Immobilization Assay Research Articles

Monoclonal antibodies to immunodominant surface-exposed protein antigens of Treponema pallidum.

Specific murine monoclonal antibodies directed against immunodominant surface-exposed protein antigens of Treponema pallidum with molecular weights of 15,500, 33,000, 44,000, and 46,000 were isolated. Of seventeen monoclonal antibodies characterized by Western blotting, three were directed against 15,500, three against 33,000, nine against 44,000, and two against 46,000 molecular weight protein antigens of Treponema pallidum. Three of the monoclonal antibodies were reactive in the haemagglutination assay, 11 in the in vitro immobilization assay, and 13 in the immunofluorescence assay. It is suggested that the different monoclonal antibodies could be useful in isolating immunodominant protein antigens of Treponema pallidum and in obtaining information on their biological relevance for use in the diagnosis of syphilis.

Monoclonal Antibody to a Human Germ Cell Membrane Glycoprotein That Inhibits Fertilization

Immunization of male or female animals of different species with homogenates of mature sperm, testis, or their extracts results in infertility. Antigenicity of sperm also has been implicated in the involuntary infertility of humans. The advent of hybridoma technology has made it possible to identify and purify sperm-specific antigens that are involved in fertility and infertility. The present authors selected one (MA-24) of 40 putative clones, secreting antibodies to sperm, for detailed analysis. This clone was determined to be of the 2a subclass of immunoglobulin G (lgG2). It was negative in the agglutination and immobilization assays, two methods that were used to test for antibodies to sperm in the serum of humans with involuntary infertility. MA-24 was germ cell-specific and did not bind to lymphocytes, erythrocytes, fibroblasts, lymphoblastoid cells, K562 leukemia cells, or other somatic cells by immunofluorescence. The MA-24 clone showed binding to the postacrosome, midpiece, and tail of viable and methanol-fixed human spermatozoa by immunofluorescence. It reacted with a single 23-kD protein band when a detergent-solubilized membrane preparation of human testis was used. The tissue-specific, but not species-specific, MA-24 antibody to the 23-kD protein completely blocked binding and penetration of zona-free hamster ova by human sperm. MA-24 also significantly inhibited fertilization in vitro of mouse eggs by murine sperm. The antigen (FA-1) to which the MA-24 antibody is directed has been isolated from human ejaculated sperm and testis by immunoaffinity chromatography. Preliminary data implicated FA-1 in human involuntary immunoinfertility. Sera from infertile patients with immunological involvement showed a strong binding to the purified antigen in the enzyme-linked immunosorbent assay. The binding pattern was comparable to that seen with the 0.3 M lithium diiodosalicylate extract of human sperm, a soluble preparation reported to contain surface antigen acting in fertilization. This finding indicates that the FA-1 antigen is immunogenic in humans. The mechanism or mechanisms by which MA-24 inhibits fertilization are not clear. Since both binding and penetration are inhibited in fertilization systems in vitro, these antibodies may hinder interactions between sperm and ovum or receptor recognition. The block is not due to agglutination or immobilization of sperm. MA-24 did not bind to ova, which shows the sperm specificity of the antibody. In the murine fertilization assay performed in vitro, MA-24 inhibited fertilization only in ova with intact zonae, and not when zonae were removed, which indicates that the inhibition occurs during the interaction between sperm and zona. In the heterogametic assay, however, MA-24 completely blocked binding and penetration of human sperm in zona-free hamster ova, an assay that has been reported to measure capacitation indirectly. These antibodies may be directed to a sperm component (enzymatic or nonenzymatic) that is vital for capacitation.

Treponemicidal levels of amoxicillin in cerebrospinal fluid after oral administration.

Seven patients in various stages of syphilis were treated by oral administration of amoxicillin (6 g daily) and probenecid (2 g daily) for 15 days. The treponemicidal level of amoxicillin was studied by a Treponema pallidum immobilization assay and found to be 0.070 micrograms/ml, as compared with 0.003 micrograms of penicillin/ml. Taking into account the WHO-recommended level of 0.018 micrograms/ml of penicillin, the minimal level of amoxicillin was estimated as 0.42 micrograms/ml. This level was obtained in serum and cerebrospinal fluid (CSF) of all patients treated with the above mentioned oral combination. It is concluded that treponemicidal levels of amoxicillin can be obtained in CSF after oral administration. The amoxicillin regimen described may be a valuable alternative to single-dose parenteral penicillin in the treatment of syphilitic patients with suspected CNS involvement, provided that reasonable compliance can be obtained.

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from [formula omitted] plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent D. discoideum cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [ 3H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.

Solubilization and isolation of the human placenta receptor for epidermal growth factor-urogastrone.

We describe the solubilization and purification of both the photoaffinity-labeled and unlabeled human placenta receptor for epidermal growth factor-urogastrone (EGF-URO). The photolabeled receptor can be purified 300- to 500-fold from membrane extracts by combined immunoaffinity and lectin-agarose affinity chromatography. This isolation approaches theoretical purity. Upon gel filtration and wheat germ agglutinin-Sepharose chromatography, the photolabeled receptor and the EGF-URO binding activity co-migrate, as measured by a newly developed lectin-agarose immobilization assay of receptor binding. Sequential ion exchange, Cibacron blue-Sepharose, wheat germ agglutinin-Sepharose, and gel filtration chromatography yield a 110-fold purification of the EGF-URO binding activity; this purified fraction contains protein constituents that exhibit electrophoretic mobilities parallel to those of the photolabeled receptor constituents, that have apparent molecular weights of 180,000 and 160,000. This molecular weight range is consistent with a measured apparent Stokes radius for both the photolabeled and unlabeled receptor of 5.1 nm (Sephacryl S-200 gel filtration), although an apparent radius of 4.3 nm is estimated by gel filtration on Sepharose-6B. The apparent molecular weight of the photolabeled receptor is unaffected by 2-mercaptoethanol. This work provides a basis for further detailed studies of this growth factor receptor.

The occurrence of antigenic determinants common to flagella of different salmonella strains.

AbstractFive Salmonella proteins, hitherto regarded as antigenically distinct, have been shown to share common antigenic determinants. It is proposed that the flagellar (H) antigens be designated as either Hv (variable) or Hc (common); in these terms the Kauffmann‐White schema describes the Hv determinants only. Antibodies to Hc determinants could be demonstrated by precipitation reactions detected by the Ouchterlony gel diffusion technique and by passive hemagglutination assays.To account for the finding that antibody to Hc determinants is not detected by bacterial immobilization assays it was proposed that Hv determinants are distributed along the flagella so that antibody can bind with two sites on the one flagella, thus disrupting the wave motion; in contrast, antibody to Hc determinants can only bind to one flagella at a time and therefore acts as a crosslink and does not cause immobilization.

Serum antibody to Salmonella flagellar antigens. I. Methods of antibody assay.

SummaryIn this report two methods are described for the assay of antibody to Salmonella flagellar (H) antigens. One depends on the agglutination of tanned sheep erythrocytes sensitized with polymerized flagellin, tanned red cell haemagglutination (TRC), and the other is a microprecipitin test employing nanogram quantities of I125 labelled monomeric flagellin. When compared with the Standard bacterial immobilization assay, the TRC is more sensitive in detecting natural antibody, human antibody and early primary antibody in rats. The microprecipitin test is most useful as an inhibition assay for determining the antigenicity of flagellar antigen preparations.

γM and γG Antibodies in Mice: the Response to S. Adelaide and the Effect of Splenectomy

Summary The primary antibody response to S. adelaide has been studied in mice using a flagellar immobilization assay. The change in spleen weight is bimodal with time: the first peak occurring on day 4 of the response and the second on day 10. Spleen homogenate supernatants also exhibit two distinct antibody peaks, but there is no direct correlation between the size of a spleen and its antibody content. The primary antibody response consists of the early, transient production of γM antibodies followed by the production of γG antibodies. This response is quantitatively reduced by the removal of spleens from mice before antigen injection. Splenectomy does not, however, alter the proportion of γM to γG as compared to intact mice.