Glycosaminoglycan chains from α5β1 integrin are involved in fibronectin-dependent cell migrationDedicated to the memory of Professor Carl P. Dietrich.

α5β1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The α5β1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into α5β1 integrin also supports that integrin is a proteoglycan. Also, cells grown with xyloside adhered on fibronectin with no alteration in α5β1 integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, α5β1 integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of α5β1 integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of α5β1 integrin are involved in cellular migration on fibronectin.

The Interaction of TSH Receptor Autoantibodies with 125I-Labelled TSH Receptor

Detergent-solubilized porcine TSH receptor (TSHR) has been labeled with 125I using a monoclonal antibody to the C-terminal domain of the receptor. The ability of sera containing TSHR autoantibody to immunoprecipitate the labeled receptor was then investigated. Sera negative for TSHR autoantibody (as judged by assays based on inhibition of labeled TSH binding to detergent-solubilized porcine TSHR) immunoprecipitated about 4% of the labeled receptor, whereas sera with high levels of receptor autoantibody immunoprecipitated more than 25% of the labeled receptor. The ability to immunoprecipitate labeled TSHR correlated well with ability of the sera to inhibit labeled TSH binding to the receptor (r = 0.92; n = 63), and this is consistent with TSHR autoantibodies in these samples being directed principally to a region of the receptor closely related to the TSH binding site. Preincubation of labeled TSHR with unlabeled TSH before reaction with test sera inhibited the immunoprecipitation reaction, providing further evidence for a close relationship between the TSHR autoantibody binding site(s) and the TSH binding site. This was the case whether the sera had TSH agonist (i.e., thyroid stimulating) or TSH antagonist (i.e., blocking) activities, thus, providing no clear evidence for different regions of the TSHR being involved in forming the binding site(s) for TSHR autoantibodies with stimulating and with blocking activities. The ability of TSHR autoantibodies to stimulate cyclic AMP production in isolated porcine thyroid cells was compared with their ability to immunoprecipitate labeled porcine TSHR. A significant correlation was observed (r = 0.58; n = 50; P < 0.001) and the correlation was improved when stimulation of cyclic AMP production was compared with inhibition of labeled TSH binding to porcine TSHR (r = 0.76). Overall, our results indicate that TSHR autoantibodies bind principally to a region on the TSHR closely related to the TSH binding site, and this seems to be the case whether the autoantibodies act as TSH agonists or antagonists.

A new automated turbidimetric immunoassay for quantifying alpha 1-antitrypsin in serum.

This rapid, sensitive equilibrium turbidimetric immunoassay for quantification of alpha 1-antitrypsin involves a monospecific antibody, polyethylene glycol 6000 to accelerate and enhance the immunoprecipitation reaction, and Tween 20 surfactant to decrease and stabilize the sample-blank values. Turbidity at 334 nm is measured by an automated discrete analyzer. Grossly lipemic, icteric, or hemolyzed samples can be assayed. Correlation with results by radial immunodiffusion (RID) was excellent (r = 0.97, n = 84). Analytical recovery averaged 97.7 (SD 2.9)%. Within-run CVs ranged from 1.6 to 1.9%, between-day CVs from 2.0 to 3.5%. Reference values for healthy adults (n = 147) were determined by parametric estimation (for an assumed normal distribution of untransformed data). The lower limit (g/L) with its 0.90 confidence interval is 1.23 (range 1.18-1.28), the upper limit is 2.15 (2.10-2.20), and the mean is 1.69 g/L.

Viral immunoblotting: A sensitive method for detecting viral-specific oliogoclonal bands in unconcentrated cerebrospinal fluid

A new method for detecting viral antibodies in cerebrospinal fluid is described. The technique has many advantages over previously published methods in that it is highly sensitive eliminating the need to concentrate the CSF, takes 5 h to complete, avoids the use of radionucleides, and most importantly circumvents problems associated with prozone effects which occur in immunoprecipitation reaction since the viral antigen is immobilized on nitrocellulose membranes.

A unique protein in normal human cerebrospinal fluid.

On analysis of cerebrospinal fluid (CSF) samples from normal volunteer donors by high-resolution zone electrophoresis on agarose gel, an electrophoretically homogeneous protein band consistently appeared in the gamma-globulin region. Application of immunofixation electrophoresis in attempts to identify the band with use of monospecific antibodies against individual human serum proteins and against heavy- and light-chain immunoglobulins as well as polyvalent antisera did not produce a positive immunoprecipitation reaction with the protein band. The serum samples from these subjects did not show similar bands. Therefore, we conclude that this protein band is a normally occurring protein that is unique to CSF.

Turbidimetric immunoassay of serum C-reactive protein.

This rapid, reliable equilibrium turbidimetric immunoassay for serum C-reactive protein involves a potent monospecific antibody. Polyethylene glycol-6000 to accelerate and enhance the immunoprecipitation reaction, and Tween-20 surfactant to lower and stabilize the sample blank values. Grossly lipemic, icteric, or hemolyzed sera can be assayed. Values up to about 220 mg/L, for which the standard curve is linear, can be measured without sample dilution. Results by the proposed method and by radial immunodiffusion (r 0.989) or laser nephelometry (r = 0.957) correlated well. Analytical recovery averaged 101.3%. Within-, between-, and day-to-day CVs ranged from 0.9% to 3.5%, 0.8% to 5.5%, and 1.9% to 4.8%, respectively. The method is demonstrably superior to radial immunodiffusion or nephelometry. Any spectrophotometer that can measure turbidimetrically at 340 nm can be used.

Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.

RADIOIMMUNOASSAYS EMPLOYING DOUBLE ANTIBODY TECHNIQUES

ABSTRACT Double antibody methods are applicable to all radioimmunoassay systems. At least three approaches exist. The first, in which immunoprecipitating serum is used to precipitate soluble antigen-antibody complexes, is the most commonly used. Since the effects of interfering factors, especially those present in serum, become minimal as equilibrium is approached, care must be taken to insure that the period of incubation is of sufficient length for the immunoprecipitation reaction to reach equilibrium. In general, all efforts should focus on achieving reaction conditions which maximize stability and optimize immunoprecipitation. A second approach involves use of the first antiserum in an insolubilized form after prior precipitation with the immunoprecipitating antiserum. This approach is attractive since most non specific factors cannot interfere with the immunoprecipitation step to a significant extent once the formation of antigen-antibody aggregates is well underway. The third approach, in which immunoprecipitating antiserum is conjugated to a solid matrix, in principle represents a more ideal method of separating bound hormone from free. If this approach is ever to be utilized, a method of conjugating antiserum to a suitable matrix such that the antibody retains most of its immunoreactivity must be devised.