R Factor-Mediated Resistance to Aminoglycoside Antibiotics in Pseudomonas aeruginosa

Conjugal transferability of drug resistance was examined, in eleven Pseudomonas aeruginosa strains which were isolated in Frankfurt. Four R factors were demonstrated from three strains using P. aeruginosa as recipients but they were nontransferable to Escherichia coli K12. Two R factors, i.e., R_ms146 and R_ms147, mediated resistances to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), kanamycin (KM), lividomycin (LV), gentamicin C complex (GM) and 3′,4′-dideoxykanamycin B (DKB). They mediated the formation of aminoglycoside-inactivating enzymes, i.e., SM phosphotransferase, SM adenylyltransferase, KM and LV phosphotransferase 1, and GM and DKB 6′-N-acetyltransferase. TC resistance conferred by these R factors was due to impermeability of the drug. P. aeruginosa Ps 142 carried two kinds of R factor in one cell, R_ms148 (SM) and R_ms149 (SM·SA·GM·CPC) (CPC, carbenicillin). R_ms148 (SM) was transferable at a high frequency of 10^–1 and mediated the formation of SM phosphotransferase. R_ms149 mediated the formation of drug-inactivating enzymes, i.e., GM 3-N-acetyltransferase and β-lactamase, but did not inactivate SM. SM resistance was probably due to impermeability of the drug.     

Deletion mutants from R plasmids with increased copy number.

Rms 201-12 and Rms 201-46 are R mutants with increased copy number, and are derived from a conjugative plasmid Rms 201 that encodes resistance to five drugs, ampicillin (Apc), tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfonamides (Sa). The mutants expressed increased levels of resistance to Apc, Sm, and Cm, and a decreased level of resistance to Tc than those of the parent Rms 201 plasmid. When the Rms 201-12+ or Rms 201-46+ cells were inoculated onto plates containing a high concentration of Tc, colonies developed on the plate at a frequency of 10-3 to 10-4 after overnight incubation. The cells grown on the Tc plate carried a tet (Tc gene)-deleted R mutant besides tet-possessing Rms 201-12 or Rms 201-46, and we isolated the tet-deleted R mutant by purifying the R+ cells on drug-free plates. On the other hand, various deletion mutants possessing tet were isolated by prolonged culture of the cells. We have presented a circular gene order of Rms 201 by comparing the genetic markers of all deletion mutants derived from Rms 201, Rms 201-46, and Rms 201-12. The gene(s) regulating the copy number was closely linked to the rep gene. The gene(s) specifying entry exclusion was jointly lost with the tra region.     

Thermosensitive Replication of a Kanamycin Resistance Factor

A strain of Proteus vulgaris isolated from the urinary tract of a patient with postoperative pyelonephritis and resistant to sulfonamide, streptomycin, tetracycline, and kanamycin (KM) was found to transfer only KM resistance by cell-to-cell conjugation. The genetic determinant controlling the transferable KM resistance was considered to be an R factor and was designated R (KM). Successive transfer of KM resistance was demonstrated also from Escherichia coli 20S0, which received the R (KM) factor, to other substrains of E. coli K-12 or Salmonella typhimurium LT-2. The transfer of the R (KM) factor was strongly affected by the temperature at which the mating culture was kept. The transfer frequency of R (KM) at 25 C was about 10(5) times higher than at 37 C. The R (KM) factor was spontaneously eliminated from the host bacterial cells when P. vulgaris was cultured at 42 C, but no elimination occurred at 25 C. This elimination of the R (KM) factor at elevated temperature was also observed when the R (KM) factor infected E. coli and S. typhimurium. On the other hand, a normal R factor could not be eliminated from the same E. coli host strain by cultivation at the higher temperature. We consider the thermosensitive transfer and the spontaneous elimination of the R (KM) factor at higher temperature to depend upon thermosensitive replication of the R (KM) factor.     

In vivo formation of transmissible resistance factor by recombination between nontransmissible resistance factor and Col B factor.

Germ-free swine were artificially contaminated with tetracycline (TC) sensitive strains of Escherichia coli and Klebsiella pneumoniae. One of these strains, E. coli 3306, was infected with a plasmid carrying kanamycin (KM) resistance, i.e., T-kan factor. Another strain, E. coli P-5, carried a conjugally transferable Col B factor. Among the nine strains used, only E. coli P-38 became TC-resistant after TC administration. Three types of TC-resistance E. coli P-38 strains were found; (a) one strain carried nontransferable TC resistance and could not produce colicin, (b) one strain carried TC resistance with a high transmission frequency which could not produce colicin, and (c) one strain carried TC resistance with a low transmission frequency that could produce colicin B. Genetic studies disclosed that the transmissible TC resistance factors, i.e., Rms105 (group b) and Rms104 (group c), were formed by recombination between Col B factor and nontransmissible TC-resistance (tet) determinant which appeared in E. coli P-38 mutants.     

Salmonella typhimurium LT2 mutation affecting the deletion of resistance determinants on R plasmids.

Plasmid Rms312, specifying resistance to tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), sulfonamide (Su), and mercury chloride (Mer), deletes both Tc and Cm Sm Su Mer determinants at a high frequency in Salmonella typhimurium LT2. S. typhimurium mutants that were stable carriers of Rms312 were isolated by alternate culture of R-bearing cells in a medium containing either tetracycline or chloramphenicol. In one of these mutants the deletion frequency of drug resistance determinants was decreased by about 100-fold not only Rms312, but also in R100, R1, and R6-5. This mutation caused a slight reduction of ultraviolet resistance but did not affect generalized genetic recombination, indicating that the mutation is different from recA. The mutation, designated dor (deletion of r-determinants), was mapped to a position near 57 units in the new linkage map of S. typhimurijm LT2 (K. E. Sanderson and P. E. Hartman, Microbiol. Rev. 42:471-519, 1978). The dor mutation had no effect on IS1-mediated illegitimate deletion, indicating that the dor mutation is different from the del mutation described by Nevers and Saedler (P. Nevers and H. Saedler, Mol. Gen. Genet. 160:209-214, 1978).     

Recombination Between a Thermosensitive Kanamycin Resistance Factor and a Nonthermosensitive Multiple-Drug Resistance Factor

The thermosensitive kanamycin (KM) resistance factor, R(KM)(t), and a nonthermosensitive multiple-drug resistance factor, R(100), were simultaneously introduced into Escherichia coli and Salmonella typhimurium. The temperature sensitivity of both R factors remained unchanged as long as they replicated independently. Under certain conditions, however, a new thermosensitive R factor harboring resistance markers for kanamycin, streptomycin (SM), and sulfanilamide (SA) was obtained by recombination between the R(KM)(t) and R(100) factors. R factors carrying resistance markers for KM and SA, or for SM and SA, were obtained from the recombinant R(KM SA SM)(t) by spontaneous segregation. Though the R(100) factor has been known as an fi(+) (positive for F-mediated fertility inhibition of its host) type and it does not restrict any coexisting phages, the thermosensitive recombinants of R(100) with R(KM)(t) and their segregants were found to be fi(-) and to restrict the replication of all T-even phages, as does the R(KM)(t) factor. Double infection immunity was not observed between the R(KM)(t) and R(100) factors.     

Epistatic effects of immunoglobulin GM and KM allotypes on outcome of infection with hepatitis C virus

Immunoglobulin GM and KM allotypes-genetic markers of gamma and kappa chains, respectively-are associated with immune responsiveness to several infectious pathogens and with survival in certain viral epidemics. We hypothesized that GM and KM allotypes affect the outcome of hepatitis C virus (HCV) infection. To test this hypothesis, we serologically allotyped 100 persons with well-documented clearance of HCV infection and 198 matched persistently infected persons. None of the GM or KM phenotypes by itself was associated with the clearance or persistence of HCV infection. Particular combinations of these phenotypes, however, were significantly associated with the outcome of HCV infection. Subjects with GM 1,17 5,13 and KM 1,3 phenotypes were over three times (odds ratio [OR], 3.57; 95% confidence interval [CI], 1.44 to 8.87) as likely to clear the infection as the subjects who lacked these phenotypes. This GM phenotype had a similar association with clearance in the absence of KM 3 (OR, 2.75; 95% CI, 1.21 to 6.23). The presence of GM 1,3,17 23 5,13 phenotype (in the absence of KM 3) was associated with persistence (OR, 0.21; 95% CI, 0.06 to 0.77), while its absence (in the presence of KM 1,3) was associated with the clearance of infection (OR, 2.03; 95% CI, 1.16 to 3.54). These results show epistatic interactions of genes on chromosomes 14 (GM) and 2 (KM) in influencing the outcome of an HCV infection. Further investigations involving candidate genes (GM, KM, HLA, and Fcgamma receptors) and cellular and humoral immune responses to HCV epitopes are needed to understand the mechanisms underlying these associations.     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

A mutant defective in partitioning of composite plasmid Rms201.

Escherichia coli harboring mutant plasmids defective in maintenance stability (from the conjugative plasmid Rms201) showed a wide distribution of ampicillin resistance levels, as well as increased frequency of plasmid loss from the cell. The amounts of covalently closed circular deoxyribonucleic acid of mutant plasmid Rms268 and parental plasmid Rms201 per chromosome were 5.3 and 6.1%, respectively. The beta-lactamase activities of strains W3630(Rms268) and W3630(Rms201) were 0.56 and 0.44 U/mg of protein, respectively. Frequency of plasmid loss from W3630(Rms268) was about 0.8 to 1.2% per cell generation, 100 times more than that of the wild-type strain. Ampicillin resistance levels of the colonies harboring the mutant plasmid showed a wide distribution, from low (100 micrograms/ml) to high (1,600 micrograms/ml). A miniplasmid (pMS268) with a mass of 7 X 10(6) daltons and encoding ampicillin resistance was isolated from Rms268. Frequency of pMS268 loss from W3630(pMS268) was about 0.8 to 1.9% per cell generation. W3630(pMS268) also showed a wide range of distribution in the levels of ampicillin resistance. These results indicated that the copies of Rms268 in E. coli did not segregate evenly between daughter cells at cell division and that the gene involved was located on the miniplasmid.     

Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)⁺ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)⁺ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 10⁶ and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10⁶. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.     

Regular segregation of composite plasmid Rms201.

Copy number mutants Rms201ts15 and Rms201ts16 were isolated at 30 degrees C from a temperature-sensitive replication mutant (Rms201ts14) of the conjugative plasmid Rms201. The numbers of plasmids per chromosome of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) grown at 30 degrees C were 2.2, 7.4, and 20, respectively. The synthesis of covalently closed circular plasmid deoxyribonucleic acid stopped in Rms201ts14, Rms201ts15, and Rms201ts16 immediately after a "shift-up" in temperature (42 degrees C). At 42 degrees C, antibiotic-sensitive derivatives appeared after a certain lag time: the lag times of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) were 2.5, 5, and 6.8 generations, respectively. After these times, plasmid-positive cells in the populations decreased at a rate of about 50% per generation in all of the mutants. From these results we conclude that plasmid segregation (partition) of Rms201 occurs by regular segregation (partition).     

Biochemical properties of a penicillinase from Escherichia coli carrying Rms 298.

We obtained two R plasmids, i.e., Rms195 and Rms298, from a clinical isolate, E. coli GN5503. Penicillin beta-lactamase (PCase) was extracted from ML1410 Rms195+ and Rms298+, and was purified by chromatography. Rms195 PCase was identical to the type I PCase mediated by R-TEM, RI and Rms212. The isoelectric point of Rms298 PCase was 5.9 and its molecular weight was 21,000 +/- 1,000. The substrate profile and physiochemical properties indicate that Rms298 PCase belongs to the type IV PCase mediated by Rms139 isolated from Pseudomonas aeruginosa.     

Epistatic effects of genes encoding tumor necrosis factor-α, immunoglobulin allotypes, and HLA antigens on susceptibility to non-insulin-dependent (type 2) diabetes mellitus

 Non-insulin-dependent diabetes mellitus (NIDDM) is a complex disease with a very high degree of heritability. Linkage and segregation analyses have not been very productive in identifying genes responsible for polygenic diseases such as NIDDM, and the majority of the genes determining susceptibility to this disorder remain to be identified. Using a case-control study design, we investigated the possible roles of genes coding for HLA class II antigens, tumor necrosis factor-α (TNF-α), and immunoglobulin (Ig) allotypes (GM and KM) in a group of Caucasians from Belgium (214 NIDDM patients and 200 controls). All genetic markers were determined by polymerase chain reaction-based methods. We demonstrate that particular homozygous genotypes of TNF-α and GM and KM allotypes epistatically interact with HLA-DQα1^{Arg 52} and contribute to an increased relative risk of NIDDM. Received: 3 January 1999 / Accepted: 18 March 1999     

Denaturation mapping of R factor deoxyribonucleic acid.

The R factor NR1 consists of two components: a resistance transfer factor which harbors the tetracycline resistance genes (RTF-TC) and the r-determinants component which harbors the other drug resistance genes. Using partial denaturation mapping it is possible to distinguish the RTF-TC region from the r-determinants region of the composite R factor NR1 DNA which has a contour length of 37 mum and a density of 1.712 g/ml. The r-determinants region was a relatively undenatured 8.5-mum segment of the molecule when the deoxyribonucleic acid was partially denatured at pH 10.7. An RTF-TC genetic segregant of NR1 which had lost the r-determinants component had a contour length of 28.7 mum and a density of 1.710 g/ml. Characterization of an RTF-TC using partial denaturation mapping at pH 10.7 confirmed that the relatively undenatured 8.5-mum r-determinants segment of the composite R factor had been deleted. Circular, transitioned NR1 DNA molecules (1.716 to 1.718 g/ml), whose contour lengths were consistent with an RTF-TC plus an integral number of tandem copies of r-determinants, were also characterized by denaturation mapping. The relatively undenatured region in these molecules had a length equal to an integral number of copies of r-determinants and was located at the same site in the partially denatured RTF-TC as the single copy of r-determinants in the 37-mum composite NR1. This indicates that there is a unique integration site for r-determinants in the RTF-TC component. The R factor UCR122, a TC deletion mutant of NR1, was also characterized by denaturation mapping. The translocation of the TC resistance gene(s) on the denaturation map permitted the alignment of the denaturation map with the heteroduplex map of Sharp et al. (u073). Linear and circular monomeric and presumed multimeric r-determinants DNA molecules (p = 1.718 g/ml) were partially denatured at a higher pH (11.10). The r-determinants multimers showed a repeating 8.3-mum (monomeric) partial denaturation pattern indicating a head-to-tail arrangement of monomers in these poly-r-determinant molecules.     

Loss and repair of conjugal fertility and infectivity of the resistance factor and sex factor in Escherichia coli

Hirota, Yukinori (University of Osaka, Osaka, Japan), Toshio Fujii, and Yukinobu Nishimura. Loss and repair of conjugal fertility and infectivity of the resistance factor and sex factor in Escherichia coli. J. Bacteriol. 91:1298-1304. 1966.-The drug-resistance factor, R, and the sex factor, F, have homologous traits, including contagious transmission, mediation of sexuality of the host cell, and autonomous replication in their host bacteria. Cooperation between F and R factors was found with a mutant R factor, which is nontransmissible in F(-) bacteria, becoming transmissible when introduced into bacteria carrying F. Conversely, the chromosome of a sterile male strain carrying the mutant sex factor, F(r), becomes transmissible when an R factor is introduced into the cell. The genetic determinants of R factors have been analyzed by isolation of mutant R factors, by sexual conjugation of the host bacteria, and by transduction of R factors with phage P1kc. The fertility determinant of the R factor, m, is inseparable from the determinant for its infectivity, but can be separated from the loci for autonomous replication of the R factor. R and F thus carry genetic determinants governing the same functions.     

Auxin inhibition of decapitation-induced branching is dependent on graft-transmissible signals regulated by genes Rms1 and Rms2

Decapitation-induced axillary bud outgrowth is a vital mechanism whereby shoots are able to continue normal growth and development. In many plants, including wild-type garden pea (Pisum sativum L.), this process can be inhibited by exogenous auxin. Using the ramosus (rms) increased branching mutants of pea, we present evidence that this response to auxin is dependent on graft-transmissible substance(s) regulated by the genes Rms1 and Rms2. The response to exogenous auxin is massively diminished in decapitated rms1 and rms2 mutant plants. However, basipetal auxin transport is not reduced in intact or decapitated mutants. Grafting rms1 or rms2 shoots onto wild-type rootstocks restored the auxin response, indicating that Rms1 and Rms2 gene action in the rootstock is sufficient to enable an auxin response in mutant shoots. We conclude that Rms1 and Rms2 act in the rootstock and shoot to control levels of mobile substance(s) that interact with exogenous auxin in the inhibition of bud outgrowth after decapitation. At least for rms1, the reduced auxin response is unlikely to be due to an inability of auxin to decrease xylem sap cytokinin content, as this is already low in intact rms1 plants. Consequently, we have genetic evidence that auxin action in decapitated plants depends on at least one novel long-distance signal.     

Mutational Analysis of Branching in Pea. Evidence That Rms1 and Rms5 Regulate the Same Novel Signal

The fifth increased branching ramosus (rms) mutant, rms5, from pea (Pisum sativum), is described here for phenotype and grafting responses with four other rms mutants. Xylem sap zeatin riboside concentration and shoot auxin levels in rms5 plants have also been compared with rms1 and wild type (WT). Rms1 and Rms5 appear to act closely at the biochemical or cellular level to control branching, because branching was inhibited in reciprocal epicotyl grafts between rms5 or rms1 and WT plants, but not inhibited in reciprocal grafts between rms5 and rms1 seedlings. The weakly transgressive or slightly additive phenotype of the rms1 rms5 double mutant provides further evidence for this interaction. Like rms1, rms5 rootstocks have reduced xylem sap cytokinin concentrations, and rms5 shoots do not appear deficient in indole-3-acetic acid or 4-chloroindole-3-acetic acid. Rms1 and Rms5 are similar in their interaction with other Rms genes. Reciprocal grafting studies with rms1, rms2, and rms5, together with the fact that root xylem sap cytokinin concentrations are reduced in rms1 and rms5 and elevated in rms2 plants, indicates that Rms1 and Rms5 may control a different pathway than that controlled by Rms2. Our studies indicate that Rms1 and Rms5 may regulate a novel graft-transmissible signal involved in the control of branching.     

Drug resistance of enteric bacteria. VII. Recombination of R factors with tetracycline-sensitive mutants

Hashimoto, Hajime (Gunma University, Maebashi, Japan), and Susumu Mitsuhashi. Drug resistance of enteric bacteria. VII. Recombination of R factors with tetracycline-sensitive mutants. J. Bacteriol. 92:1351-1356. 1966.-The transmissible drug-resistance factor R is able to confer resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM), and sulfonamide (SA) on a host bacterium when infected by cell-to-cell contact. Tetracycline-sensitive mutants were isolated from either CM- or SM-sensitive mutants of an R factor. Among 30 mutants isolated, 10 were point mutants which could recombine with each other, forming recombinant R factors able to grow on plates containing 50 mug/ml of TC. The recombination frequency of TC-resistant recombinants was 10(-2) to 10(-3) in bacterial cells carrying two types of TC-sensitive R factors by superinfection with both factors. Segregational patterns of the various markers on the R factor, i.e., chl, str, sul, and m, the locus determining R mating, and their linkage order, were investigated among TC-resistant recombinants of the R factor. When TC was used as the selective drug, the tet locus mapped on the R factor as an end marker. In view of the fact that these results are inconsistent with the linkage order of various markers reported previously, a circular genetic structure for the R factor which includes five tet-s and three chl-s loci is presented.     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.