Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method.

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).

LUPUS ANTICOAGULANT (LA) COEXISTENT WITH TRANSIENT PROTHROMBIN (FII) INHIBITOR: FTI DEFICIENCY DUE TO CLEARANCE OF THE B/MUNOCOMPLEX

23 y.o. man with acute nephritis and bleeding (epistaxis, ecchymosis) at presen-taticn. Family and personal past history negative for bleeding. Laboratory data consistent with SLE. Coagulation tests: FT Ratio (R) 1.8, aPTT R 2.4, FII:C <1%, FIIR:Ag 996, other coagulation factors normal. Tissue thromboplastin inhibition test (TTIT) R 2.8, congenital FII deficiency (696) R 1.6.1. FII survival time (Fll-ccncentrate infusion - 60 U/kg) t1/2: 9 hours.2. FII neutralizing activity (FTI:C normal plasma (NP) + buffer 5996; NP + patient plasna {PtP) 5096): absent.3. Irmunoccrplex formaticn4. FII inhibitor characterization (purified FII coupled to CNBr-activatedSepharose →PtP incubation with Fll-Sepharose→specific antiFII irrrrunoglobulins (Ig)* elution at acid pH→identification by double iimunodifftision): precipitin line with anti IgA, anti IgG2, anti k, anti 1.5. LA characterization (after FII inhibitor disappearance): TTTT on mixtures NP + PtP or N Ig in equal volumes.Diagnosis: SIE, LA (IgG); polyclonal (IgA, IgG2, k, 1) not neutralizing FII inhibitor; hypoprothrxmbinemia due to clearance of the irrrrunocorrplex.FII inhibitor was transient. Bleeding was rapidly controlled by replacement therapy. LA persits after FII inhibitor disappearance.

Sugar composition and serological specificity of Erwinia carotovora lipopolysaccharides

Sugar composition and serological specificity of the lipopolysaccharides (LPS) purified from 16 Erwinia carotovora strains in six different serogroups were determined. All the LPS preparations contained 2-keto-3-deoxyoctonoic acid, glucosamine, heptose, and glucose, and most contained galactose. Either rhamnose or fucose was present in LPS from 12 of the strains, and the presence or absence of these deoxy sugars was consistent for all strains within a serogroup. LPS from two strains contained mannose. One unidentified sugar was present in all LPS preparations, but six other unidentified sugars varied in different LPS preparations. All LPS preparations reacted in an enzyme-linked, immunosorbent assay (ELISA) with antisera produced against whole cells of a type strain in the same serogroup as the strain from which the LPS was extracted. Several cross-reactions among strains that previously were observed in immunodiffusion tests with whole-cell preparations were also observed in ELISA with purified LPS. Some of the LPS preparations also reacted in immunodiffusion with a precipitin line of identity with whole-cell preparations. The results indicate that E. carotovora serogroups probably are based on the LPS 0-antigen.

Functional antigens of Trichuris muris released during in vitro maintenance: their immunogenicity and partial purification

SUMMARYThe immunogenicity of the secretory exo-antigen (EXA) originating in the stichosome of Trichuris muris has been investigated. High levels of immunity to challenge were produced using EXA (100 μg) by subcutaneous administration. Vaccination with a range of doses of EXA in Freunds' Incomplete Adjuvant (FIA) showed a dose response; 1 μg of EXA reduced the worm burden by 50% and doses of 10 μg or more reduced the worm burden by 80–90% at Day 9 post-infection. Worms recovered from vaccinated mice were significantly smaller than those from controls. A precipitin line identifiable with EXA was shown to immunize mice against T. muris challenge by vaccination. The protective antigen contained in EXA has been partially isolated using gel filtration, ion exchange filtration and isoelectric focusing and has been shown to be a protein of 30000 molecular weight having a pi of 6·8–7·3.

C-protein from rabbit soleus (red) muscle

A new form of skeletal-muscle C-protein has been isolated from rabbit soleus (red) muscle. This new form of C-protein has been purified to homogeneity by a procedure similar to that used to purify C-protein from white skeletal muscle. In soleus muscle, only this new form of C-protein could be detected, whereas in psoas (white) muscle, only the previously identified form of C-protein was detected. The content of C-protein in rabbit soleus muscle is comparable with that found in psoas muscle. Other rabbit skeletal muscles composed of a mixture of fibre types contained at least two forms of C-protein. C-Protein derived from red skeletal muscle bound to myosin isolated from either red or white tissue, with maximum binding occurring at a ratio of approximately 13 microgram of red C-protein/100 microgram of myosin. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that C-protein isolated from red skeletal muscle has a molecular weight approx. 7% greater than that of C-protein isolated from white skeletal muscle. The amino acid content of both forms of C-protein was similar but major differences in the mol % of isoleucine and threonine were found. Antiserum against C-protein from white rabbit skeletal muscle formed a single precipitin line with rabbit C-protein on double in agar. This antiserum did not form a precipitin line when diffused against red C-protein from rabbit skeletal muscle. Also, this antiserum bound specifically to the A-band region of myofibrils isolated from psoas (white) muscle, but it did not bind to myofibrils prepared from soleus (red) muscle.

Specific Precipitating Antibodies To VIII: CAg In Two Patients With Haemophilia

IgG and Fab fragments were prepared from the plasma of two haemophilia A patients whose anti- V111 : C titres were 2,000 and 400 Bethesda U./ml. 125-I-IgG and Fab specific for VIII :CAg were purified by a solid phase procedure and the reactivity of these two antibodies with VIII: CAg was studied by immunoprecipitation in agarose. With double diffusion and autoradiographic methods, both the IgG and Fab antibodies showed a precipitin line against normal plasma, serum, haemophilia A+ plasma, cryopre ci pi t ate , purified F.VIII/vWF or VIII: C free of vWF. No precipitin line was observed in haemophilia A- or severe von Willebrand’s disease. With electroimmunoassay (radi o-Laurell) using both IgG and Fab antibodies, results of VIII: C A g were in agreement in all samples with those obtained by immunoradiometric assay using the same antibodies. The specificity of the immunoprecipitation observed in agarose with IgG or Fab fragments was assessed by modifying the pH (7.5 to 9.5) of the buffer, the ionic composition (0.15, 0.5 and 1M sodium chloride, 0.15 and 0.5M potassium iodide, 0.15 and 0.5M sodium thiocyanate) of the washing fluid,or by carbamy1ation of the anti-VIII: CAg IgG. In all cases, specific precipitation was observed. In addition, 125-I-IgG or Fab isolated by the same technique from normal plasma gave no precipitinline with VIII: CAg. This study demonstrates that, contrary to previous evidence, 1) human anti-VIII: CAg antibodies are both precipitating as well as neutralizing when studied by highly sensitive techniques; and 2) monovalent Fab can also precipitate VIII: C Ag. The results of this study raise some questions about the lattice theory of immunoprecipitation.