Biomedical subjects

P L Lim

Publications and source records attributed to P L Lim.

49 records · Page 3Linked to original sources

PubMed1988

A spectrophotometric method for evaluating a latex agglutination assay of Salmonella typhi lipopolysaccharide.

A spectrophotometer set at a wavelength of 400 nm was used to read reaction mixtures containing a 0.01% suspension of antibody-sensitized latex particles and different amounts of soluble antigen, after incubation in tubes for 30 min. The antigen used was Salmonella typhi lipopolysaccharide, and the antibody was an O-9-specific monoclonal antibody. Agglutination was indicated by a fall in the turbidimetric measurement compared to control, unagglutinated latex. It was observed that agglutination increased with increasing concentrations of antigen to a maximum at 0.25-1.0 micrograms/ml, after which, the amount of agglutination decreased at a similar rate as far as 1 mg/ml of antigen, when no agglutination occurred at all. Thus, a bi-symmetrical curve was obtained, suggestive of a precipitation reaction. At the 'equivalence point', the turbidity of the reaction mixture compared to control fell from OD400 1.1 to OD400 0.3. A similar inhibition of reaction at 'antigen excess' was observed visually in the reaction mixtures. Conventional slide tests performed in parallel could also be inhibited, but at higher antigen levels. Inhibition could be achieved with a commercially available latex used for the detection of Neisseria meningitidis antigen.

Antigens, Bacterial↗

PubMed1988

A comparison of immunological methods for the detection of Trichinella spiralis antigen.

Eight immunological methods all using the same monoclonal antibody reagent were compared for the detection of Trichinella spiralis antigen. These were based on: (1) the direct adsorption of the antigen to the immunoadsorbent (nitrocellulose membrane, polyvinyl chloride strip or microplate); (2) capture of the antigen by antibodies pre-sensitized on the immunoadsorbent; and (3) latex agglutination. The methods found suitable were: (a) capture radioimmunoassay (capture-RIA) (sensitivity: less than 0.5 microgram/ml antigen); (b) direct enzyme immunoassay (direct-ELISA) (less than 0.5 microgram/ml); (c) tube latex agglutination test (2.2 micrograms/ml); and (d) direct immunodot assay (8.8 micrograms/ml). However, the performance of the direct-ELISA was greatly affected by the presence of each of three extraneous substances (bovine serum albumin (BSA), lipopolysaccharide (LPS), normal swine muscle homogenate (NSM) added to the antigen sample. The direct immunodot assay was also affected by the presence of BSA or LPS, whereas both the capture-RIA and the tube latex agglutination methods were affected by the presence of NSM only. The findings are discussed with a view of detecting T. spiralis larvae directly from pork samples by immunological means.

Animals↗

PubMed1988

Regulation of an idiotype+ B cell lymphoma. Effects of antigen and anti-idiotopic antibodies on proliferation and Ig secretion.

The B cell surface Ig molecule plays an important regulatory role in delivering inductive/tolerogenic signals to the cell. In this paper, the effect of Ag and anti-idiotopic antibodies on the in vitro proliferation and Ig secretion of a B cell tumor was studied. The tumor (BCL1), which had been transfected with the TEPC-15 VH and VL Ig genes, expresses surface Ig and secretes antibody that binds the hapten phosphorylcholine. We found that Ag (C polysaccharide and phosphorylcholine carrier Ag) and two different anti-idiotopic antibodies, in the absence of T cells, all inhibited the proliferation of the T15+ transfectant cell line. The anti-idiotopic antibodies, but not Ag, also inhibited the secretion of T15 Ig by this cell line, suggesting different functional roles for Ag vs anti-Id in the regulation of B cell inactivation. The inhibition of secretion and proliferation appears to be cell cycle phase related. In addition, mouse rIL-4 could override the inhibition of proliferation induced in these studies. These phenomena, demonstrating that binding of surface Ig can result in the transduction of negative growth signals to a B cell tumor, can be viewed as a manifestation of immunologic tolerance. These findings collectively demonstrate that Ag and anti-Id mediate different signals to B cells via interaction with the surface Ig. Because of the monoclonal nature of the T15 transfectant and the anti-idiotypic antibodies, this system can be used to investigate the underlying molecular reactions involved in the B cell response and induction of tolerance.

Animals↗

PubMed1988

Alternative expression of secreted and membrane forms of immunoglobulin mu-chain is regulated by transcriptional termination in stable plasmacytoma transfectants.

During B cell differentiation the mu H chain of IgM is produced initially as a cell surface (m) and later as a secreted (s) form. Production of either the secreted (microseconds) or the membrane (micron) mRNA results from selection of either the promoter proximal (microseconds) or distal (micron) poly(A)/cleavage site. We have transfected lymphoid cell lines of disparate developmental stages with productively rearranged Ig mu-chain constructs to determine the mechanism by which alternative 3' end selection is achieved. Stable transfectants of lymphomas (early stage, microseconds less than or equal to micron) and a plasmacytoma (late stage, microsecond much greater than micron) produce the anticipated levels of microsecond and micron mRNA. Transcription termination between the microsecond and micron poly(A) sites occurs only in the plasmacytoma transfectants, and appears to be the rate-limiting step in the production of the final steady state microsecond/micron mRNA ratio. We propose that as the B cell becomes terminally committed to secretion, the mechanism responsible for the regulation of the steady state microsecond/micron mRNA ratio shifts from exclusively post-transcriptional to predominantly transcriptional termination.

Animals↗

PubMed1987

Isolation of specific IgM monoclonal antibodies by affinity chromatography using alkaline buffers.

Comparative studies on the efficacies of various buffers in eluting absorbent-bound antibodies revealed the denaturative effect of 3 M KSCN on all three IgM antibodies but not on an IgG protein, and the generally weak eluting power of glycine-HCl buffer, pH 2.5, on these monoclonal antibodies. A recently described medium consisting of 50% (v/v) ethylene glycol in an alkaline buffer, pH 10.5, was found to be relatively efficient for elution in all cases. However, subsequent studies on one of the IgM antibodies showed that alkali alone could effect elution, with recovery of active protein improving on increasing the pH, till the maximum (38%) at pH 11.0, after which denaturation occurred. Addition of ethylene glycol to the medium facilitated the elution; however, at pH greater than 10.0, the solvent potentiated the denaturative effect of the medium. Since pH 11.0 was found to be the highest pH in which all three IgM antibodies examined were stable, 0.1 M glycine-NaOH buffer, pH 11.0, may be a useful eluent for IgM (and other) antibodies in general.

Antibodies, Monoclonal↗

PubMed1987

Detection of group D salmonellae in blood culture broth and of soluble antigen by tube agglutination using an O-9 monoclonal antibody latex conjugate.

Latex particles sensitized with a Salmonella O-9 monoclonal antibody were used to detect group D salmonellae in blood culture broths. The system was found to be 100% accurate when used on 104 clinical specimens that included 13 S. typhi isolates. Enteric fever could thus be diagnosed as early as 1 day after the initiation of culture in some (18%) cases. The latex particles were also used in an experimental situation to detect soluble S. typhi lipopolysaccharide by a tube agglutination method. This was found to be more than four times as sensitive as the conventional slide agglutination method because at least 50 ng of antigen per ml in either buffer or urine was detected, albeit with less efficiency when serum was used.

Antibodies, Monoclonal↗

PubMed1986

Monoclonal IgM/A hybrid antibodies: artifacts due to anti-idiotype (T15) antibodies in commercial anti-alpha sera.

A mouse hybridoma antibody, with specificity for Trichinella spiralis, was found to react against both anti-mu and anti-alpha sera obtained from two to three commercial sources. It also had a unique electrophoretic mobility, bound to staphylococcal protein A, and was comprised of a single type of heavy chain (mu-like) and light chain (kappa). However, Northern blot analysis of RNA extracted from the corresponding hybridoma cells revealed only mu but no alpha message. Subsequent studies on the protein, as well as on a similar "hybrid" antibody previously described, using antisera raised to the antibody but which had been absorbed against normal mu and alpha determinants, suggested an idiotype common to both antibodies: this was intuitively guessed, and later confirmed, to be the phosphorylcholine-specific T15 idiotype. It was further shown that anti-T15 antibodies prevailed in the commercial anti-alpha sera and these (rather than alpha-chain specific antibodies) were responsible for the cross-reactivities of the sera with the hybrid antibodies.

Animals↗

PubMed1985

Intestinal absorption of bacterial antigens in normal adult mice. I. Preparation and characterization of the antigens.

The preparation of flagellin (FLA) from Salmonella adelaide and Boivin antigen (BA) from Vibrio cholerae is described. These two antigens differed chemically from each other and showed marked differences in their degradability by mouse intestinal juice in vitro and in their fate in the intact mouse when given intravenously. Unlike BA, FLA was rapidly degraded in intestinal juice and readily sequestered and degraded by the liver. The suitability of these antigens in oral absorption studies is discussed.

Animals↗

PubMed1985

Intestinal absorption of bacterial antigens in normal adult mice. II. A comparative study of techniques.

Marked differences were seen in the oral absorption of Boivin antigen (BA) and flagellin (FLA) using antigens labelled by 3H-dinitrophenylation. Thus, after feeding 3H-DNP-FLA, a large proportion of the label was rapidly absorbed, concentrated and degraded by the liver so that no antigenic material could be recovered from the circulation. In contrast, the absorption of 3H-DNP-BA was low and slow; the absorbed material appeared stable and not readily taken up by the liver, so that 8-20% of the non-dialysable radioactivity found in the plasma was precipitable by specific antibodies. Such observations could not be made when 125I-labelled antigens were used: deiodination readily occurred in vivo, and there was re-utilization of the released label. On the other hand, using everted gut sacs, it was possible to recover 125I-labelled antigenic material after transport. A technique of measuring unlabelled antigen absorbed in the intact animal is also described, which detects antigen by combination with radioiodinated antibodies injected intravenously into the animal.

Animals↗

PubMed1983

Diagnosis of enteric fever by inhibition assay using peroxidase-labelled monoclonal antibody and Salmonella typhi lipopolysaccharide.

An enzyme-linked immunoassay was developed for diagnosing enteric fever. The test measured the inhibition of binding between a labelled, monoclonal IgM antibody and the insolubilized antigen, Salmonella typhi lipopolysaccharide (LPS). Good discrimination was seen between 32 proven typhoid cases (88.0 +/- 4.4% inhibition) and non-typhoid cases. The latter consisted of 27 febrile patients bacteriologically and serologically (Widal test) found to be negative for typhoid (26.3 +/- 10.8% inhibition), 46 patients screened for syphilis (VDRL test) but found negative (31.2 +/- 13.3% inhibition), and 27 healthy blood donors (44.6 +/- 13.9% inhibition). The test also efficiently detected all 5 known typhoid carriers (90.6 +/- 3.4% inhibition). The antibody binds antigen 9 in the LPS; however, this reaction was inhibited by antibodies directed against both this antigen and an adjacent antigen, 12. Anti-12 antibodies presumably inhibit by steric hindrance and their importance in the test is discussed. Thus, the assay potentially detects (only) those systemic infections caused by salmonellae that possess antigen 9 or 12 (viz. S. typhi and S. paratyphi A).

Adult↗

PubMed1982

The effect of antibody on the intestinal absorption of macromolecules and on intestinal permeability in adult mice.

Oral immunization was shown to reduce the passage of bacterial antigen (BA) through the intestinal wall. The extent of the reduction found was dependent on the technique used for antigen assay, but with 125I-BA in an everted gut sac system, the amount of transported antigen was less than 60% of the amount in a control non-immune system. The inhibitory effect was due to coproantibodies which probably function by complexing and trapping the antigen in the mucous layer. This inhibition of antigen transport seems a prerogative of IgA antibodies since serum-derived antibodies caused concomitant adverse effects.

Aging↗

PubMed1982

Processing of orally absorbed immunogen.

The absorption of immunogen from the adult mouse intestine was demonstrated. This was shown indirectly by the ability of oral doses of a bacterial antigen to prime an animal for an anamnestic, systemic response, and directly by the ability of absorbed material from the plasma in vivo or everted gut sacs in vitro to prime normal mice when injected intravenously. In all cases, the resultant, systemic response in the recipient animals was qualitatively different (presumably due to IgA production) from that obtained in mice primed intravenously with the native antigen.

Administration, Oral↗

PubMed1978

Some interesting isolates from a diagnostic laboratory.

Citrobacter koseri, Plesiomonas shigelloides, Edwardsiella tarda, Yersinia enterocolitica, Alkalescens dispar, Vibrio parahaemolyticus, and Vibrio alginolyticus were seven interesting microorganisms isolated recently in our diagnostic laboratory.

Bacteria↗