Fc and C3bi receptors and the differentiation antigen BH2-Ag are randomly distributed in the plasma membrane of locomoting neutrophils

Reports from several laboratories suggest that neutrophils arrested during locomotion preferentially bind immune complexes at the front of the cell. Such asymmetry of binding has been interpreted as indicating an active modulation of phagocytic receptors to the anterior of the cell. To investigate this further, we have used digital analysis of fluorescence images to determine the binding patterns of mAbs directed against the Fc receptors, the receptors for the C3bi fragment of C3, and a neutrophil-specific antigen. We found that all three proteins are distributed nearly identically along the length of migrating neutrophils, and their distribution very closely parallels the anterior to posterior distribution of the plasma membrane. The use of mAbs offered an important advantage in that the binding of antireceptor antibodies, unlike the binding of ligands, should be independent of potential changes in the affinity of the receptors. We conclude that the anterior distribution of the phagocytic receptors in the plasma membrane of locomoting neutrophils parallels the overall increase in membrane area at the front of a migrating cell and that specific translocation of phagocytic receptors does not occur.     

Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils

To address the question whether leukocyte integrins are able to generate signals activating neutrophil functions, we investigated the capability of mAbs against the common beta chain (CD18), or the distinct alpha chains of CR3, LFA-1, or gp150/95, to activate neutrophil respiratory burst. These investigations were performed with mAbs bound to protein A immobilized to tissue culture polystyrene. Neutrophils plated in wells coated with the anti-CD18 mAbs IB4 and 60.3 released H2O2; H2O2 release did not occur when neutrophils were plated in wells coated with an irrelevant, isotype-matched mAb (OKDR), or with mAbs against other molecules (CD16, beta 2-microglobulin) expressed on the neutrophil surface at the same density of CD18. Four different mAbs, OKM1, OKM9, OKM10, 60.1, which recognize distinct epitopes of CR3 were unable to trigger H2O2 or O2- release from neutrophils. However, mAbs against LFA-1 or gp150/95 triggered both H2O2 and O2- release from neutrophils. Stimulation of neutrophils respiratory burst by both anti-CD18, and anti-LFA-1 or gp150/95 mAbs was totally inhibited by the microfilaments disrupting agent, cytochalasin B, and by a permeable cAMP analogue. While the capability to activate neutrophil respiratory burst was restricted to anti-LFA-1 and gp150/95 mAbs, we observed that mAbs against all members of leukocyte integrins, including CR3, were able to trigger neutrophil spreading. These findings indicate that, in neutrophils, all three leukocyte integrins can generate signals activating spreading, but only LFA-1 and gp150/95 can generate signals involved in activation of the respiratory burst. This observation can be relevant to understand the mechanisms responsible for the activation of neutrophil respiratory burst by tumor necrosis factor-alpha, which has been shown to be strictly dependent on expression of leukocyte integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. Wright. 1989. J. Cell Biol. 109:13411349.     

Involvement of beta 2-integrin in ROS-mediated neutrophil apoptosis induced by Entamoeba histolytica

Cellular adhesion through beta 2-integrin (CD18) is an important step in signal transduction leading to apoptosis of human neutrophils, and NADPH oxidase-derived reactive oxygen species (ROS) are essential for neutrophil apoptosis induced by Entamoeba histolytica. Therefore, we investigated the role of beta 2-integrin-mediated signals in ROS-dependent neutrophil apoptosis induced by E. histolytica. Entamoeba-induced apoptosis was inhibited by pre-incubation of cells with mAb to CD18, but not CD29, suggesting that beta )-integrin plays an important role in this response. Moreover, Entamoeba-induced ROS generation in neutrophils was inhibited by mAbs against CD18 or CD11b, but not by mAbs against CD11a, CD11c, or CD29. A combination of d-galactose plus anti-CD18 mAb had a larger inhibitory effect than d-galactose alone on Entamoeba-induced apoptosis and ROS generation. Furthermore, Entamoeba-induced apoptosis and ROS generation were inhibited by pre-treatment of cells with an inhibitor of phosphatidylinositol-3-kinase (PI-3-kinase). These results indicate that beta 2-integrin and PI-3-kinase are crucial signaling molecules in ROS-dependent apoptosis of neutrophils induced by E. histolytica.     

Outer membrane protein III of Neisseria gonorrhoeae: variations in biological properties of antibodies directed against different epitopes

Monoclonal antibodies (mAbs) have been raised to gonococcal outer membranes. A panel of six mAbs was identified by several criteria as reacting with outer membrane protein III (P.III). Competitive radioimmunoassays showed that the mAbs could be grouped into three pairs recognizing different epitopes on P.III. These epitopes are equally present on all pathogenic Neisseria. The mAbs demonstrated differing protective effects in model systems. Those directed against one epitope were particularly effective in protecting Chang conjunctiva epithelial cells against gonococcal challenge. mAbs against this epitope and another promoted complement-mediated bactericidal activity, while those directed against the third epitope were ineffective. Thus the biological effects of mAbs directed against P.III vary according to the epitope recognized.     

Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies

Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.     

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.     

Responses of neutrophils to anti-integrin antibodies depends on costimulation through low affinity Fc gamma Rs: full activation requires both integrin and nonintegrin signals

The relative contribution of integrin and nonintegrin signals to neutrophil activation is incompletely understood. Immobilized anti-integrin Abs were previously shown to induce robust activation of neutrophils without any additional stimulus, suggesting that cross-linking of integrins is sufficient for full activation of the cells. However, the possible contribution from other receptors has not been tested in this system. In this study, we show that neutrophil responses to anti-integrin Abs requires costimulation through low-affinity Fc gamma Rs. Murine neutrophils lacking the FcR gamma-chain or Fc gamma RIII failed to respond to immobilized Abs against beta(1), beta(2), or beta(3) integrins and the activation of wild-type cells could be prevented by blocking Abs against Fc gamma RII/III. Plate-bound anti-CD18 Abs initiated a respiratory burst from human neutrophils, but this response was abrogated when the F(ab')(2) of the same Abs were used or the cells were preincubated with Fc gamma RIIA-blocking Abs. Lack of Fc gamma RIII or administration of Fc gamma R-blocking Abs had no effect on responses of TNF-stimulated cells plated on fibrinogen or rICAM-1. TNF restored the respiratory burst of Fc gamma RIII-deficient neutrophils plated on anti-CD18 mAbs. The p38 MAPK inhibitor SB203580 attenuated the responses of neutrophils to anti-CD18 mAbs or TNF stimulation on a fibrinogen surface. Taken together, these results indicate that activation of low-affinity Fc gamma Rs is required for neutrophil responses induced by anti-integrin Abs and suggest that a second coactivation signal (e.g., through TNF or FcR ligation) is indispensable for full integrin-mediated activation of neutrophils. These second signals are interchangeable and they may converge on the p38 MAPK.     

Nanofibrous microfiltration membrane based on cellulose nanowhiskers

A multilayered nanofibrous microfiltration (MF) membrane system with high flux, low pressure drop, and high retention capability against both bacteria and bacteriophages (a virus model) was developed by impregnating ultrafine cellulose nanowhiskers (diameter about 5 nm) into an electrospun polyacrylonitrile (PAN) nanofibrous scaffold (fiber diameter about 150 nm) supported by a poly(ethylene terephthalate) (PET) nonwoven substrate (fiber diameter about 20 μm). The cellulose nanowhiskers were anchored on the PAN nanofiber surface, forming a cross-linked nanostructured mesh with very high surface-to-volume ratio and a negatively charged surface. The mean pore size and pore size distribution of this MF system could be adjusted by the loading of cellulose nanowhiskers, where the resulting membrane not only possessed good mechanical properties but also high surface charge density confirmed by the conductivity titration and zeta potential measurements. The results indicated that a test cellulose nanowhisker-based MF membrane exhibited 16 times higher adsorption capacity against a positively charged dye over a commercial nitrocellulose-based MF membrane. This experimental membrane also showed full retention capability against bacteria, for example, E. coli and B. diminuta (log reduction value (LRV) larger than 6) and decent retention against bacteriophage MS2 (LRV larger than 2).     

钩端螺旋体外膜蛋白LipL32单克隆抗体的制备及鉴定

LipL32是钩端螺旋体外膜中含量最丰富的蛋白,有很好的免疫原性,且在致病性钩端螺旋体中高度保守。在非变性条件下纯化LipL32重组蛋白,与佐剂混匀后免疫BALB/c小鼠,取脾脏细胞与NS-1骨髓瘤细胞融合,然后用间接酶联免疫吸附试验(ELISA)和有限稀释法分别进行筛选和细胞克隆,获得29株能稳定分泌抗LipL32单克隆抗体的杂交瘤细胞株,它们能识别8个LipL32抗原表位。用这些细胞株制备腹水型抗体,并对特性进行鉴定。用生物素标记这些抗体后两两配对,采用双抗夹心ELISA检测提取自15株致病性钩端螺旋体及其他致病菌的抗原,以评估单克隆抗体的灵敏度和特异度。筛选出1对灵敏度高和特异度好的单克隆抗体,ELISA检测rLipL32的最低浓度为1ng/ml。结果提示,该钩端螺旋体外膜蛋白LipL32单克隆抗体及检测方法有很好的应用前景。     

Resting murine neutrophils express functional alpha 4 integrins that signal through Src family kinases

There is mounting evidence that alpha(4) (CD49d) integrins are involved in neutrophil recruitment and function during inflammatory responses. We report that all resting murine neutrophils derived from bone marrow or peripheral blood express easily detectable levels of alpha(4) integrins on their surface. These alpha(4) integrins were functional, as demonstrated by stimulation of respiratory burst when neutrophils adhered to surfaces coated with the murine vascular cell adhesion molecule-1 (mVCAM-1). Adhesion occurred via alpha(4) integrins, as preincubation of neutrophils with an anti-alpha(4)-specific Ab inhibited attachment to mVCAM-1. Direct cross-linking of the alpha(4) integrin subunit by surface-bound mAbs also elicited superoxide release and release of the secondary granule marker, lactoferrin. The functional responses that occurred downstream of alpha(4) integrin cross-linking required signaling by Src family kinases. Neutrophils derived from hck(-/-)fgr(-/-)lyn(-/-) triple-knockout or hck(-/-)fgr(-/-) double-knockout mice failed to undergo respiratory burst when plated on mVCAM-1. Triple mutant neutrophils were also defective in release of both superoxide and lactoferrin when plated on surfaces coated with mAbs directed against alpha(4). Correlated with impaired alpha(4)-induced functional responses, triple-mutant neutrophils also failed to spread and tightly adhere to anti-alpha(4) mAb-coated surfaces. This is the first direct evidence that functional alpha(4) integrins are expressed by murine PMNs, and that these surface molecules can mediate cellular responses such as tight adhesion, spreading, sustained respiratory burst, and specific granule release in vitro. Moreover the alpha(4) integrins, like all other integrins tested, use the Src family kinases to transduce intracellular signals.     

Immunization method for multi-pass membrane proteins using highly metastatic cell lines

A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6–3.0 × 10⁸ cells/spleen [n = 6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7–11.4 × 10⁸ cells/spleen (n = 10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins.     

New insight into the Nox4 subcellular localization in HEK293 cells: first monoclonal antibodies against Nox4

Nox4, a member of Nox family of NADPH oxidase expressed in nonphagocytic cells, is a major source of reactive oxygen species in many cell types. But understanding of the role of Nox4 in the production of ROS and of regulation mechanism of oxidase activity is largely unknown. This study reports for the first time the generation and characterization of 5 mAbs against a recombinant Nox4 protein (AA: 206-578). Among 5 novel mAbs, 3 mAbs (8E9, 5F9, 6B11) specifically recognized Nox4 protein in HEK293 transfected cells or human kidney cortex by western blot analysis; mAb 8E9 reacted with intact tet-induced T-REx™ Nox4 cells in FACS studies. The other 2 mAbs 10B4 and 7C9 were shown to have a very weak reactivity after purification. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4, while mAbs 6B11 and 5F9 are directed to its cytosolic tail. Contrary to mAb 6B11, mAb 5F9 failed to detect Nox4 at the plasma membrane. Cell-free oxidase assays demonstrated a moderate but significant inhibition of constitutive Nox4 activity by mAbs 5F9 and 6B11. In conclusion, 5 mAbs raised against Nox4 were generated for the first time. 3 of them will provide powerful tools for a structure/function relationship of Nox4 and for physiopathological investigations in humans.     

Allergens Induce the Release of Lactoferrin by Neutrophils from Asthmatic Patients

BackgroundDespite the evidence that Lactoferrin (Lf) is involved in allergic asthma processes, it is unknown whether neutrophils can be one of the main cellular sources of this key inflammatory mediator directly in response of an IgE mediated stimulus. The present study was undertaken to analyze this question.MethodsNeutrophils from healthy subjects (n = 34) and neutrophils from allergic asthmatic patients (n = 102) were challenged in vitro with specific allergens to which the patients were sensitized, PAF, or agonist mAbs against IgE-receptors, and the levels of Lf were measured in the culture supernatant. The levels of serum IgE together with the severity of symptoms were also analyzed.ResultsLf was released into the culture supernatant of neutrophils from allergic asthmatic patients in response to allergens and PAF. This response was highly allergen-specific, and did not happen in neutrophils from healthy donors. Allergen effect was mimicked by Abs against FcεRI and galectin-3 but not by FcεRII. The levels of released Lf correlated well with the levels of serum specific IgE and severity of asthma symptoms. These observations represent a novel view of neutrophils as an important source of Lf in allergic asthma. Importantly, the levels of released Lf by neutrophils could therefore be used to evaluate disease severity in allergic asthmatic patients.     

Uniformity of protective antigens among isolates of the cattle tick, Boophilus microplus

Abstract. Gut membrane antigens were extracted from ten isolates of the cattle tick Boophilus microplus; the antigen extracts were probed with bovine antisera and three murine monoclonal antibodies (mAbs) in Western blots and dot-ELISA. The antisera had been obtained from cattle which were vaccinated with larval and gut extracts of B.microplus , and which were subsequently protected (84% and 94% respectively) against challenge with B.microplus. One of the mAbs (QU13) has been demonstrated to precipitate protective antigens from the midgut of B.microplus. Gut antigens from all ten isolates displayed similar reactivity profiles against bovine antisera and also against mAbs in Western blots. The end-point titres of antigens in dot-ELISA showed four-fold variation between isolates against bovine antisera, and also against mAb QUI 3. Larval membrane antigen extracted from N-strain B.microplus reacted with QU13 in dot-ELISA, indicating that protective antigens are common to both larval and adult stages of B.microplus. It was concluded that protective antigens recognized by QUI3 and antigens recognized by sera from protected cattle were conserved between the ten isolates examined, and between life-cycle stages.     

Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.     

Immunocytochemical identification of metallothionein- positive cells in rheumatoid synovium and analysis of their cell lineage

Metallothionein is a ubiquitous low molecular weight metalloprotein with powerful protective properties against oxygen radical-mediated cytotoxicity associated with inflammatory processes. In rheumatoid arthritis, the inflammatory damage to the synovium appears to be mediated by free radicals released by the high concentration of neutrophils found in the synovial fluid of the inflamed joint. Synovial tissue obtained during routine surgery on rheumatoid and non-rheumatoid joints was subjected to an indirect immunoperoxidase protocol for the immunolocalization of metallothionein using mouse monoclonal anti-metallothionein antibody E9, reactive against the two major isoforms of mammalian metallothionein. A layer of large dendritic-like cells situated subsynovially in the rheumatoid synovium stained very positively for the metalloprotein, both cytoplasmically and in their nuclei. These cells were not found in non-rheumatoid osteoarthritic or in undamaged synovial tissue associated with traumatic joint injury. An attempt was made to investigate their lineage using a series of antibody markers against epithelial cells, endothelial cells, smooth muscle, mesothelial cells, fibroblasts, neutrophils, dermal dendrocytes, macrophages, low and high molecular weight cytokeratin as well as a cell proliferation marker. From our results, it is suggested that these metallothionein-positive cells are probably myofibroblasts similar to the highly motile cells present in granulation tissue. They may originate from perivascular areas of synovium and their movement into the inflamed synovium may reflect the cytoprotective role of metallothionein acting as a free radical scavenger against oxidative damage     

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.     

Cartilage oligomeric matrix protein specific antibodies are pathogenic

Introduction

Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP-specific monoclonal antibodies (mAbs).

Methods

B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated and the pathogenicity of mAbs was investigated by passive transfer experiments.

Results

B cell immunodominant epitopes were localized within 4 antigenic domains of the COMP but with preferential response to the epidermal growth factor (EGF)-like domain. Some of our anti-COMP mAbs showed interactions with the native form of COMP, which is present in cartilage and synovium. Passive transfer of COMP-specific mAbs enhanced arthritis when co-administrated with a sub-arthritogenic dose of a mAb specific to collagen type II. Interestingly, we found that a combination of 5 COMP mAbs was capable of inducing arthritis in naive mice.

Conclusions

We have identified the specificities of mAbs to COMP and their contribution to the development of arthritis. These findings will further improve our understanding of the autoantibody mediated immunopathologies occurring widely in rheumatoid arthritis (RA), as well as in other autoimmune disorders.     

Critical role for cross-linking of trimeric lectin domains of surfactant protein D in antiviral activity against influenza A virus

Collectins are multimeric host defence lectins with trimeric CRDs (carbohydrate-recognition domains) and collagen and N-terminal domains that form higher-order structures composed of four or more trimers. Recombinant trimers composed of only the CRD and adjacent neck domain (termed NCRD) retain binding activity for some ligands and mediate some functional activities. The lung collectin SP-D (surfactant protein D) has strong neutralizing activity for IAVs (influenza A viruses) in vitro and in vivo, however, the NCRD derived from SP-D has weak viral-binding ability and lacks neutralizing activity. Using a panel of mAbs (monoclonal antibodies) directed against the NCRD in the present study we show that mAbs binding near the lectin site inhibit antiviral activity of full-length SP-D, but mAbs which bind other sites on the CRD do not. Two of the non-blocking mAbs significantly increased binding and antiviral activity of NCRDs as assessed by haemagglutination and neuraminidase inhibition and by viral neutralization. mAb-mediated cross-linking also enabled NCRDs to induce viral aggregation and to increase viral uptake by neutrophils and virus-induced respiratory burst responses by these cells. These results show that antiviral activities of SP-D can be reproduced without the N-terminal and collagen domains and that cross-linking of NCRDs is essential for antiviral activity of SP-D with respect to IAV.     

Total coliform detection in drinking water: comparison of membrane filtration with Colilert and Coliquik

The Colilert (CL) and Coliquik (CQ) systems were compared in a presence-absence format against the Standard Methods membrane filtration (MF) technique to determine whether differences existed in total coliform detection. Approximately 750 water samples were collected from distribution systems, covered and uncovered storage reservoirs, well sites, and the influent to drinking water treatment plants. Samples were analyzed for total coliforms and heterotrophic bacteria with MF, CL, and CQ. The agreements between CL and MF and between CQ and MF were both greater than 94.8%, which indicates that both may be acceptable methods for total coliform detection. Disagreement between the CL and CQ methods was primarily due to false-negative results. Furthermore, laboratory and field inoculation methods were compared for CL, more than 98% agreement was obtained. This finding indicates that sampling and immediate field inoculation may be an alternative to the traditional laboratory inoculation.