An immunofiltration apparatus for accelerating the visualization of antigen on membrane supports.

Although proteins can be transferred and bound to a membrane with several different methods such as capillary transfer, electroblotting, etc., only the "shaker/incubation" method is commonly used for visualization of the proteins. We have tested an apparatus for the immunofiltration of solutions through nitrocellulose membrane which greatly accelerates the kinetics for the visualization of proteins. As a model system, avidin, bound on nitrocellulose membrane, was detected with 5-min filtering steps of a 1:2000 dilution of ascites of murine monoclonal antibody against avidin followed by a 1:5000 dilution of goat anti-mouse IgG-horseradish peroxidase and 0.5 mg/ml chloronapthol in the presence of 0.01% peroxide. The same solutions used with 5-min incubation steps with the shaker/incubation method could not detect avidin at almost 10 times that amount. Further studies with monoclonal antibodies specific for native C-reactive protein and modified-CRP, 15.1D6 and 13.3H12 respectively, showed that immunofiltration did not result in altered specificity compared to the shaker/incubation method. Also, data are presented showing the advantages of a 10-slot top for the immunofiltration of solutions through distinct areas of a membrane.

An appraisal of polystyrene-(ELISA) and nitrocellulose-based (ELIFA) enzyme immunoassay systems using monoclonal antibodies reactive toward antigenically distinct forms of human C-reactive protein.

The purpose of this study was to compare and contrast two enzyme immunoassay systems: the enzyme-linked immunosorbent assay (ELISA), which utilizes polystyrene microtiter plates as the adsorptive surface and the enzyme-linked immunoflow assay (ELIFA), which utilizes nitrocellulose membranes. The principal parameter under scrutiny was the denaturing or unfolding effects caused by the interaction of the protein with the adsorptive surfaces in each assay system. These effects were monitored by utilizing two conformationally distinct forms of human C-reactive protein (CRP), the native form of CRP and a denatured form (M-CRP), with a corresponding panel of monoclonal antibodies (MAbs) specific to either CRP or M-CRP. The results show that the ELIFA system was less sensitive than the ELISA system but that the ELIFA assay can be completed in less time than the ELISA. Also, adsorption of native CRP to the polystyrene surface in the ELISA system resulted in conformational changes of the adsorbed native CRP protein such that M-CRP reactive determinants were available for binding with anti-M-CRP MAbs, whereas native CRP adsorbed to the nitrocellulose membrane in the ELIFA system resulted in very limited conversion of CRP to M-CRP reactive epitopes. These results have important implications for development of immunoassays and screening of MAbs for proteins whose conformations may be affected by adsorption to various surfaces.

Follicle-stimulating hormone pulse amplitude decreases with the onset of the breeding season in the mare.

The relationship between daily mean FSH concentrations in serum and the pattern of FSH detected by frequent sampling for 12-h periods (samples every 15 min) was examined in five mares during the transition into the breeding season. The five mature anestrous mares were exposed to a natural increase in daylength. Blood samples were collected daily from February 1 until the first ovulation of the breeding season (April 14 +/- 3.7 days, Mean +/- SEM). Periods of frequent blood collection were performed every two weeks. Blood samples were obtained daily by jugular venipuncture or jugular cannula (frequent samples). Mean daily concentrations of FSH in serum determined by RIA decreased during seasonal transition. Patterns of FSH in serum detected by frequent sampling were pulsatile. FSH pulse amplitude decreased during seasonal transition, and the decrease in amplitude was associated with the decrease in mean serum FSH concentrations. This decrease in FSH pulse amplitude may reflect an involvement of a follicular product from developing follicles or a change in hypothalamic stimulation of pituitary FSH release.

Effect of pulsatile gonadotrophin release on mean serum LH and FSH in peri-parturient mares.

Changes in the pattern of LH and FSH in serum were studied in 6 mares foaling during the summer. Samples were collected frequently (every 15 min) for 24 h twice before foaling, -33 +/- 2 and -12 +/- 2 days, and for 12 h after foaling, on Day 0 and Day 4. Simultaneous pulses of FSH and LH were observed before foaling (r2 = 0.99). Before foaling, gonadotrophin pulses were infrequent (6 in 264 h of observation). On the day of foaling, LH and FSH pulse frequency increased (P less than 0.005) with 2-4 pulses per mare. The amplitudes of pulses of LH and FSH were higher before parturition than for those observed on Day 0 (P less than 0.001). The presence of FSH pulses was associated with an increased mean value of FSH for those days, including Day 0 (P less than 0.02). The presence of resolvable pulses of LH was not associated with an increase in mean LH values on those days (P greater than 0.6). The present results demonstrate that LH and FSH release is pulsatile in the periparturient mare. In addition, mean values of FSH in serum were elevated when pulses were detected, whereas mean concentrations of LH remained low. These results are consistent with the concept of one hypothalamic releasing hormone for LH and FSH in the mare.

Synthesis of human beta-endorphinyl-thiolglycine and its use for the preparation of affinity columns of beta-endorphin.

The peptide human beta-endorphinyl-thiolglycine (I) has been synthesized by the solid-phase method. The citraconyl derivative of peptide I was coupled to aminohexyl-Sepharose by reaction with silver nitrate/N-hydroxysuccinimide in water. The citraconyl groups were removed in aqueous acetic acid and the resulting resin was used in affinity chromatography for the purification of antisera to beta-lipotropin and beta-endorphin.