Liquid chromatographic purification and detection of anabolic compounds.

The role of liquid chromatography within methods of analysis for steroids, related compounds and beta-agonists in biological samples is discussed. Special attention is given to the application of liquid chromatography in sample preparation and extract clean-up. Different forms of liquid chromatography, including immunoaffinity chromatography, are compared and evaluated. Methods for confirmation based on gas chromatography-mass spectrometry and cryotrapping Fourier transform infrared spectrometry are discussed.

Monoclonal antibodies raised against Pap fimbriae recognize minor component(s) involved in receptor binding.

Pap fimbriae were purified from a recombinant strain and used for the production of monoclonal antibodies (MAbs). These MAbs were screened in a fimbriae ELISA with eight different P fimbriae as well as 1A and 1C fimbriae. Five MAbs were specific for Pap fimbriae whereas one MAb did react with Pap, F7(2) and F11 fimbriae. Previously, we described two F11 MAbs which also reacted with Pap, F7(2) and F11 fimbriae. In a whole bacteria ELISA it was shown that the MAbs, which recognized Pap, F7(2) and F11 fimbriae, reacted with recombinant strains which did not express Pap or F11 fimbriae, but still expressed the globoside binding properties. Not one of the five MAbs which are specific for Pap fimbriae reacted with these globoside binding recombinant strains. In a haemagglutination and adherence assay it was shown that only the MAbs which recognized the Pap, F7(2) and F11 fimbriae inhibited the adhesive properties of the globoside binding recombinant strain. Therefore it is concluded that in the present study MAbs are presented which recognize the minor components responsible for adhesion.

Monoclonal antibodies for serotyping the P fimbriae of uropathogenic Escherichia coli.

Monoclonal antibodies (MAbs) against seven serologically different P fimbriae (F7(1), F7(2), F8, F9, F11, F12, and F13) of uropathogenic Escherichia coli were tested for their ability to detect the P fimbriae on wild-type strains. In a plate agglutination test the MABs could detect the fimbriae on strains which expressed cloned fimbriae but not on wild-type strains. In a coagglutination test and in a whole-bacterium enzyme-linked immunosorbent assay the MAbs recognized the fimbriae on strains with cloned fimbriae and on wild-type strains. However, the coagglutination test has some disadvantages: only immunoglobulin G MAbs can be used, and the results cannot be read in an objective way. From these results, we concluded that the whole-bacterium enzyme-linked immunosorbent assay is the most convenient method for the determination of P fimbriae on wild-type E. coli strains. With this fast and easy method it is possible to do epidemiological studies on the distribution of P fimbriae among clinical isolates of uropathogenic E. coli and to extend the O:K:H serotype with the F serotype.

Monoclonal antibodies that recognize the P fimbriae F71, F72, F9, and F11 from uropathogenic Escherichia coli.

The Escherichia coli P fimbriae F71, F72, F9, and F11 from four cloned strains were purified, and polyclonal antisera were raised in rabbits. Cross-reactions of these antisera with eight different cloned and purified fimbriae were measured in an enzyme-linked immunosorbent assay. These antisera showed a reaction with the homologous fimbriae and also with most heterologous fimbriae. Monoclonal antibodies (MAbs) directed against the same four native fimbriae were produced by the fusion of spleen cells from immunized BALB/c mice with SP2/0 myeloma cells. The resulting four series of MAbs were also screened in an enzyme-linked immunosorbent assay with eight different cloned and purified fimbriae. Four different F71 hybridomas produced MAbs which recognized only epitopes on F71 fimbriae. Two F72 MAbs recognized epitopes on F72 and F9 fimbriae, whereas another F72 MAb recognized an epitope on only F72 fimbriae. Three MAbs raised against F9 reacted only with epitopes on F9 fimbriae. Six MAbs against F11 fimbriae could be divided into two groups: on the one hand two MAbs recognizing F11, pyelonephritis-associated pilus, Pap, and F72 fimbriae and on the other hand four MAbs recognizing F11 and "Clegg" fimbriae. None of the MAbs reacted with 1A or 1C fimbriae. In a hemagglutination inhibition assay it was shown that none of the MAbs produced inhibited the adhesive properties of homologous cloned strains.