Monoclonal antibodies requiring coating buffer with low pH for efficient antigen capture in sandwich ELISA: the rarities or practically important phenomena?

This article reexamines some opinions concerning pH requirements for optimal immobilization of monoclonal antibodies (mAbs) by passive adsorption in antigen capture ELISA. It was discovered that substitution of "classical" sodium phosphate (pH 7.5) and carbonate (pH 9.5) coating solutions by acid (pH 2.8) buffers maximized antigen capture 4 out of 10 different tested anti-HBsAg mAbs, resulting in a 1.5-2.5 increase of binding curve coefficients. By measuring both mAbs amounts and functionality, the enhancement effect was attributed to the better preservation of solid phase antibodies activity.

Beta-casomorphins-7 in infants on different type of feeding and different levels of psychomotor development.

Casomorphins are the most important during the first year of life, when postnatal formation is most active and milk is the main source of both nutritive and biologically active material for infants. This study was conducted on a total of 90 infants, of which 37 were fed with breast milk and 53 were fed with formula containing cow milk. The study has firstly indicated substances with immunoreactivity of human (irHCM) and bovine (irBCM) beta-casomorphins-7 in blood plasma of naturally and artificially fed infants, respectively. irHCM and irBCM were detected both in the morning before feeding (basal level), and 3h after feeding. Elevation of irHCM and irBCM levels after feeding was detected mainly in infants in the first 3 months of life. Chromatographic characterization of the material with irBCM has demonstrated that it has the same molecular mass and polarity as synthetic bovine beta-casomorphin-7. The highest basal irHCM was observed in breast-fed infants with normal psychomotor development and muscle tone. In contrast, elevated basal irBCM was found in formula-fed infants showing delay in psychomotor development and heightened muscle tone. Among formula-fed infants with normal development, the rate of this parameter directly correlated to basal irBCM. The data indicate that breast feeding has an advantage over artificial feeding for infants' development during the first year of life and support the hypothesis for deterioration of bovine casomorphin elimination as a risk factor for delay in psychomotor development and other diseases such as autism.

Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: evidence that transporter undergoes endoproteolytic cleavage.

BACKGROUND: Serotonin transporter (SERT) is important target molecule for many antidepressive drugs and substances of abuse and is implicated in psychiatric disorders. We performed immunoblotting analysis of human and rat SERT in platelets and brain using the panel of eight site-specific SERT monoclonal and polyclonal antibodies (mAbs and pAbs). METHODS: SDS-PAGE/Western blotting was conducted using peroxidase-labeled DEAE and affinity purified SERT antibodies under conditions preventing SERT post-extraction degradation. RESULTS: Immunoreactive polypeptides of 14, 22, 32, 35, 37, 56, 68, and approximately 150-200 kDa were revealed in human platelet extracts using N-terminal and C-terminal SERT antibodies. In rat brain, C-terminal mAbs detected 68, 56, and 37 kDa proteins, in postmortem human brain predominated 35-37 kDa proteins. The immunoreactivity was abolished after antibody preadsorption with antigens. N-terminal pAbs recognized the 68 kDa protein, affinity purified on C-terminal mAbs, confirming its identity as full-size human SERT (the predicted size approximately 70.5 kDa). CONCLUSIONS: The explanation of the results of immunoblotting most likely is a site-specific SERT endoproteolytic cleavage and a marked difference in glycosylation rather than nonspecific protein degradation, cross-reactivity with other epitopes or SERT alternative splicing.

Analysis of the binding of bispecific monoclonal antibodies with immobilized antigens (human IgG and horseradish peroxidase) using a resonant mirror biosensor.

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.