite their current vaccination. Vet surgeons may use a serological test to verify their clinical observations. The gold standard is the Microscopic Agglutination Test (MAT). After infection, the dog produces agglutinating antibodies against the lipopolyosidic antigens shared by the infectious strain but also, after vaccination, against the lipopolyosidic antigens shared by the serovars used in the bacterins (Leptospira species serovars Icterohaemorrhagiae and Canicola in most countries). MATs were performed in a group of 102 healthy field dogs and a group of 6 Canicola-challenged dogs. A diagnosis algorithm was constructed based on age, previous vaccinations, kinetics of the agglutinating antibodies after infection or vaccination and the delay after onset of the disease. This algorithm was applied to 169 well-documented sera (clinical and vaccine data) from 272 sick dogs with suspected leptospirosis. Totally, 102 dogs were vaccinated according to the usual vaccination scheme and 30 were not vaccinated. Leptospirosis was confirmed by MAT in 37/102 (36.2 per cent) vaccinated dogs and remained probable in 14 others (13.7 per cent), thus indicating the permanent exposure of dogs and the weakness of the protection offered by the current vaccines to pathogenic Leptospira. [ABSTRACT FROM AUTHOR]

The sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true-negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinically positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as the gold standard test. Highest sensitivity was shown for the CIDC antigen (100 per cent) followed by the c.c.pro antigen (99.39 per cent). However, the USDA antigen showed substantially less (p<0.05) sensitivity (62.19 per cent). Highest specificity was found for the USDA antigen (100 per cent) followed by the CIDC (97.5 per cent) and c.c.pro antigen (96.5 per cent). Positive and negative predictive values (assumed glanders prevalence of <0.1 per cent) for each antigen were calculated to be 95.88 and 99.48 (c.c.pro), 97.04 and 100 (CIDC), 100 and 76.33 per cent (USDA), respectively. Almost perfect agreement (0.96) was found between CFT using either c.c.pro or CIDC and IB. [ABSTRACT FROM AUTHOR]

In 2007, a survey was conducted on the prevalence of antibodies to 19 Leptospira serovars in goats in Poland . Sera were collected from adult females of all 49 breeding goat herds in Poland by applying simple random sampling. In total, 736 sera were tested by the microscopic agglutination test. A herd was considered seropositive if at least one goat with a titre of 100 or more was detected. Herd-level seroprevalence of Leptospira was 89.8 per cent and individual-level seroprevalence varied from less than 1.0 to 85.0 per cent among the herds. Antibodies to Leptospira serovars Zanoni, Bratislava, Autumnalis, Australis and Javanica were most frequently detected. Although 40.3 per cent of seropositive goats had high antibody titres (≥400), suggesting recent infection, no relationship with abortions or other clinical manifestation of leptospirosis in goats was detected. [ABSTRACT FROM AUTHOR]

Background and Purpose: The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications.Experimental Approach: An anti-EGFR/EGFRvIII llama single-domain antibody (EG(2)) and two higher valency format constructs, bivalent EG(2)-hFc and pentavalent V2C-EG(2) sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies.Key Results: Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG(2) to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG(2) and EG(2)-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG(2), V2C-EG(2) and EG(2)-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG(2)-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG(2)-hFc selective accumulation/retention in anatomically defined tumour regions.Conclusions: Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma. [ABSTRACT FROM AUTHOR]

Unexpected positive results from the widely used IDEXX ELISA for the detection of antibodies to porcine reproductive and respiratory syndrome virus (PRRSV) may confound investigations of the disease. Supplementing the ELISA with blocking agents and the use of lgG purified from serum samples had no effect on the unexpected positive results, suggesting that they were due to an antibody-antigen reaction. Simple competitive and blocking ELISAs were developed by modifying the IDEXX ELISA, and they and an indirect fluorescent antibody test (IFAT) were used to examine PRRSV antibodies in 33 antibody-negative, 88 antibody-positive and 73 unexpectedly positive sera. All the unexpectedly positive sera were negative by IFAT, and 89·0 per cent were negative by both the competitive and blocking ELISAs. The competitive ELISA (97·7 per cent) and the blocking ELISA (96·5 per cent) detected more positive sera than the IFAT (90·9 per cent). These results show that both ELISAs are capable of distinguishing positive and unexpectedly positive sera, and suggest that most of the unexpected positive signals are false-positives. [ABSTRACT FROM AUTHOR]

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The early diagnosis of recurrence and metastasis is critically important for decreasing the morbidity and mortality associated with oral cancers. Although liquid biopsy methods hold great promise that provide a successive "time‐slice" profile of primary and metastatic oral cancer, the development of non‐invasive, rapid, simple, and cost‐effective liquid biopsy techniques remains challenging. In this study, an ultrasensitive and selective electrochemical liquid biopsy is developed for oral cancer screening based on tracking trace amounts of cancer biomarker by functionalized asymmetric nano‐channels. Detection via antigen–antibody reactions is assayed by evaluating changes in ionic current. Upon the recognition of cancer biomarker antigens in bio‐fluids, the inner wall of nano‐channel immobilized with the corresponding antibodies undergoes molecular conformation transformation and surface physicochemical changes, which significantly regulate the ion transport through the nano‐channel and help achieve sensitivity with a detection limit of 10–12 g mL−1. Furthermore, owing to the specificity of the monoclonal antibody for the antigen, the nano‐channel exhibits high selectivity for the biomarker than for structurally similar biological molecules present in bio‐fluids. The effectiveness of this technique is confirmed through the diagnosis of clinical cases of oral squamous cell carcinoma. This study presents a novel diagnostic tool for oral cancer detection in bio‐fluids. [ABSTRACT FROM AUTHOR]

Cotton (Gossypium hirsutum L. cv. Stoneville No. 406) fibres possess several types of cell wall-bound (1→3)-β-glucanase activities and a significant amount of the (1→3) -β-glucan, callose. When fibre cell wall fragments are incubated in vitro, free glucose is released. Such autolysis is barely measurable at the primary cell wall stage of development when the endogenous (1→3)-β-glucan content is low. At the beginning of secondary cell wall formation, when the (1→3)-β-glucan is at a maximum, the rate of autolysis increases markedly and then remains more or less constant. Nojirimycin, a specific inhibitor of β-glucosidase, decreases the amount of free glucose released and results in the production of a corresponding amount of (1→3)-β-linked oligosaccharides. [ABSTRACT FROM AUTHOR]

Non-Iymphoid cells (NLC) were obtained by thoracic-duct cannulation of mesenteric lymphadenectomized (MLNx) congenitally alhymic nude rats. The cells were similar to NLC collected directly from testicular lymph and from the thoracic duct of MLNx euthymic rats, la+ NLC were characterized by a highly irregular, veiled or dendritic surface. Otherwise the cells were heterogeneous with regard to ultrastructure, non-specific acid esterase activity, and surface antigens. Roughly a third of the cells had surface Thy-1 antigen. A negative correlation was found between the presence of Thy-1 and non-specific acid esterase activity. Compared with macrophages and granulocytes they showed limited phagocytosis of simple lest substances. However, Ia+ NLC often contained lysosomes and some were occasionally observed in the act of phagocytosing and ingesting other cells, thus demonstrating phagocytic ability. Since la+ NLC were abundant in peripheral lymph but rare in central lymph, they must be held back by the draining lymph nodes. Their relationship to NLC within lymph nodes is discussed. [ABSTRACT FROM AUTHOR]

The effects of propamidine and their dose dependency, on utilization of the third and fifth complement components in immune haemolysis have been compared. While C3 utilization is not disturbed that of CS is markedly inhibited by propamidine in concentrations as low as 0.5 mist Both, binding of CS to surface-fixed C3b and cleavage of CS by convertases C42 and C3bBb, are also inhibited in the presence of propamidine. Since neither C3 cleavage by these enzymes nor even C5 cleavage by the cobra venom factor-supported convertase CFVBb is significantly reduced a general convertase-inhibiting effect of propamidine is ruled out. Rather the effect on utilization of C5 is the result of interference with binding of CS to C3b and hence impairment of its accessibility to the convertases. These findings thus further support the role of surface-fixed C3b in C5 activation proposed earlier. [ABSTRACT FROM AUTHOR]

The effect of synthetic complexes of polyriboinosinic and polyribocytidylic acids (poly-IC) on the production of homocytotropic antibodies by guinea-pigs was studied. It was observed that poly-IC ires a definite adjuvant effect on the production of both IgG1 and IgE. The adiuvant effect was present when poly-IC was injected 24 hours after the antigen, but was absent when the polynucleotide was injected 48 hours after the antigen. In comparison with lipopolysaccharide (LPS) the adjuvant effect of poly-IC was weaker, more than 0.5 mg/kg of this polynucleotide being required to show an adjuvant effect, whereas only 20 µg/kg of LPS was enough to show a positive adjuvant effect. Furthermore, considerably less antibody was detected in the animals stimulated with the polynucleotide. [ABSTRACT FROM AUTHOR]

Evidence is presented which suggests that T cell-dependent non-specific factor (NSF) cannot be the sole mediator of 'co-operation' for all types of thymus-dependent antibody response. NSF can replace the requirement for T cells in responses to hapten conjugated to particulate carriers, but cannot do so in responses to hapten conjugates of soluble thymus-dependent carriers. [ABSTRACT FROM AUTHOR]

The in vitro polyclonal pokeweed mitogen (PWM)-induced activation of human B lymphocytes is enhanced by addition of autologous or allogeneic irradiated T cells. This model for B/T-cell cooperation may be used to define and describe the balance between T helper and T suppressor phenomena. The present study investigates the helper and suppressor capacities of mononuclear cells isolated from peripheral blood of infectious mononucleosis patients during the acute disease and the reconvalescence period. During the acute disease we found a functional lack of T helper capacity; furthermore, the T cells were able to suppress the PWM and T-cell-dependent B-cell proliferation of healthy donor cells. The suppression was non-cytotoxic; i.e. not due to destruction of the responder cells. This phenomenon of non-cytotoxic suppression was found for all seven patients studied and disappeared during the reconvalescence period, indicating that the T lymphocytosis seen in infectious mononucleosis includes an expansion of T suppressor cells. [ABSTRACT FROM AUTHOR]

A rapid, simple technique measures small amounts of total protein applied on an agarose gel slab. Alternating current through the gel may reduce diffusion of the protein, which is chemically fixed. The intensity of the Coomassie-Blue-stained spots is a function of the amount of protein determined by comparison with simultaneously run standards. Densitometric and visual estimates of protein in spinal fluids by the stained protein assay correlated with Lowry values highly significantly (n = 28; P < 0.001, Spearman test of rank correlation). As little as 0.2 μg protein was measured; this figure should be compared with 10 jug for the Lowry method. The reproducibility of the method was ± 5%. [ABSTRACT FROM AUTHOR]

Rabbits made immunologically unresponsive by neonatal administration of HSA, HGG or BSA were given a course of intravenous injections of the respective antigens, adsorbed on alum, after a lapse of 13-27 months since the last administration of antigen. 8/12 responded to HSA, 4/5 to HGG, 9/10 to BSA, as judged by immune elimination of antigen, but this was delayed in onset and slow compared with that in previously untreated rabbits. The antibody formed was small in quantity and usually failed to precipitate with antigen. The sedimentation coefficients of 131I-labelled antigens, in the presence of excess antibody, were measured by ultracentrifugation through a sucrose density gradient. These showed that only small complexes were formed in some of the non-precipitating antisera. In one instance the diffusion coefficient of the complex was also measured, by a technique based on diffusion through agar gel. The calculated molecular weight of the complex, 330,000 indicated the presence of only two combining sites on the antigen. Combination of the anti-HSA sera with an HSA fragment was also measured. Whereas the amount of the fragment bound by ordinary hyperimmune anti-HSA sera was about one-fifth the HSA bound, the amounts bound by the test sera were relatively much less. Some non-precipitating sera failed to bind the fragment, although they bound HSA. These findings indicate that following neonatally induced immunological unresponsiveness the capacity to respond to antigen returns piecemeal in respect of different parts of the antigenic mosaic, and that it may be severely restricted. The theoretical implications are discussed. [ABSTRACT FROM AUTHOR]

A state of protein-overloading paralysis has been induced to bovine γ globulin (BGG) in adult CBA mice. Paralysis was demonstrated by a lack of immune elimination of 131I labelled BGG after prior challenge with Freund's adjuvant containing BGG. The degree of paralysis was found to diminish when the time between the paralysing injection and challenge injection was increased. This loss of the paralysed state could be prevented by injections of antigen whilst the mice were still paralysed. The superficial nature of the differences between paralysis (unresponsiveness induced in adults) and tolerance (unresponsiveness induced in neonatals) to BGG in CBA mice is discussed. The possibility that suppression of antibody production in previously immunized animals given a massive dose of antigen, is a distinct phenomenon from the induction of paralysis/tolerance in animals not previously immunized, is briefly discussed. [ABSTRACT FROM AUTHOR]

A state of immunological unresponsiveness to bovine serum albumin (BSA) or human gamma globulin (HGG) can be produced in guinea pigs by contact with these proteins either before and soon after birth, or even soon after birth alone. As judged by failure of treated animals to show immune elimination of the antigen, or anaphylaxis on reinjection, the animals were completely unresponsive when tested up to 5 months after birth. Administration of 6-mercaptopurine to guinea pigs, even in lethal doses, did not prevent the development of antibodies to HGG. After administration of BSA or HGG, labelled with 131I, to pregnant guinea pigs, their offspring at term contained significant amounts of the labelled proteins. HGG was catabolized in new-born guinea pigs with the same half-life as in adults. [ABSTRACT FROM AUTHOR]

Cold agglutinins were separated from the sera of eleven patients suffering from the cold-antibody type of acquired haemolytic anaemia by the dissociation of the specific antigen-antibody complexes. The electrophoretic mobility of the cold antibody was found to correspond to that of γl globulin in each case. Ultracentrifugal studies carried out on eluted cold antibodies prepared from the sera of seven patients demonstrated that the antibodies were macromolecules (S20,w∼18.1). In one instance the sedimentation coefficient of the cold antibody was demonstrated to be similar to that of electrophoretically separated ‘γ1 globulin’ prepared from the same serum, indicating their identical physical state. The results of treating the sera of nineteen patients with the cold-antibody type of acquired haemolytic anaemia with mercapto-ethanol further confirmed that the cold antibodies were all macromolecules, whether derived from idiopathic cases or cases secondary to some other known disorder. [ABSTRACT FROM AUTHOR]

Rabbit spleen cells, which have been treated in vitro with certain rabbit or guinea-pig antisera (e.g. against human serum albumin) and washed, are capable of specifically adsorbing radio-isotope labelled antigen. The antibody responsible for this effect appears to be distinct from the main precipitating anti-body in the serum and the term ‘cytophilic antibody’ has been suggested. The activity of the cytophilic antibody is not destroyed by heating at 56° C. for half an hour. The cytophilic antibody is not adsorbed by red cells. Spleen cells which have been treated with methanol before treatment with antiserum take up less antigen than non-methanol-treated cells tested in the same way. However, the addition of flesh normal spleen cells to the methanol-antibody-treated cells before the addition of antigen increased the uptake of the latter. [ABSTRACT FROM AUTHOR]

Cold antibodies were separated from the sera of six patients suffering from the cold-haemagglutinin syndrome and from one patient with acquired haemolytic anaemia secondayr to lymphosarcoma by dissociation of the specific antigen-antibody complexes. The eluted antibodies were studied (a) by immuno-electrophoresis along with the parent sera against horse anti-human serum and (b) by double diffusion in agar gel along with electrophoretically separated ‘γ1 globulin’† against anti-19S ‘γ-globulin’ rabbit sera. The protein forming the cold antibody was localized in the β2-M position on immuno-electrophoresis in each instance. It was found by double diffusion in agar gel to be immunologically identical with the protein forming the abnormal ‘γl-globulin’ electrophoretic peak in the parent serum. The results of these experiments indicate that the cold antibodies derived from patients with the cold-antibody type of acquired haemolytic anaemia are macro-molecular globulins. [ABSTRACT FROM AUTHOR]

The distribution of antigen and antibody in radial double diffusion systems was studied by means of materials labelled with 131I or 14C. These studies showed that, once a precipitate begins to form, the assumption that antigen and antibody obey the laws of free diffusion is invalid. They also showed that, in the systems used, no antigen and very little antibody diffused past the zone of visible precipitation. It was found that accurate estimates of the diffusion constants of antigens could be obtained by allowing antigen and antibody to diffuse from troughs set at right angles and by measuring the angle of the precipitin line. Examples of the use of this method, and a theoretical treatment are given. An alternative method for estimating the size of antigens is to use the ‘molecular sieve’ properties of gelatin gels, which are sharply graded with the concentration of the gel. [ABSTRACT FROM AUTHOR]

A critical review is given of Goldberg's (1952, 1953) theory of Antigen-Antibody reactions in vitro. Goldberg's assumptions and the mathematical development of his theory are described in detail. Comparisons of Goldberg's predictions with experimental results are given in graphical form. Some criticisms are made of Goldberg's use of equations in regions in which the derivations given by him are not valid. [ABSTRACT FROM AUTHOR]

Discusses the association between naturally occurring mutations in the hepatitis B virus (HBV) DNA and the outcome of chronic infection. Types of mutations in HBV; Occurrence of mutations at position 1762/1764 in the pregenomic promoter; Emergence of Pre-C mutations during seroconversion from hepatitis B e antigen (HBeAg) to the corresponding antibody; Reduction of the HBeAg expression on the transcriptional level.

Objective: To evaluate the effect of the periodontal treatment on Aggregatibacter actinomycetemcomitans JP2 clone, and the IgG serum levels against its outer membrane protein (Omp29) and A. actinomycetemcomitans serotypes in aggressive periodontitis (AgP). Subjects and Methods: Seventeen patients with generalized (GAgP), 10 with localized (LAgP), and 10 healthy controls were included. AgP participants were submitted to periodontal treatment—scaling and root planing plus antibiotics (SRP+A). Periodontal parameters, for example, probing depth (PD) and clinical attachment loss (CAL), were evaluated at baseline and at 1‐year. Serum IgG against Omp29 and serotypes a, b, and c were determined by ELISA. The levels of A. actinomycetemcomitans JP2 clone were determined in subgingival biofilm samples by qPCR. Results: Periodontal treatment resulted in significant reductions of PD, CAL, and IgG levels against Omp29, serotypes b, and c. After therapy, IgG levels against A. actinomycetemcomitans serotypes, as well as the levels of the JP2 clone in AgP, became similar to controls. The reduction in JP2 clone count was correlated with a reduction of PD and IgG response against Omp29. Conclusion: Scaling and root planing plus antibiotics decreased IgG levels response against Omp29 and A. actinomycetemcomitans serotypes involved in the disease (b and c), while the serum response increased against tne commensal serotype (a), similar to what occurs in periodontally healthy individuals. [ABSTRACT FROM AUTHOR]

Background: Sickle cell disease (SCD) patients undergo multiple red blood cell (RBC) transfusions and are regularly exposed to low-prevalence (LP) antigens specific to individuals of African descent. This study evaluated the prevalence of antibodies against LP antigens in SCD patients and the need to identify these antibodies in everyday practice.Study Design and Methods: Plasma from 211 SCD patients was tested with RBCs expressing the following LP antigens: RH10 (V), RH20 (VS), RH23 (DW ), RH30 (Goa ), KEL6 (Jsa ), and MNS6 (He).Results: Nine LP antibodies were found in eight patients (3.8%): five anti-RH23, two anti-RH30, and two anti-MNS6. The exposure risk, calculated for each LP antigen, was below 3% per RBC unit, for all antigens tested. Thus, in this cohort of transfused SCD patients, the prevalence of LP antibodies was similar to that of antibodies against antigens of the FY, JK, and MNS blood group systems. These findings also reveal the occurrence of anti-RH23 in SCD patients. No anti-RH20 or anti-KEL6 were found, despite the high frequency of mismatch situations.Conclusion: These results highlight the immunogenicity of these LP antigens, and the evanescence of antibodies against LP antigens. They also highlight the importance of appropriate pretransfusion testing for patients frequently transfused, who are likely to be exposed to multiple types of blood group antigens. [ABSTRACT FROM AUTHOR]

Background: Passive therapy with convalescent plasma provides an early opportunity to intervene in Ebola virus disease (EVD). Methods for field screening and selection of potential donors and quantifying plasma antibody are needed.Study Design and Methods: Recombinant Ebola virus glycoprotein (EBOV GP) was formatted into immunoglobulin G-capture, competitive, and double-antigen bridging enzyme immunoassays (EIAs). EVD survivors in Freetown, Sierra Leone, were recruited as potential plasma donors and assessed locally using sera alone and/or paired sera and oral fluids (ORFs). Uninfected controls comprised unexposed Gambians and communities in Western Area, Sierra Leone. Antibody neutralization in selected sera was measured retrospectively in a pseudotype virus assay.Results: A total of 115 potential donors were considered for enrollment: 110 plasma samples were concordantly reactive in the three EIAs; three were concordantly unreactive and two were reactive in two of three EIAs (98.2% agreement; 95% confidence interval [CI], 93.9%-99.8%). In 88 donors with paired ORF and plasma, G-capture EIA reactivity correlated well in the two analytes (R2  = 0.795). Plasma and ORF from 44 Gambians were unreactive. ORF samples from 338 of 339 unexposed Western Area community controls were unreactive (specificity, 99.7%; 95% CI, 98.4%-99.7%); ORF samples from 113 of 116 Kerry Town EVD survivors were reactive (sensitivity, 97.4%; 95% CI, 92.5%-99.5%). Strong reactivity in G-capture and/or competitive EIAs identified donors with high plasma EBOV GP antibody levels in the double-antigen bridging assay, correlating with high levels of neutralizing antibody.Conclusions: In-field testing can qualify convalescent donors for providing high-titer antibody. [ABSTRACT FROM AUTHOR]

Background: Influenza immune globulin, manufactured from plasma of convalescent or vaccinated donors has been proposed as a potential therapy for severe influenza. In 2009, a program was initiated to collect plasma from donors who self-identified as having had H1N1 influenza or having received the H1N1 pandemic vaccine. The goal of this study was to determine the efficiency of donor screening by self-identification without antibody testing, and to evaluate demographic predictors of high-titer donations.Study Design and Methods: Plasma samples from self-identified or control donors were randomly selected to evaluate hemagglutination inhibition (HAI) antibody responses. HAI titers were correlated with donor age, gender, location, and influenza exposure history.Results: Both self-identified vaccinated and convalescent donor groups had higher geometric mean titers (GMTs) against A/California/07/2009 (H1N1) virus compared to the control donors (39.9, 24, and 8.5, respectively). The proportion of samples with titers ≥64 in vaccinated, convalescent, and control donors were 54%, 37%, and 10%, respectively. Donations with titers ≤16 were predominant in control donors (80%) and substantial in convalescent (47%) and vaccinated (40%) donors. Titers did not correlate with donor age, gender, or geographical location. GMTs for vaccinated donors were significantly higher than for convalescent donors and in both groups significantly higher than in the control.Conclusion: Targeted collection of plasma containing high levels of anti-influenza antibodies from self-identified donors was effective, but could be further improved by reducing the number of low-titer donations. More selective donor screening and/or testing for influenza antibodies could increase the potency of an influenza antibody-rich immune globulin (FLUIGIV). [ABSTRACT FROM AUTHOR]

The article presents a case study of a 65-year-old woman with HbH disease who was admitted to a hospital with breathlessness. She was diagnosed with cholecystitis and administered with antibiotics. She was later diagnosed with delayed haemolytic transfusion reaction (DHTR)-triggered hyperhaemolysis. The recommended treatment for hyperhaemolysis is discussed, which includes methylprednisolone, folic acid, and hydroxycobalamin.