Jens Krøll

Tandem Crossed Immunoelectrophoresis

Fig. 4.1. Tandem-crossed immunoelectrophoretic comparison of plasma and serum. Experimental Antigens: Normal human heparin plasma and serum were isolated from the same blood-letting. 3 samples of undiluted plasma (P) and serum (S) were placed in the application holes as indicated. 1st dimension: The first dimensional run was done at 8 V/cm for 1 hour (anode to the right). Antibodies: A polyvalent rabbit antiserum against human plasma proteins was obtained from Dakopatts, Copenhagen. The content of antiserum in the immunoelectrophoresis gel was 10% (V/V). 2nd dimension: The immunoelectrophoretic run was done at 1 . 5 V/cm for 18 hours (anode at top). Electrophoresis gels and contact bridges were made from 1% (W/V) agarose in barbital buffer ( p H 8.6, I:0.02) containing heparin 10 I.U./rnl. The same buffer but of higher ionic strength (P0.75) PRINCIPLE

Crossed‐Line Immunoelectrophoresis

This method combines the principles of crossed immunoelectrophoresis (Chapter 13) and line immunoelectrophoresis (Chapter 16). A rectangular line antigen-sample gel is placed along the first-dimensional crossed electrophoresis gel. The antigens contained in these gels are electrophoresed together into the antiserum gel. The resulting pattern of peaks standing on the respective precipitin lines permits a direct

DNA-binding proteins in Yoshida ascites tumor fluid

DNA-binding proteins were isolated from Yoshida ascites tumor fluid by chromatography on DNA-cellulose. This fraction represents 1-2% of the total ascites protein. Most of the DNA-binding proteins will bind to phosphocellulose as well. The proteins migrate by agarose gel electrophoresis at pH 8.6 as alpha and beta globulins. Quantitative immunoelectrophoresis revealed the presence of 12-18 proteins. SDS-polyacrylamide electrophoresis indicated molecular weights ranging from 3-10(4) to 10(6). Seven of the proteins were identified by specific immunoprecipitation as beta1-Eglobulin, beta2-glycoprotein I, fibrinogen split product E (fibrinogen E), coagulation factor XIII (factor XIII), alpha2-macroglobulin, IgG and IgM. Alpha1-antichymotrypsin might also be represented. In nuclear extracts of the tumor cells only factor XIII was present. With the exception of fibrinogen E and P5 all recognized DNA-binding proteins are present in normal rat plasma. With increasing tumor age the concentration of fibrinogen E, factor XIII, P5 and IgM increased both in ascites fluid and in plasma, while the concentration of other DNA-binding-proteins decreased or remained constant. Evidence is presented that the DNA- and phosphocellulose binding ascites protein fraction inhibit tumor cell growth. No inhibition was induced by corresponding protein fractions isolated from normal rat plasma.

Immunological identity between bovine preparations of thiol: protein-disulphide oxidoreductase, glutathione-insulin transhydrogenase and protein-disulphide isomerase

Five preparations of bovine thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase (EC 1.8.4.2) and one preparation of bovine liver protein-disulphide isomerase (EC 5.3.4.1) from four different laboratories showed immunological identity in double immunodiffusion and rocket-line immunoelectrophoresis. Consequently, thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase and protein-disulphide isomerase, formerly classified as two separate enzymes, should be considered as alternative activities of the same enzyme.

Specific antisera produced by immunization with precipitin lines.

Individual plasma proteins were precipitated, identified and isolated on the basis of line immunoelectrophoresis. A monospecific antibody response was induced by immunization of rabbits with less than 50 ng of precipitated antigen. Preservation of monospecificity was obtained by reimmunization with precipitates developed against the specific antisera.

Identification of immunoprecipitates in line immunoelectrophoresis.

A technique has been developed for the identification of immunoprecipitates in complex line-immunoelectrophoretic patterns. It is based on localized adsorption of individual antigens by monospecific antisera. The technique is suited for the following purposes as well: (1) testing the specificity and titer of uncharacterized antisera and (2) evaluation of binding properties of chromatographic media.

Renin binding proteins in plasma: Binding of renin to some of the plasma protease inhibitors, to lipoproteins, and to a non-trypsin-binding unidentified plasma protein

Renin is found in mouse plasma as high molecular weight forms, in addition to the fully active 40 000 dalton form. By using freshly 125 I-labelled 40 000 dalton pure submaxillary mouse renin, no binding to plasma proteins was demonstrable. However, unfolding and refolding of the labelled renin by guanidine facilitated binding to specific mouse and human plasma proteins. By using antibodies against individual human plasma proteins, the specific binding proteins were identified to be the plasma protease inhibitors: alpha2-macroglobulin, inter-alpha-trypsin inhibitor, alpha2-antithrombin. Binding was also demonstrated to alpha1- and beta1-lipoproteins, albumin and to a non trypsin binding unidentified plasma protein. No binding to 56 other tested proteins was demonstrable. It is concluded that the native 40 000 renin does not bind, but that a conformational change of the renin molecule most likely is necessary before binding occurs. It is discussed whether or not inactive or high molecular weight forms of renin in plasma are 40 000 renin bound to plasma protease inhibitors and lipoprotein.

Immunoelectrophoretic quantitation of trace proteins

A method is suggested for increasing the sensitivity of quantitative immunoelectrophoresis to a level comparable with radioimmunoassay. This is achieved by electrophoretic elution of antigen from 0.1-10 ml samples placed in glass reservoirs on the electrophoresis plate.

Identification of Bovine Serum Proteins by Quantitative Immunoelectrophoretic Methods

Line immunoelectrophoresis of bovine serum revealed a minimum of 37 bovine serum proteins. In line‐absorption immunoelectrophoresis, 13 of these proteins were identified by their cross‐reactivity with monospecific antisera against human serum proteins. One additional protein was identified by monospecific antisera against bovine immunoglobulins. Crossed immunoelectrophoresis revealed a minimum of 40 bovine proteins and demonstrated their electrophoretic mobility. In crossed‐line immunoelectrophoresis all 37 line precipitates were related to their corresponding peak precipitates and the electrophoretic mobility of the 14 proteins identificd was determined.

Quantitation of proteins by densitometric scanning of immunoelectrophoretic precipitates.

Densitometric scanning of stained immunoelectrophoretic precipitates of nine human serum proteins showed that the optical density of the precipitates is approximately proportional to the molar concentration of antigen in the sample. Deviations from this correlation were noted for the macroglobulins (mol. wt. greater than 15 x 10(4)). The resolving capacity of the line immunoelectrophoretic procedure permits densitometric valuations of composite patterns.