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Biomedical subjects

K S Joshi

Publications and source records attributed to K S Joshi.

8 recordsLinked to original sources
PubMed2006

Nonionic block copolymer antifoams.

Aqueous dispersions of alkoxylated alcohol block copolymer (BCP) drops are investigated as antifoams. A model aqueous nonionic surfactant solution of Polysorbate 20 and an industrial white water suspension are used as foaming systems. Visual evidence obtained using a two-bubble technique involving a CCD camera coupled with high magnification lenses clearly revealed the role of BCP droplets in the bubble coalescence process. The enhancement of bubble coalescence decreased as the temperature increased from 25 to 60 degrees C, which is due to the corresponding decrease in the rigidity associated with the weak interfacial structure and reduced viscosity of the BCP drops. The antifoaming efficiency measured in the macroscopic recirculation foam column increased with temperature from about 13 to 26 degrees C (attaining a maximum) and decreased as temperature increased further. Oscillatory thermo-rheometric measurements showed a sudden increase in the storage modulus (G') by several orders of magnitude, indicating gel formation initiated at about 13 degrees C and having a maximum at around 26 degrees C for an aqueous solution of the BCP above a critical concentration of around 20 wt %. Results obtained using small-angle X-ray scattering, micro-differential scanning calorimetry, and proton nuclear magnetic resonance confirmed the existence of ordered gel-like structures. Furthermore, macroscopic tests using a sparged air foam column showed a significant increase in antifoaming efficiency when highly hydrophobic particles are embedded in the BCP drops dispersed in water.

Journal Article↗
PubMed1993

The immunochemistry of sandwich ELISAs--VI. Greater than 90% of monoclonal and 75% of polyclonal anti-fluorescyl capture antibodies (CAbs) are denatured by passive adsorption.

Quantitative data are presented showing that the method most commonly used to immobilize antibodies in microtiter immunoassays functionally inactivates most of the antibodies. These results were collected using five affinity purified polyclonal antibodies (pAbs) and six monoclonal antibodies (mAbs) specific for fluorescein (FLU) as capture antibodies (CAbs). These CAbs were tested for their ability to capture FLU4.2-BSA after immobilization by passive adsorption, the Protein-Avidin-Biotin-Capture (PABC) system or using previously adsorbed anti-globulins. Results indicate that under optimal conditions, < 10% of monoclonal capture antibody equivalents (CAbeqv) and congruent to 22% of polyclonal CAbeqv remain functional after passive adsorption. Immobilization via the PABC system improved the performance of mAbs by more than five-fold but had less than a two-fold effect on pAbs. Many CAbs immobilized using an anti-globulin retained full activity including the ability to bind two molecules of FLU4.2-BSA/molecule of CAb. The latter result is not necessarily a recommendation for the use of anti-globulin immobilization, since the number of functional CAbeqv per well is not significantly greater than that which can be achieved using passive adsorption.

Adsorption↗
PubMed1992

The physical and functional behavior of capture antibodies adsorbed on polystyrene.

Six monoclonal and two polyclonal antibodies to fluorescein (FLU) were affinity purified and immobilized on Immulon 2 polystyrene as capture antibodies (CAbs): (a) by passive adsorption at pH 9.6, (b) via a streptavidin bridge to a biotinylated carrier molecule, and (c) via an antiglobulin which had been previously adsorbed passively to the polystyrene. Data show that less than 3.0% of the binding sites of monoclonal CAbs and approximately 5-10% of those of polyclonal CAbs were capable of capturing antigen (FLU4.2-BSA) after passive adsorption. Immobilization of CAbs via an antiglobulin or a streptavidin bridge, resulted in the preservation of antibody binding sites to greater than 70% for some monoclonals although immobilization via the streptavidin bridge resulted in the highest number of functional sites/well. The data presented are consistent with studies on other adsorbed proteins which demonstrate that passive adsorption on polystyrene results in the loss of protein function. Furthermore, these data show that generally less than half of the binding sites of antibodies available in solution are available after solid-phase immobilization even when non-adsorptive methods are employed. Some polyclonal anti-FLU also have lower average avidity following passive adsorption compared with CAbs immobilization via a streptavidin bridge. Immunochemical studies revealed that adsorbed polyclonal-CAbs performed like monoclonals when tested with multivalent antigens (FLU10-IgA) but in an expected heterogeneous manner in Scatchard plots when tested using univalent FLU-insulin. This observation implied cross-linking of immobilized CAbs by the multivalent antigen. Because only 5-10% of adsorbed polyclonal CAbs are active, the survivors must be non-randomly distributed in clusters to explain the cross-linking. This was confirmed by scanning electron microscopy which gave rise to the hypothesis that antibodies which retain activity after adsorption, are those present in clusters, i.e., the functional adsorbed CAb is an antibody cluster. Data presented in this report on the behavior of adsorbed CAbs, and reviewed from the work of others for various adsorbed proteins, indicate that the method of passive adsorption at pH 9.6, which is widely used in popular microtiter ELISAs, and which has in many ways revolutionized immunoassay, is a method of protein denaturation. Assayists that utilize passive adsorption of proteins on hydrophobic supports as part of their research need to be cognizant of this phenomenon, while inventors of immunoassay should develop alternative methods of immobilization which do not destroy 90% of the functional activity of solid-phase reactant.

Adsorption↗
PubMed1990

Humoral immunity in root caries in an elderly population. 1.

IgA, IgG and IgM antibody activity (ELISA Units/ml) to Streptococcus mutans, Actinomyces viscous and Escherichia coli CF8 in serum, parotid saliva and whole saliva was measured using the amplified ELISA (a-ELISA) while the concentration (microgram/ml) of each isotype of immunoglobulin as well as albumin and lactoferrin, was determined using sandwich ELISAs. Selection of suitable reagents from those commercially available was based on specificity tests using purified human immunoglobulin; most polyclonal reagents required further absorption to attain class specificity. Cross-absorption studies indicated the absence of patient antibodies that were cross-reactive among the bacteria studied, except for IgM in some cases. Expression of response in ELISA Units (E.U.) per microgram of immunoglobulin, i.e. specific activity, revealed that IgG specific activity was significantly higher in parotid saliva than in either whole saliva or serum for all bacteria studied; serum and whole saliva did not differ except for the higher specific activity in whole saliva to E. coli. The value of one E.U. was determined using the Comparative Antibody-immunoglobulin Capture Assay (CACA). Using this novel method, we estimated that about 0.05 percent of serum IgA was specific for Streptococcus mutans, 0.008 for Actinomyces viscosus and 0.004 for Escherichia coli CF8. The percentage of specific IgM antibodies was higher than for IgA and IgG. The concentration of IgA anti-Streptococcus mutans, Actinomyces viscosus and Escherichia coli levels are approximately 92 ng/ml, 25 ng/ml and 16 ng/ml in whole saliva and 46 ng/ml, 9.4 ng/ml and 6.3 ng/ml in parotid saliva.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomyces↗
PubMed1989

Multiparametric evaluation of retinoic acid-induced terminal differentiation of blastoid cells from acute non-lymphocytic leukemia patients in vitro.

Recent findings that retinoic acid (RA) induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line HL-60 in vitro and blast cell maturation in patients suffering from acute non-lymphocytic leukemia (ANLL) prompted an investigation on the ability of this agent to induce terminal maturation in blast cells from ANLL patients in vitro. We tested the ability of RA at 3 x 10(-6) M, 3 x 10(-7) M and 3 x 10(8-) M concentrations to induce differentiation in blastoid cells from 16 patients with ANLL using cytochemical and cytologic parameters, in addition to cytofluorometric methods. Leukemic cells in primary culture from all the patients underwent cytochemical and biochemical changes after treatment with RA. However, the extent of differentiation-positive cell clones (D+ clones) varied from patient to patient. Morphologic maturation was observed in a significant number of bone marrow samples. Leukocyte alkaline phosphatase and NBT reduction ability of cells, which are biochemical markers of granulocytic differentiation, were also significantly increased with a simultaneous decrease in DNA and RNA synthesis (which was estimated using a Phywe ICP-11 impulse flow cytometer).

Adolescent↗
PubMedDate not supplied

The immunochemistry of sandwich ELISAs--V. The capture antibody performance of polyclonal antibody-enriched fractions prepared by various methods.

Studies compare the performance of antibody-enriched serum fractions prepared by various methods, when adsorbed on polystyrene microtiter wells as capture antibodies (CAbs) and tested against multivalent antigens. The criteria of performance in the RIA used included antigen capture capacity (AgCC) and the nmol of functional capture sites per microtiter well (CAbt). Affinity purified polyclonal (pAb) and monoclonal antibodies (mAb) were employed as reference CAbs. AgCC was highest for enriched fractions prepared using caprylic acid and a high-pressure SpG affinity column. The performance of capture antibodies is expressed by an equation which was empirically derived and experimentally tested; CAbt x AgCC/ng adsorbed IgG. In terms of this parameter, CAb-enriched fractions prepared with caprylic acid performed best. The data reported also provide insight into solid-phase ligand immunochemistry. Adsorbed polyclonal CAb performed with remarkable homogeneity in percent bound and in Scatchard plots. Values obtained for CAbt from Steward-Petty plots were directly correlated with the length of the LBR of log-log percent bound plots but indicated that less than 10% of the potential capture sites of polyclonal CAbs remained functional after adsorption; mAb showed a more serious loss of activity. The loss of CAbt was a general phenomenon for all preparations tested although relative to their antibody content, certain antibody-enriched fractions retained a higher proportion of CAbt than their affinity-purified counterparts. Comparative studies in which the activity of adsorbed mAb and pAb was compared to the same antibodies immobilized by a non-adsorptive procedure, indicated that adsorbed CAbs also express lower affinity. The studies we report offer a single parameter criterium for comparatively evaluating CAb performance while simultaneously revealing the need to develop immobilization procedures that can preserve CAbt and antibody affinity so that immunoassays with wide dynamic ranges and high AgCC can be developed without waste of antibody.

Adsorption↗
PubMedDate not supplied

Terminal differentiation of human leukemic blasts in response to 12-0-tetradecanoyl-13-phorbol acetate (TPA).

Phorbol ester, 12-0-Tetradecanoyl-13-Phorbol-Acetate (TPA), induces a terminal macrophage-like differentiation of cells from human acute myelogenous leukemia cell lines. We report here that blastoid cells obtained from acute nonlymphocytic leukemia (M1-M2) undergo differentiation-related changes characteristic of macrophage lineage after exposure to TPA. Blast cells from a patient with ANLL-M1-M2 underwent morphological, functional and histochemical changes after treatment with 1 x 10(-7) and 1 x 10(-8) M TPA. The changes included adhesion to the plastic substrate, 2-4 fold increase in the number of NBT positive cells and an increase in the number of alpha-naphthyl-acetate esterase (alpha-NAE) positive cells. These differentiation changes after treatment with TPA were followed by decrease in proliferative index and G1 cells containing high RNA as estimated by flow cytometry. Of the thirteen cases of undifferentiated or unclassified leukemias studied, two failed to respond to TPA. These data suggest that leukemic blasts retain their ability to express a variety of differentiated functions on induction by TPA. Our data gives evidence suggesting that the "switch" into the differentiation pathways occurred after inhibition of proliferation and reduction in the percentage of G1 high RNA containing cells.

Adolescent↗