Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGβ, hCGβn, hCGβcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGβ-, 8 hCGα-, and 13 αβ-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1 %; epitope β1), 12 with low (<1.0 %; epitopes β2/4), and 13 with high (>>1 %; epitopes β3/5) hLH cross-reactivity. The majority of hCGβ epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of β-sheet loops 1 and 3 (epitopes β2–6; 27 mAbs) and the second around the cystine knot (e.g., epitopes β1, β7, and β10; 9 mAbs). Four mAbs recognized epitopes on hCGβcf-only (e.g., epitopes β11 and β13) and six mAbs epitopes on the remote hCGβ-carboxyl-terminal peptide (epitopes β8 and β9 corresponding to amino acids 135–144 and 111–116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGβ-only, or hCG together with hCGβ or hCG together with hCGβ and hCGβcf. Sandwich assays that measure hCG plus hCGβ and eventually hCGβcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGβcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope β1 (Asp10, Asp60, and Gln89) in combination with epitopes β2 or β4 located at the top of β-sheet loops 1 + 3 of hCGβ involving aa hCGβ20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-013-0994-6) contains supplementary material, which is available to authorized users.

ABSTRACTmRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated. One clone, pM1-11, contains all three exons of human c-myc. The second clone, pM4-10, represents a relatively rare transcript that initiated in the farst intron and includes the coding exons 2 and 3. The cDNA insert in pMl-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells. Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction. The protein produced in E. coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system. In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa. The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation. Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.Activation of the cellular myc gene has been associated with neoplasia in a variety of species. In transformed cells, the c-myc gene can be altered by several mechanisms that result in abnormal or elevated expression of c-myc RNA (1, 2) and, presumably, in increased levels of c-myc protein. Analysis of genomic clones of the human c-myc gene shows that it contains three exons (3). Exon 1 represents a long 5' noncoding leader region, whereas all of the coding sequences are located in exons 2 and 3. The coding sequences are highly conserved among species and with v-myc, the viral oncogene homolog harbored by avian myelocytomatosis virus (4, 5). Immunoprecipitation studies show that myc-specific antisera recognize proteins of =60 kDa in human cells (6-8). The highly conserved nature of the c-myc protein in many vertebrate species suggests that c-myc must supply a crucial function in eukaryotic cells. Such a function has not been elucidated, although studies on v-myc and c-myc indicate that this protein is found predominantly in the nucleus and binds to double-stranded DNA (9-13).Studies on the structure and function of the c-myc gene product have been hampered by the difficulty in obtaining sufficient amounts of protein for biochemical analyses from human cells that express elevated levels of c-myc RNA (unpublished observations). In this report, we describe the isolation and characterization of cDNA clones containing the complete coding region of human c-myc. The cDNA clones were used to express the c-myc gene in heterologous cells under regulated conditions. The c-myc gene products synthesized in Escherichia coli and in Saccharomyces cerevisiae were compared and found to exhibit host-cell-specific modifications. MATERIALS AND METHODSCell Strains. E. coli strain KRR123 was derived from strain RR1 (14) by phage P1 transduction of the lon9 allele from str...

Chromosomal DNA from strain UT400, a previously described deletion mutant of Escherichia coli K-12 that lacks outer membrane protein a, failed to hybridize with plasmid DNA (pGGC110) containing the structural gene for protein a. We designate the genetic locus for protein a, located at approximately 12.5 min of the E. coli chromosome, ompT.

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