+ Translate

Preparation of rabbit anti immuno globulin e for use in radio immunoassays of total immuno globulin e and specific immuno globulin e antibodies

, : Preparation of rabbit anti immuno globulin e for use in radio immunoassays of total immuno globulin e and specific immuno globulin e antibodies. Journal of Immunological Methods 20: 185-200

Detailed methodology for preparing and testing isolated rabbit anti-human Ig[immunoglobulin]E suitable for radioimmunoassays of total IgE and specific IgE antibodies was presented. A method for obtaining a product free of anti-idiotype antibodies was also described. IgE was isolated from E-myeloma serum (PS) by 40 and 50% saturated ammonium sulfate fractional precipitation, followed by DEAE Sephadex ion-exchange chromatography. The purified product was digested with papain, and the Fc fragments were separated from the Fab fragments by G-150 gel filtration and ion-exchange chromatography. Rabbits were immunized with IgE (Fc), and the antisera with the highest precipitin titers were pooled. A globulin fraction was prepared from the pooled antiserum by 50% saturated ammonium sulfate precipitation, and the fraction was absorbed using an immunosorbent prepared from whole human serum having a very low IgE level; this globulin fraction was also absorbed with immunosorbents prepared from D-myeloma protein and from IgE (Fab). Following absorption, the antibodies were demonstrated by micro-Ouchterlony technique to have no cross-reaction with IgG, IgA, IgM, IgD or any serum protein other than IgE. More than 200 mg of isolated anti-IgE (Fc) was prepared from antiserum globulin by consecutive affinity chromatography on columns of insolubilized IgE. Elution peaks appeared following the application of 0.1 M glycine-HCl, 1.0 M NaCl buffer at pH 2.5, as well as following a subsequent flush with 0.1 M phosphate, 1.0 M NaCl buffer at pH 7.4. The eluates were tested, pooled, radiolabeled with iodine-125 and were demonstrated to be effective in RAST [radioallergosorbent test] analyses. Antibodies to idiotypic determinants of the IgE molecule were eliminated by preparing the affinity chromatography column from a 2nd E-myeloma protein (HL) rather than from the E-myeloma protein (PS) used for immunization of the rabbits. Anti-IgE preparations which were free of anti-idiotype antibodies displayed less nonspecific binding to IgG and IgD coated discs than preparations containing such antibodies.

(PDF 0-2 workdays service)

Accession: 006173758

Submit PDF Full Text: Here

Submit PDF Full Text

No spam - Every submission is manually reviewed

Due to poor quality, we do not accept files from Researchgate

Submitted PDF Full Texts will always be free for everyone
(We only charge for PDFs that we need to acquire)

Select a PDF file:

Related references

Bazin, H.; Platteau, B.; Beckers, A.; Pauwels, R., 1978: Differential effect of neo natal injections of anti mu or anti delta antibodies on the synthesis of immuno globulin m immuno globulin d immuno globulin e immuno globulin a immuno globulin g 1 immuno globulin g 2a immuno globulin g 2b and immuno globulin g 2c immuno globulin classes. The effect of neonatally initiated injections of or antibodies on Ig[immunoglobulin]M, IgD, IgA, IgE, IgG1, IgG2a, IgG2b and IgG2c subclasses were described. In chickens, mice and rats, suppression was demonstrated...

Thimel H., 1975: Preparation of mono specific immuno globulin fractions immuno globulin g immuno globulin m and anti immuno globulin sera anti immuno globulin g anti immuno globulin m for quantitative serum immuno globulin measurement in the dog. Zentralblatt fuer Veterinaermedizin Reihe B 22(5): 393-402

Droessler, K.; Haedge, D.; Ambrosius, H., 1982: Investigations on structural and functional properties of immuno globulin g 1 and immuno globulin g 2 antibodies in guinea pigs 2. production and identification of specific anti sera against immuno globulin g 1 and immuno globulin g 2 isotypes and the fc fc' and fab fragments of immuno globulin g 2. Rabbit antisera against specifically purified guinea pig IgG1 and IgG2 antibodies and against several papain-fragments of IgG2 were produced. These antisera were made specific by absorption with purified Ig of the other subclass and assayed for mo...

Van Knapen F.; Franchimont J.H.; Verdonk A.R.; Stumpf J.; Undeutsch K., 1982: Detection of specific immuno globulins immuno globulin g immuno globulin m immuno globulin a immuno globulin e and total immuno globulin e levels in human trichinosis by means of the enzyme linked immuno sorbent assay. Sera from 58 cases of confirmed human trichinosis were examined over an 11 mo. period. The enzyme-linked immunosorbent assay (ELISA) was used for the detection of class-specific serum antibodies (IgG, IgM, IgA, IgE) and for total serum IgE. The in...

Sprouse R.F.; Caldwell C.W.; Everett E.D., 1981: Radio immunoassays that use iodine 125 labeled protein a for determination of histoplasma capsulatum specific immuno globulin g immuno globulin a and immuno globulin m class antibodies in histoplasmosis. A radioimmunoassay for determination of human IgG, IgA and IgM antibodies to H. capsulatum is reported. Yeast-phase antigen from H. capsulatum was adsorbed onto polyvinyl chloride microtiter plates as the solid phase. Human antibodies were allowed...

Abraham, G.N.; Welch, E.; Trieshmann, H.W.Jr, 1978: Human tri clonal anti immuno globulin g gammopathy part 2 determination of the antigenic specificity patterns of the immuno globulin g immuno globulin a and immuno globulin m auto antibodies for the subclasses of immuno globulin g. The specificity and reactivity patterns of monoclonal Ig[immunoglobulin]G, IgA and IgM anti-IgG autoantibodies isolated from the serum of 1 patient (Gil) were determined for IgG of the 4 .gamma.-chain subclasses. The hemagglutination produced by t...

Robbins J.B., 1971: Specific secondary biological properties of purified rabbit immuno globulin m immuno globulin g and immuno globulin m immuno globulin g antibodies to brucella abortus and bordetella pertussis. Infection & Immunity: 220-227

Van-Munster, P.J.J.; Nadorp, J.H.S.M.; Schuurman, H.J., 1978: Human antibodies to immuno globulin a a radio immunological method for differentiation between anti immuno globulin a antibodies and immuno globulin a in the serum of immuno globulin a deficient individuals. In the sera of 12 of 27 individuals with IgA deficiency (serum level below 0.02 mg IgA/ml) class-specific anti-IgA antibodies were demonstrated by hemagglutination. These sera showed false-positive results in a solid-phase inhibition radioimmunoas...

Ottesen, E.A.; Weller, P.F.; Lunde, M.N.; Hussain, R., 1982: Endemic filariasis on a pacific island 2. immunologic aspects immuno globulin complement and specific anti filarial immuno globulin g immuno globulin m and immuno globulin e antibodies. Individuals (68) from a Pacific island hyperendemic for subperiodic bancroftian filariasis [Wuchereria barcrofti] were selected from a larger study population to include the entire clinical spectrum of filarial infection in that region and also an...

Hammarstrom L.; Smith C.I.E., 1983: Immuno globulin g 2 deficiency in a healthy blood donor concomitant lack of immuno globulin g 2 immuno globulin a immuno globulin e and specific anti carbohydrate antibodies. Lack of serum IgG2, IgA and IgE was found in a health male adult blood donor. No secretory IgA could demonstrated. In vitro activation of lymphocytes did not induce IgA secreting cells although no class specific suppressor cells could be found. No...