Checkerboard Immunoblotting Research Articles

Antibodies to oral pathobionts and colon cancer risk in the CLUE I cohort study.

Periodontitis has been associated with an increased risk for gastrointestinal cancers. The objective of our study was to investigate the association of antibodies to oral bacteria and the risk of colon cancer in a cohort setting. Using the CLUE I cohort, a prospective cohort initiated in 1974 in Washington County, Maryland, we conducted a nested case-control study to examine the association of levels of IgG antibodies to 11 oral bacterial species (13 total strains) with risk of colon cancer diagnosed a median of 16 years later (range: 1-26 years). Antibody response was measured using checkerboard immunoblotting assays. We included 200 colon cancer cases and 200 controls matched on age, sex, cigarette smoking status, time of blood draw and pipe or cigar smoking status. Controls were selected using incidence density sampling. Conditional logistic regression models were used to assess the association between antibody levels and colon cancer risk. In the overall analysis, we observed significant inverse associations for 6 of the 13 antibodies measured (P-trends <.05) and one positive association for antibody levels to Aggregatibacter actinomycetemcomitans (ATCC 29523; P-trend = .04). While we cannot rule out a role for periodontal disease in colon cancer risk, findings from our study suggest that a strong adaptive immune response may be associated with a lower risk of colon cancer. More studies will need to examine whether the positive associations we observed with antibodies to A. actinomycetemcomitans reflect a true causal association for this bacterium.

Distinct Microbial Signatures between Periodontal Profile Classes

Precise classification of periodontal disease has been the objective of concerted efforts and has led to the introduction of new consensus-based and data-driven classifications. The purpose of this study was to characterize the microbiological signatures of a latent class analysis (LCA)–derived periodontal stratification system, the Periodontal Profile Class (PPC) taxonomy. We used demographic, microbial (subgingival biofilm composition), and immunological data (serum IgG antibody levels, obtained with checkerboard immunoblotting technique) for 1,450 adult participants of the Dental Atherosclerosis Risk in Communities (ARIC) study, with already generated PPC classifications. Analyses relied on t tests and generalized linear models with Bonferroni correction. Men and African Americans had higher systemic antibody levels against most microorganisms compared to women and Caucasians (P < 0.05). Healthy individuals (PPC-I) had low levels of biofilm bacteria and serum IgG levels against most periodontal pathogens (P < 0.05). Subjects with mild to moderate disease (PPC-II to PPC-III) showed mild/moderate colonization of multiple biofilm pathogens. Individuals with severe disease (PPC-IV) had moderate/high levels of biofilm pathogens and antibody levels for orange/red complexes. High gingival index individuals (PPC-V) showed moderate/high levels of biofilm Campylobacter rectus and Aggregatibacter actinomycetemcomitans. Biofilm composition in individuals with reduced periodontium (PPC-VI) was similar to health but showed moderate to high antibody responses. Those with severe tooth loss (PPC-VII) had significantly high levels of multiple biofilm pathogens, while the systemic antibody response to these microorganisms was comparable to health. The results support a biologic basis for elevated risk for periodontal disease in men and African Americans. Periodontally healthy individuals showed a low biofilm pathogen and low systemic antibody burden. In the presence of PPC disease, a microbial-host imbalance characterized by higher microbial biofilm colonization and/or systemic IgG responses was identified. These results support the notion that subgroups identified by the PPC system present distinct microbial profiles and may be useful in designing future precise biological treatment interventions.

Serum Antibody Responses to Periodontal Microbiota in Chronic and Aggressive Periodontitis: A Postulate Revisited

The authors revisited the 1999 International Workshop postulate of robust serum antibody responses to infecting agents in localized aggressive periodontitis (LAgP) and weak responses in generalized aggressive periodontitis (GAgP). Antibody responses were further examined in localized and generalized chronic periodontitis (LCP and GCP). The study includes 119 patients (60 males and 59 females, aged 11 to 76 years), 18 with LAgP, 37 with GAgP, 37 with LCP, and 27 with GCP. Multiple subgingival plaque samples/patient (1,057 in total) were analyzed with respect to 11 bacterial species using checkerboard DNA-DNA hybridizations, and serum immunoglobulin (Ig)G levels were measured against the same bacteria using checkerboard immunoblotting. Further, infection ratios (antibody level over the average bacterial colonization by the homologous species) were computed for each patient. Comparisons of bacterial colonization, serum IgG levels, and infection ratios were made across the diagnostic categories using multivariable linear regression models adjusting for age and race/ethnicity. There were no statistically significant differences in serum IgG levels to Aggregatibacter actinomycetemcomitans among the four diagnostic categories. IgG levels to several species, including Porphyromonas gingivalis, Treponema denticola, and Campylobacter rectus, were highest in patients with GAgP and significantly different from LCP and GCP, but not from LAgP. Comparisons based on infection ratios showed no statistically significant differences for any species between GAgP and LAgP. This study provides evidence against the 1999 Workshop's postulate of weak serum antibody responses in patients with GAgP and shows that serum IgG responses in GAgP are comparable to those in LAgP, but higher than in GCP or LCP for several species.

Determinants of serum IgG responses to periodontal bacteria in a nationally representative sample of US adults

To assess the distribution of elevated antibody titres to multiple periodontal bacteria, including established/putative pathogens and health-related species, by selected demographic, behavioural, and oral- and general health-related characteristics. Data from 8153 >or=40-year-old participants from the third National Health and Nutrition Examination Survey were used, including 1588 edentulous individuals. We used checkerboard immunoblotting to assess serum IgG levels to 19 periodontal species. Thresholds for elevated antibody responses were defined for each species using the 90th percentile titre in periodontal healthy participants, using two alternative definitions of periodontitis. Edentulous individuals showed lower antibody responses than dentate participants, notably for titres to "red complex" species and Actinobacillus actinomycetemcomitans. Elevated titres to Porphyromonas gingivalis were twice as prevalent in participants with periodontitis than in periodontal healthy individuals. Non-Hispanic blacks and Mexican-Americans were more likely to display elevated titres for P. gingivalis compared with non-Hispanic whites (22.9%versus 19.4%versus 9.5%). Current smokers were significantly less likely to exhibit high titres to multiple bacteria than never smokers. Demographic, behavioural, and oral- and general health-related characteristics were strong determinants of systemic antibody responses to periodontal bacteria in a nationally representative sample of US adults.

Relationships Among Gingival Crevicular Fluid Biomarkers, Clinical Parameters of Periodontal Disease, and the Subgingival Microbiota

The objectives of this study were to measure levels of gingival crevicular fluid (GCF) biomarkers and subgingival bacterial species in periodontally healthy subjects and subjects with periodontitis to explore the relationships among these biomarkers, the subgingival microbiota, and the clinical parameters of periodontal disease. Clinical periodontal parameters were measured at six sites per tooth in 20 subjects with periodontitis and 20 periodontally healthy subjects. GCF and subgingival plaque samples were obtained from the mesio-buccal aspect of every tooth. GCF levels of interleukin (IL)-1beta and IL-8 and matrix metalloproteinase 8 were measured using checkerboard immunoblotting, and the levels of 40 bacterial taxa were quantified using checkerboard DNA-DNA hybridization. A subset of "clinically healthy" sites from each group was analyzed separately. The significance of the differences between groups was determined using the unpaired t test or the Mann-Whitney test. Correlations among immunologic, microbiologic, and clinical data were determined using the Spearman rank correlation coefficient. There were positive correlations among mean clinical parameters, mean levels of the three biomarkers, and the proportions of orange and red complex species (P <0.05). Clinically healthy sites from subjects with periodontitis had higher levels of IL-1beta and IL-8 and higher proportions of orange and red complex species (P <0.05) than clinically healthy sites from periodontally healthy subjects. Red complex species were positively associated with the expression of all biomarkers (P <0.05), whereas purple and yellow complex species had negative correlations with IL-1beta and IL-8 (P <0.05). Clinically healthy sites from subjects with periodontitis have higher levels of GCF biomarkers and periodontal pathogens than clinically healthy sites from periodontally healthy subjects. Different microbial complexes demonstrated distinct associations with specific GCF biomarkers.

Serum Antibodies to Periodontal Bacteria as Diagnostic Markers of Periodontitis

Assessment of periodontal conditions in epidemiologic studies usually requires a clinical examination, which is resource-intensive. We investigated the ability of serum immunoglobulin G (IgG) antibodies to periodontal bacteria to reflect clinical periodontal status. We used checkerboard immunoblotting to assess serum IgG levels to 19 species, including established/putative periodontal pathogens and non-pathogenic bacteria, in 5,747 dentate adults aged > or = 40 years who participated in the third National Health and Nutrition Examination Survey between 1988 and 1994. Three earlier described alternative definitions of periodontitis were used, based on specific combinations of probing depth and attachment level values. Optimized elevated titer thresholds and corresponding sensitivities and specificities were calculated for each definition. Titers significantly associated with periodontitis were identified in univariable and multivariable logistic regression models. Parsimonious models were subsequently developed using age, gender, race/ethnicity, education, smoking, and diagnosed diabetes. In unadjusted models, high titers to Porphyromonas gingivalis were most strongly associated with periodontitis across all definitions (odds ratio, 2.07 to 2.74; P <0.05). In parsimonious models including demographic data, smoking, and diagnosed diabetes, high P. gingivalis titers were consistently associated with periodontitis, whereas high Eubacterium nodatum titers were associated with periodontal health in two of three definitions. Receiver operating characteristic curves for the parsimonious multivariable models showed that the area under the curve ranged between 0.72 and 0.78. Serum IgG titers to selected periodontal species, combined with demographic and behavioral characteristics, resulted in a moderately accurate classification of periodontal status in epidemiologic studies. The external validity of these findings must be examined further.

Heterogeneity of systemic inflammatory responses to periodontal therapy

We investigated the effect of comprehensive periodontal therapy on the levels of multiple systemic inflammatory biomarkers. Thirty patients with severe periodontitis received comprehensive periodontal therapy within a 6-week period. Blood samples were obtained at: 1-week pre-therapy (T1), therapy initiation (T2), treatment completion (T3), and 4 weeks thereafter (T4). We assessed the plasma concentrations of 19 biomarkers using multiplex assays, and serum IgG antibodies to periodontal bacteria using checkerboard immunoblotting. At T2 and T4, dental plaque samples were analysed using checkerboard hybridizations. At T3, PAI-1, sE-selectin, sVCAM-1, MMP-9, myeloperoxidase, and a composite summary inflammatory score (SIS) were significantly reduced. However, only sE-selectin, sICAM, and serum amyloid P sustained a reduction at T4. Responses were highly variable: analyses of SIS slopes between baseline and T4 showed that approximately 1/3 and 1/4 of the patients experienced a marked reduction and a pronounced increase in systemic inflammation, respectively, while the remainder were seemingly unchanged. Changes in inflammatory markers correlated poorly with clinical, microbiological and serological markers of periodontitis. Periodontal therapy resulted in an overall reduction of systemic inflammation, but the responses were inconsistent across subjects and largely not sustainable. The determinants of this substantial heterogeneity need to be explored further.

Application of the Checkerboard Immunoblotting Technique to the Quantification of Host Biomarkers in Gingival Crevicular Fluid

The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high-throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples. Monoclonal antibodies were used to bind GCF interleukin (IL)-1beta and -8 and matrix metalloproteinase (MMP)-8 to the surface of membranes. Biotinylated antibodies were used to detect bound antigens in a checkerboard format. Signals were developed using chemiluminescence, captured on film, and quantified using software for array analysis. The assay was tested for potential cross-reactions among the three pairs of antibodies. Eleven CBIBs were processed to determine the analytical sensitivity of the assay. Forty GCF samples were analyzed using CBIB and enzyme-linked immunosorbent assay (ELISA) in parallel, and the significance of the correlations among the results was tested using the Pearson correlation coefficient. Nine hundred thirty-one GCF samples were collected from 20 periodontally healthy subjects and 20 periodontitis subjects and analyzed using CBIB to test the assay's sensitivity and dynamic ranges using clinical samples. The CBIB was capable of distinguishing among the three analytes. The sensitivity and dynamic ranges of the assay were suitable for the detection of the three targets in the majority of GCF samples. There were highly statistically significant (P <0.0001) positive correlations between CBIB and ELISA data for all three biomarkers. The periodontitis subjects had statistically significantly higher mean levels of IL-1beta and -8 compared to healthy subjects. The CBIB technique is a sensitive and specific assay for the high-throughput quantification of MMP-8 and IL-8 and -1beta in GCF.

Assessing the levels of immunoglobulins in the saliva of diabetic individuals with periodontitis using checkerboard immunodetection

Current methods for determining salivary antibodies are cumbersome for large-scale screenings. To test checkerboard immunodetection for monitoring salivary antibodies and to profile them in diabetic individuals with periodontitis. Salivary anti-Porphyromonas gingivalis, anti-Actinobacillus actinomycetemcomitans and total IgA levels of 10 individuals were compared using checkerboard immunoblotting and enzyme-linked immunosorbent assay (ELISA). Close correlation between both methods was found in anti-P. gingivalis IgA and total IgA, but not in anti-A. actinomycetemcomitans IgA, because of high background levels in ELISA. Thereafter, checkerboard immunodetection was used to compare salivary antibodies of 20 adult type II diabetic with 32 non-diabetic individuals with (n=22) or without (n=10) periodontitis. Patients with periodontitis (regardless of their diabetic condition) expressed increased levels of total IgA in both whole and parotid saliva, but reduced levels of anti-A. actinomycetemcomitans IgA in whole saliva. Consequently, the proportion of anti-A. actinomycetemcomitans IgA in the total IgA was lower in saliva of patients with periodontitis compared with healthy controls. Checkerboard immunodetection was reliable and economical for screening saliva samples for multiple antibody reactions. Our results support previous reports which suggested that patients with periodontitis are able to secrete high levels of salivary Ig, but are hampered in targeting their salivary response toward A. actinomycetemcomitans.

Periodontal infection profiles in type 1 diabetes

We investigated the levels of subgingival plaque bacteria and serum IgG responses in patients with type 1 diabetes and non-diabetic controls of comparable periodontal status. Fifty type 1 diabetes patients (mean duration 20.3 years, range 6-41) were age-and gender-matched with 50 non-diabetic individuals with similar levels of periodontal disease. Full-mouth clinical periodontal status was recorded, and eight plaque samples/person were collected and analysed by checkerboard hybridization with respect to 12 species. Homologous serum IgG titres were assessed by checkerboard immunoblotting. In a sub-sample of pairs, serum cytokines and selected markers of cardiovascular risk were assessed using multiplex technology. Among the investigated species, only levels of Eubacterium nodatum were found to be higher in diabetic patients, while none of the IgG titres differed between the groups, both before and after adjustments for microbial load. Patients with diabetes had significantly higher serum levels of soluble E-selectin (p=0.04), vascular cell adhesion molecule-1 (VCAM-1; p=0.0008), adiponectin (p=0.01) and lower levels of plasminogen activator inhibitor-1 (PAI-1; p=0.02). After controlling for the severity of periodontal disease, patients with type 1 diabetes and non-diabetic controls showed comparable subgingival infection patterns and serum antibody responses.

Associations between IgG antibody to oral organisms and carotid intima–medial thickness in community-dwelling adults

Aims The aims of this study are to describe the relationships between IgG antibodies to 17 oral organisms and atherosclerosis as indexed by carotid intima–medial wall thickness (IMT) and to evaluate the role of smoking. Methods and results Our study is based on a subset of participants in the Atherosclerosis Risk in Communities (ARIC) Study, who received a complete periodontal examination during visit 4 (1996–1998). The outcome was mean carotid IMT ≥ 1 mm assessed by B-mode ultrasound. The exposures were serum IgG antibody levels against 17 periodontal organisms using a whole bacterial checkerboard immunoblotting technique. Evaluation of all 17 antibodies indicated that antibody to Campylobacter rectus resulted in the best-fitting model (OR = 2.3, 95% CI = 1.83–2.84) and individuals with both high C. rectus and Peptostreptococcus micros titers had almost twice the prevalence of IMT ≥ 1 mm than those with only a high C. rectus antibody (8.3% versus 16.3%). Stratification by smoking indicated that all microbial models significant for smokers were also significant for never smokers except for Porphyromonas gingivalis ( p = 0.08). Conclusions This is the first study to report a relationship between IgG antibody reactive to oral organisms and subclinical atherosclerosis with significant relationships evident in both ever and never smokers.

Longitudinal stability of serum immunoglobulin G responses to periodontal bacteria

The value of seroepidemiology in the study of periodontal infections has not been adequately explored. This study examined serum immunoglobulin (IgG) responses to periodontal bacteria in patients with periodontitis and periodontitis-free individuals over a 30-month period. Eighty-nine patients with chronic periodontitis and 42 control subjects with no deep periodontal pockets and no or minimal attachment loss (30-72 years old, 43% men) were included. Patients were examined at baseline, after completed periodontal therapy 4 months post-baseline, and at 30 months, and controls, at baseline and 30 months. IgG antibodies to 19 periodontal species were determined by checkerboard immunoblotting. On average, patients displayed at baseline up to 800-fold higher titers than controls to all but three species. Over the 30-month period, titers remained stable at low levels in controls. In patients, periodontal conditions improved from a baseline mean probing depth of 3.6 mm, bleeding on probing of 62% and an average of 21.5 pockets of=6 mm/person, to 2.5 mm mean pocket depth, 30% bleeding on probing, and 1.2 deep pockets, at 30 months. Over time, antibody titers showed a modest decline in patients, but remained significantly elevated at 30 months in comparison with controls. Antibody-level changes over time were not significantly different between subjects that did and did not receive adjunctive systemic antibiotics. Conspicuous differences in IgG titers to periodontal bacteria exist between periodontitis patients and periodontally healthy controls. Despite successful periodontal therapy, titers remained elevated over a 30-month period, suggesting that serology may mark the history of past periodontal infection.

Gene expression signatures in chronic and aggressive periodontitis: a pilot study.

This pilot study examined gene expression signatures in pathological gingival tissues of subjects with chronic or aggressive periodontitis, and explored whether new subclasses of periodontitis can be identified based on gene expression profiles. A total of 14 patients, seven with chronic and seven with aggressive periodontitis, were examined with respect to clinical periodontal status, composition of subgingival bacterial plaque assessed by checkerboard hybridizations, and levels of serum IgG antibodies to periodontal bacteria assayed by checkerboard immunoblotting. In addition, at least two pathological pockets/patient were biopsied, processed for RNA extraction, amplification and labeling, and used to study gene expression using Affymetrix U-133 A arrays. Based on a total of 35 microarrays, no significantly different gene expression profiles appeared to emerge between chronic and aggressive periodontitis. However, a de novo grouping of the 14 subjects into two fairly robust clusters was possible based on similarities in gene expression. These two groups had similar clinical periodontal status and subgingival bacterial profiles, but differed significantly with respect to serum IgG levels against the important periodontal pathogens Porphyromonas gingivalis, Tannerella forsythensis and Campylobacter rectus. These early data point to the usefulness of gene expression profiling techniques in the identification of subclasses of periodontitis with common pathobiology.

Rapid evaluation of serum and gingival crevicular fluid immunoglobulin G subclass antibody levels in patients with early-onset periodontitis using checkerboard immunoblotting.

A method was developed to evaluate the presence of immunoglobulin G (IgG) subclass (1-4) antibody to Actinobacillus actinomycetemcomitans, serotype b (strain Y4) in patients with early-onset periodontitis on a single nitrocellulose membrane. Sera from 30 early-onset periodontitis patients and gingival crevicular fluid samples from 2 patients were collected and tested with four different preparations of A. actinomycetemcomitans (Y4). The principle steps of the assay are: a) binding of the bacterial antigen (Y4) and the anti-human IgG antibody (capture antibody) in parallel lanes on nitrocellulose membranes; b) incubation of known concentrations of the IgG subclasses 1, 2, 3 and 4, as well as a dilution of serum and/or gingival crevicular fluid from patients in lanes perpendicular to the antigen lanes; c) incubation of the membranes with the corresponding peroxidase conjugated anti-human IgG subclass secondary antibody; d) detection of positive signals by enhanced chemiluminescence. The blots were evaluated by visual comparison to a series of blots containing known concentrations of IgG subclasses. The method was used to rapidly screen a relatively large number of patient sera and gingival crevicular fluid samples for IgG subclasses in a cost-effective assay. The predominant IgG subclass found in early-onset periodontitis was IgG2.

Simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by checkerboard immunoblotting (CBIB)

A simple technique, checkerboard immunoblotting (CBIB), is described for simultaneous quantitation of specific IgE antibodies against several allergens in human sera. Using as little as 50 microliters of each of the 20 sera examined against 20 different allergens, it was possible in a single run to achieve 400 tests. To guarantee high specificity and sensitivity of the assay, this new application of CBIB employs purified allergens, cyanogen bromide-activated nitrocellulose membrane, and Phosphorimager technology. Results are expressed both qualitatively (five classes) and quantitatively in kilo units per liter equilibrated against the World Health Organization (WHO) standard for IgE. There was excellent agreement between the results of CBIB and the results of Pharmacia Cap System, an alternative method widely used for measuring serum-specific IgE. The CBIB method certainly could be useful in any laboratory interested in allergy clinical research for easy screening and relative quantitation of allergen-specific human IgE.

Simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by checkerboard immunoblotting (CBIB)

Simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by checkerboard immunoblotting (CBIB)

Anaphylaxis after treatment with recombinant factor VIII

Treatment of hemophilia patients with recombinant factor VIII concentrates has not previously been associated with anaphylaxis. A 5-week-old boy with severe hemophilia A developed dyspnea, cyanosis, hypotension, and a diffuse urticarial rash following treatment with a recombinant factor VIII (Recombinate). To identify the cause of anaphylaxis in this patient, the vial lot was examined for the presence of endotoxin, and a checkerboard immunoblotting technique was used to test serum and/or plasma samples from the patient and mother for the presence of antibodies (IgA, IgG, IgE, and IgM) to Recombinate-related antigens (recombinant factor VIII, von Willebrand factor, human serum albumin, Chinese hamster ovary proteins, bovine serum albumin, mouse monoclonal anti-human factor VIII, polyethylene glycol 3350), and to ethylene oxide, the agent used to sterilize the infusion equipment. No immune response directed against the Recombinate-related antigens or ethylene oxide that could be associated with the anaphylactic reaction was identified. Endotoxin was not present upon rabbit pyrogen testing of the therapeutic product. These studies failed to show any association between Recombinate and the onset of the allergic reaction. This seems to be the first reported case of anaphylaxis following the infusion of a recombinant form of factor VIII concentrate.

207 Rapid and simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by Checkerboard immunoblotting (CBIB)

207 Rapid and simultaneous quantitation of specific IgE against 20 purified allergens in allergic patients sera by Checkerboard immunoblotting (CBIB)

Monoclonal immunoglobulin A antibodies directed against cholera toxin prevent the toxin-induced chloride secretory response and block toxin binding to intestinal epithelial cells in vitro.

Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be important in resistance to oral challenge with virulent Vibrio cholerae, although alternative mechanisms for protection of intestinal epithelia against CT-induced fluid secretion have been proposed. The ability of anti-CT IgA to block the effects of CT on human enterocytes has not been directly tested because of the lack of a well-defined in vitro intestinal epithelial cell system to directly measure toxin action and the limited availability of purified anti-CT IgA antibodies. We have generated hybridomas that produce monoclonal IgA and IgG antibodies directed against CT by fusion of Peyer's patch cells with mouse myeloma cells after oral-systemic immunization of mice with CT and CT B-subunit protein. All of the anti-CT antibodies recognized the B subunit. Three clones (designated anti-CTB IgA-1, IgA-2, and IgA-3) which produced IgA antibodies in dimeric and polymeric forms were selected. Checkerboard immunoblotting demonstrated that IgA-1 recognized an epitope distinct from that recognized by IgA-2 and IgA-3 and that none of the antibodies were directed against the binding site of GM1, the intestinal cell membrane toxin receptor. The protective capacity of these IgAs was tested in vitro with human T84 colon carcinoma cells grown on permeable supports as confluent monolayers of polarized enterocytes. When each anti-CTB IgA was mixed with 10 nM CT and applied to the apical surfaces of T84 cell monolayers, all three IgAs blocked CT-induced Cl- secretion in a dose-dependent manner and completely inhibited binding of rhodamine-labelled CT to apical cell membranes. Thus, monoclonal anti-CTB IgA antibodies are sufficient to protect human enterocytes in vitro against CT binding and action.

Application of checkerboard immunoblotting (CBIB) to the detection of anti-viral IgG in human serum.

In the present study, we have begun to investigate the possibility of using checkerboard immunoblotting (CBIB) as a semi-quantitative screening tool for detecting human serum IgG against specific viral antigens. The viral antigens studied were Epstein-Barr, herpes simplex I and II, cytomegalovirus, varicella zoster, rubella, rubeola, and mumps. Western immunoblotting experiments using these partially purified preparations demonstrated that there were apparently no interactions between IgG from non-immune sera and the respective viral antigen preparations. The CBIB assay was evaluated using sera of known positive or negative immune status for the viral antigens. There was excellent agreement between the results of CBIB and the results of alternative methods for evaluating immune status: all discrepancies (1/18 sera for mumps, 3/18 sera for rubeola, and 1/28 sera for rubella) involved sera with borderline results, either by CBIB or by the alternative method. Therefore, although further work is required to define the method in terms of sensitivity and clinical specificity, and to refine positive/negative cutpoint criteria for certain antigen components, our preliminary experience suggests that CBIB has considerable potential in the efficient and inexpensive screening of sera for the presence of IgG against a panel of viral antigens, so as to identify subjects at risk for infection.