Immunochemical study of the specific antigens of human amniotic fluid]

Four specific antigens (trophoblastic beta 1-globulin, placental lactogen, alpha 1- and alpha 2-globulins of human placenta) were identified using antisera to the native amniotic fluid. Five antigens with the mobility of prealbumins, alpha 1-globulins, alpha 2-globulins and beta 2-globulins which bear no resemblance with the previously studied antigens were identified using antisera to the acid fraction of the amniotic fluid. Both the prealbumins and alpha 2-globulin were found in the blood serum of foetuses of different age and of newborn infants; these proteins were absent from the blood serum of pregnant women and donors. They received the names of embryonic prealbumine 1, embryonic prealbumine 2 and embryonic alpha 2-globulin. The protein with the mobility of alpha 1-globulins was found in the amniotic fluid of foetuses and in the blood serum of pregnant women only and received the name of amniotic alpha 1-globulin. The concentration of the antigens in question was studied in the developing foetuses and in the blood serum of pregnant women at different stages of pregnancy.     

Defective monocyte to macrophage maturation in human immunodeficiency virus infection

To look for possible defects in cells of the monocyte/macrophage system, blood monocytes from patients infected with human immunodeficiency virus (HIV) were cultured on hydrophobic Teflon for 7 days and their ability to differentiate into mature macrophages in the presence of serum was followed. The following parameters were studied as indicative of successful terminal maturation: (1) the expression of maturation-associated antigens (transferrin receptor, surface transferrin, the BA-2 antigen, MAX antigens), (2) the disappearance of the MOP15 antigen, and (3) a more than 20-fold increase in intracellular ferritin concentration. It was found that the patients′ blood monocytes did not differentiate in vitro but rather remained immature precursor cells. If the same holds true in vivo, the results could indicate that the pathophysiology of the acquired immunodeficiency syndrome (AIDS) may be, to a large extent, linked with the functional consequences of this impaired monocyte-to-macrophage maturation.     

Immunochemical determination of placenta-specific and interorganic antigens in placental extract and blood serum in pregnant rats

It has been demonstrated that placenta extract of rats contains up to 14 antigens. Moreover, 11 of them are interorgan proteins of wide and limited specificity, two antigens (alpha 1- and alpha 2-globulins) are attributed to acute-phase proteins typical for pregnancy. beta 1-Globulin is a specific protein of rat placenta. The content of these antigens in blood serum increases with pregnancy and reaches a maximum toward the delivery; 3-4 days after delivery beta 1-globulin disappears completely from maternal blood, whereas the concentration of acute-phase proteins drops to the initial level.     

Antidinitrophenyl antibodies in pigs and bulls

Using antigens fixed to cellulose (immunosorbents) the formation of antibodies at an average concentration of 1 mg per ml. of serum in pigs and bulls was shown after repeated immunization by dinitrophenylated γ-globulin in adjuvant. The amount of antibodies to the protein bearing the dinitrophenyl group was only exceptionally higher than 5% of the total amount of antibodies to the immunizing antigen. The dinitrophenyl group fixed directly to cellulose, binds only about one third of antibodies to the immunizing antigen. The antibodies were purified by precipitation with the immunizing antigen, followed by decomposition of the precipitate by a solution of dinitrophenol and separation of the mixture of the antibody, hapten and antigen on DEAE-Sephadex. The yield of antibodies was 60 to 70%. The antibodies were characterized as pure γ-globlin with a slightly lower electrophoretic heterogeneity than that of non-specific γ-globulin.     

Immunochemical study of the CBA mouse kidney]

Immunoelectrophoresis and agar precipitation reaction allowed one to reveal 7 antigens in CBA mouse kidney. One of them, with a relative electrophoretic mobility--0.35, is specific for the kidney. The remaining renal antigens are common to the kidney and other organs and tissues of mice. The antigens with mobility in the albumin and alpha-globulin zones proved to be serum ones. An antigen with a mobility approximating 0 is specific for the kidney and liver. An antigen with a mobility in the alpha 2-globulin zone does not seem likely to be homogeneous and, apart from broad inter-organ specificity, bears the greatest resemblance to the lungs antigen.     

Interaction of genes controlling two allotypes in chickens1

This paper presents the results of the investigations of the newly detected antigen of chicken blood serum, called K2. It was established that the K2 antigen which was identified with isoimmune serum was a β-globulin with the molecular weight over 200 000. The results of the genetic analysis based on sire-dam-offspring combinations seemed to indicate that the antigen under examination was controlled by a gene hypostatic to the gene controlling the previously described Kl allotype.     

Migration inhibition of alloimmune mouse macrophages by soluble transplantation antigens

Specific allogeneic transplantation antigens were solubilized and shown to inhibit the migration of alloimmune macrophages. Alloimmune macrophages treated with trypsin prior to antigen exposure migrated in the presence of the specific soluble antigens. The arming of nonimmune macrophages and the rearming of trypsinized immune macrophages with hyperimmune serum was readily observable using the migration inhibition test, whereas immune serum was ineffective.     

Immunochemical identification and physico-chemical characteristics of specific alpha 2 globulin of human granulocytes]

The antigen which has alpha 2-globulin mobility was identified immunochemically in leukolysate and pus extract. This antigen was localized in polymorphonuclear leukocytes by the immunofluorescence technique in the blood of healthy donors. The alpha 2-globulin of granulocytes (alpha 2GG) is not identical to lactoferrin, lysozyme, granulocyte elastase, fibronectin, fetal hemoglobin, amyloid P-component of the serum. The molecular mass of alpha 2GG is equal to 50 +/- 8 Kd. in the pus extract. The biological role of alpha 2GG is unknown.     

Immunological studies on insect metamorphosis. I. Qualitative and quantitative description of the blood antigens of the Cecropia silkworm

1. The cell-free blood of the Cecropia silkworm produces a maximum of nine bands of antigen-antibody precipitate when reacted in antiserum-agar tests with antisera prepared by injecting Cecropia extracts into rabbits. The blood antigens producing these bands of precipitate have the properties of proteins in that they are non-dialyzable, labile at 75°C., and salted out by 75 per cent saturated ammonium sulfate. One antigen was identified as a carotenoid protein. 2. Six bands of precipitate were selected for further study. Absorption tests revealed that the blood, at all stages of metamorphosis, is capable of precipitating the antibodies which produce five of these bands. This result indicates that five of the six antigens are present in the blood throughout metamorphosis. The sixth antigen is undetectable in blood from fourth instar larvae, appears in the blood late in the fifth instar, persists during the pupal stage, and disappears again during adult development. 3. When blood samples from various stages of metamorphosis were tested in antiserum-agar tubes, the rates of advance of the six bands of precipitate underwent systematic change in close correlation with the morphological stage of the silkworm's metamorphosis. These changes are interpreted in terms of concentration changes of the corresponding blood antigens. The validity of this interpretation was tested in several ways, with the conclusion that the interpretation was generally acceptable for the system under consideration. 4. All six antigens appear to increase in concentration during the last larval instar and to decrease in concentration during the period of adult development. However, each antigen has its own characteristic pattern of concentration change which differs from those of the other five. In order to explain this diversity, we conclude that the physiological mechanisms which regulate the synthesis and utilization of the blood antigens control each antigen on an individual basis.     

Effects of three strains of intestinal autochthonous bacteria and their extracellular products on the immune response and disease resistance of common carp,Cyprinus carpio

The study isolated three strains of intestinal autochthonous bacteria Aeromonas veronii BA-1, Vibrio lentus BA-2, and Flavobacterium sasangense BA-3 from the intestinal tract of the common carp (Cyprinus carpio). To reveal the effects of these three strains of bacteria on the innate immunity of carp, the lysozyme, complement C3, total serum protein, albumin and globulin levels, respiratory burst activity, phagocytic activity by blood leucocytes and the expression of IL-1b, lysozyme-C, and TNF-α were examined after feeding with seven different diets for up to 28 days. Also the survival of carp against Aeromonas hydrophila was challenged for 14 days. The carp were fed seven different diets: one control, three diets supplemented with 1 × 10⁸ cell g⁻¹ of carp intestinal bacteria BA-1 (Group D-I), BA-2 (Group D-II) and BA-3 (Group D-III), and three diets supplemented with extracellular products FA-1 (Group E-I), FA-2 (Group E-II) and FA-3 (Group E-III) which were corresponding to the strains BA-1, BA-2, and BA-3, respectively, up to 28 days. For groups D-I, D-III, E-I and E-III, the innate immune parameters of carp were significantly increased, the expression of three immune-related genes in blood was significantly up-regulated examined during 7, 14, and 21 days of feeding, and the survival rate was improved. The study indicates that the two isolated intestinal autochthonous bacteria A. veronii BA-1 and F. sasangense BA-3 could positively influence immune response and enhance disease resistance of carp against A. hydrophila infection.     

Immunochemical characteristics of alpha 2-globulin from the rat "pregnancy zone" in ontogeny

An antigen with electrophoretic mobility of alpha 2-globulins was found in the blood serum of the pregnant rats by means of electrophoresis. It is detected not only in the blood serum and tissue extracts of the pregnant rats, but occurs in low concentrations (1 microgram/ml) in the blood serum of the normal adult animals. The concentration of this protein attains the maximal values by the end of the pregnancy period. Alpha 2-globulin appears to be a protein associated with pregnancy.     

Laboratory Diagnosis of Canine Pyometra

The principal laboratory test used to confirm the pyometra diagnosis in the bitch has been the determination of the total white blood cell count in venous blood. A marked elevation is known to be a characteristic of the disease. In the present study, the white blood cell count was determined as well as the γ-globulin level. An elevation of the γ-globulin level and the total white blood cell count was very characteristic to the pyometra patients. The increase in the number of white blood cells nor the high γ-globulin level cannot be regarded specific for pyometra, therefore it was regarded important to find out a more specific test for pyometra. When sonicated E. coli bacteria were tested against sera from pyometra patients in electroimmunodiffusion, the precipitation was almost always detected when E. coli had been isolated from the uterus. This technique provides a quick method in detecting the causative E. coli infection. The present study suggests that whenever laboratory tests are used to confirm the pyometra diagnosis by the total white blood cell count, it is advantageous to analyze the total γ-globulin level in the serum as well as specific antibodies against a common E. coli antigen. Because of the reliability of the glutaraldehyde coagulation test and the simple technique, this can be suggested as the method of choice for an average small animal practice.     

Interactions between glycosaminoglycans and proteins, with particular reference to a new technique for isolating serum glycoproteins

1. Chondroitin sulphate–serum protein complexes (A, B and C), successively precipitated by adding chondroitin sulphate to serum at three arbitrary descending pH values (5·2, 4·3 and 3·1), were dissociated at pH 6·7 and chromatographed on DEAE-Sephadex, when the liberated serum proteins were simultaneously freed of chondroitin sulphate and separated into five fractions. Evidence that serum proteins were precipitated as a result of electrostatic interactions with dissociated carboxylate groups on the glycosaminoglycan is presented. 2. Serum proteins (fraction G), unable to form complexes with chondroitin sulphate, contained 4·4% of sialic acid and accounted for 4 and 26% of the total protein and protein-bound sialic acid in serum respectively. This fraction interacted electrostatically with chondroitin sulphate only when rendered more basic by removal of sialic acid residues with neuraminidase. The heat stability, solubility properties and high carbohydrate content of fraction G classified it as a seromucoid fraction. 3. Fraction G contained several glycoprotein and hexuronic acid-containing fractions, including a hitherto undetected brown-pigmented high-molecular-weight serum component, which migrated in starch gel between the origin and Sα₂-globulin and contained 3·1 and 4·1% of sialic acid and hexuronic acid respectively. 4. Glycosaminoglycan–protein interactions are discussed in relation to protein fractionation. By prior removal of less acidic proteins by these interactions, a new technique is available for isolating serum seromucoids in higher yields and under milder conditions than existing methods.     

Conversion of brain-specific complex type sugar chains by N-acetyl-beta-D-hexosaminidase B

The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.     

Glycan microarray profiling of parasite infection sera identifies the LDNF glycan as a potential antigen for serodiagnosis of trichinellosis

Diagnostic methods for parasite infections still highly depend on the identification of the parasites by direct methods such as microscopic examination of blood, stool and tissue biopsies. Serodiagnosis is often carried out to complement the direct methods; however, few synthetic antigens with sufficient sensitivity and specificity are available. Here we evaluated a glycan microarray approach to select for synthetic glycan antigens that could be used for serodiagnosis of parasitic infections. Using a glycan array containing over 250 different glycan antigens, we identified GalNAcβ1–4(Fucα1–3)GlcNAc-R (LDNF) as a glycan antigen that is recognized by antibodies from Trichinella-infected individuals. We synthesized a neoglycoconjugate, consisting of five LDNF molecules covalently coupled to bovine serum albumin (BSA), and used this neoglycoconjugate as an antigen to develop a highly sensitive total-Ig ELISA for serological screening of trichinellosis. The results indicate that glycan microarrays constitute a promising technology for fast and specific identification of parasite glycan antigens to improve serodiagnosis of different parasitic infections, either using an ELISA format, or parasite-specific glycan arrays.     

Discrepancy between flow cytometric analyses of CALLA using two monoclonal antibodies

Both the J5 and BA-3 monoclonal antibodies are considered to be specific for epitopes on the common acute lymphoblastic leukemia antigen (CALLA). Flow-cytometric analyses of three cell lines and one normal bone marrow sample using these antibodies as CALLA markers demonstrated that J5-labeled cells were always brighter than those labeled with BA-3, and that the ratio of their fluorescence intensities varied widely in the different systems. Furthermore, one of the lines, RPMI 8226, while positive for J5, appeared to be negative when labeled with BA-3, except for a slight displacement of the fluorescence distribution relative to the control. A possible explanation for the observed results is that the BA-3 binding epitope or epitopes on CALLA may vary in their number and/or accessibility to the antibody. These observations suggest that the use of a single monoclonal antibody to detect a cell surface antigen may be misleading, particularly when a negative result is obtained.     

Elimination of intestinally absorbed antigen into the bile by IgA

Polymeric serum IgA has been shown to transport antigens in the form of IgA immune complexes from the circulation into the bile. To test the hypothesis that this mechanism could aid in the clearance of intestinally absorbed antigens, mice were gastrically intubated with trinitrophenylated human serum albumin (TNP-HSA). After 3 hr, two groups were injected i.v. with either MOPC 315 (IgA, anti-TNP) or control ascitic fluid. One hour later, blood and bile samples were taken. Small amounts of antigen (0.0008% of the intubated dose) were detected in the serum of control animals by solid phase radioimmunoassay. In animals receiving anti-TNP IgA, serum levels were decreased significantly and intact antigen appeared in the bile, whereas no antigen was detectable in the bile of control mice. These results suggest that IgA-mediated hepatobiliary transport can function as one of the mechanisms for the elimination of intestinally absorbed antigens.     

Genetic markers in patients with intracranial aneurysms

HLA antigens, blood group systems (ABO, Rh, MNSs, P, Kell, Lewis and Duffy) and serum group systems (Hp, Tf, Gc, Pi, Bf, C3 and C4) were studied in a series of patients with intracranial aneurysms. A significantly increased frequency of HLA antigen A28, a significantly decreased frequency of HLA antigen B40, and a significantly decreased frequency of complement factor C4 B2 was found among the patients when compared with controls from the same geographic area.     

Further evidence for the B-cell nature of hairy cells. A study using immunostaining of splenic tissue with a wide panel of monoclonal antibodies

Phenotypic characterization of neoplastic cells from 5 patients with hairy cell leukemia (HCL) was performed with 29 monoclonal and 6 polyclonal (anti-Ig) antibodies using immunoperoxidase staining of fresh frozen splenic tissue. Monotypic Ig was expressed in 4 cases, one case was non-expressive. Strong staining was obtained in all cases by monoclonal antibodies (MAs) specific for 3 pan-B-lymphocyte antigens (by anti-B 1, To 15, anti-Leu 12). Five other B-cell related antigens detectable with appropriate MAs (BA-1, anti-B2, DAKO-C3 b R, Tü 1, 38.13) were absent in all cases. The stainings with 13 T-cell associated MAs (OKT 3, OKT 4, anti-Leu 3 a, OKT 6, OKT 8, Tü 68, OKT 10, anti-Lyt 2, Tü 71, OKT 11, anti-Lyt 3, Tü 14, Tü 33) were all negative. Stainings with 4 MAs recognizing myelocytic and/or monocytic antigens (OKM 1, anti-Mo 1, anti-Mo 2, 3 C4) were also negative. We included 14 frozen biopsies with B-type chronic lymphatic leukemia (B-CLL) into our immunohistological study in order to establish phenotypic differences between HCL and B-CLL. Five MAs (Tü 1, anti-Lyt 2, Tü 71, BA-1 and anti-B2) gave consistently negative staining in HCL cases but positive staining in most or all B-CLL cases. The study provides significant evidence for the B-cell nature of HCL and also establishes important phenotypic differences between HCL and B-CLL.     

Separation of antigens by immunological specificity: Use of cellulose-linked antibodies as immunosorbents

1. Immunosorbents were prepared by coupling activated aminocellulose with the γ-globulin concentrates of antisera prepared against ovalbumin and human serum albumin. 2. The immunosorbents were low in solubility, but high in capacity for homologous antigens. 3. The high specificity of these immunosorbents was demonstrated by their use in fractionating various mixtures of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme and ribonuclease.