An indirect two-site binding enzyme immunoassay has been developed to quantify the pregnancy associated globulin (PAG) in rats. Anti-PAG from guinea pig is adsorbed to solid phase and binds serum-PAG, which then reacts with its remaining free determinants with anti-PAG from rabbit. Sheep anti-rabbit IgG, labeled with horse radish peroxidase (HRP), binds to the rabbit anti-PAG antibody and the enzymic activity of the solid phase is measured by incubation with the appropriate chromogenic substrate. The optical density is directly related to the quantity of PAG to be measured. Reproducible results are obtained in the range from 5 to 200 micrograms/l. The test detects 12 fmoles of PAG. Female rats show a 1000-fold higher serum level of PAG than male rats. Strain differences of the distribution of the PAG serum concentration were only found between male rats.
<div>Abstract<p>Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; <i>P</i> < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; <i>P</i> < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis. (Mol Cancer Res 2007;5(2):153–63)</p></div>
The suitability of different carrier materials for absorption of serum was investigated with respect to a centralised screening for AFP serum levels in pregnant women, which needs a transmission of the samples. AFP was quantified in an indirect two-site binding enzyme immunoassay. AFP concentrations of native serum and of the eluate from the samples dried on filter paper were in a good agreement with a coefficient of correlation of 0.902. But storage of dried serum samples should not exceed one week because a two week storage diminished the coefficient of correlation to 0.766 and AFP concentrations of paper dried samples were overestimated.
