Diffusion-in-gel Enzyme-linked Immunosorbent Assay Research Articles

Improvements to the enzyme-developed radial immunodiffusion technique

An enzyme-developed radial immunodiffusion technique, previously known as the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA), has been improved in two ways: (a) antibody-containing spots have been made larger and more distinct by revealing them with a mixture of hydrogen peroxide, 3,3′-diaminobenzidine and nickel, and further intensification of the ensuing spots with silver; (b) the reliability of the method has been enhanced by chemically coupling the antigen to a layer of a polyamino acid (poly(lysine, phenylalanine)) adsorbed to the bottom of the polystyrene petri dish. The usefulness of the improved technique is illustrated by reference to the measurement of serum concentrations of IgM and IgG, and in the assessment of antibody levels against a particulate antigen (erythrocytes).

The usefulness of Diffusion-In-Gel-ELISA in clinical practice as illustrated by a Campylobacter jejuni outbreak

In this study, the DIG-ELISA (Diffusion-In-Gel Enzyme Linked Immunosorbent Assay) is presented as a tool for the determination of antibodies with improved quantitation over other solid phase assays. The method combines the diffusion of antibodies in agar with the EIA concept in Petri dishes. Diffusion of native, undiluted sera results in (1) a concentration gradient in the effort to reach equilibrium, i.e., an end point titration; (2) a separation of serum components of different sizes; (3) unlimited possibilities for migrating antibodies to bind antigenic epitopes because of the constant antigen excess. Low affinity antibodies can bind divalently and are more readily detected; and (4) elimination of dilution errors. The combination of undiluted sera and Petri dishes as solid phase also permits a large number of samples to be tested with less effort and simplifies the practical handling of EIAs, including easy coating and washing procedures, reuse of antigen and quantitation by zone areas without instrumentation. Plates can be stored for months and are available for re-examination, demonstration and transport. The whole procedure can be conducted in a closed system, i.e., when testing highly contaminated samples. The usefulness of the procedure is demonstrated by a food-borne outbreak of Campylobacter jejuni. The outbreak involved 86 persons of whom 20% were culture positive in contrast to a seropositivity of 74% with DIG-ELISA. A diffusion time of 24 h was used for diagnostic purposes. Extended diffusion times of 48 h and 72 h were utilized when consecutive series of sera showed identical values after 24 h, indicating high antibody content that resulted in a peak serum (end point). For infectious diseases with a rapid course, this assay could be used as an acute test. With the diffusion step prepared in advance, DIG-ELISA is a ready-to-use test. When frequent sampling of sera is performed in the very early phase of the disease, DIG-ELISA reveals that all Ig-classes can be present at high titer and the diagnostic potential of the immune response is better utilized. The DIG-ELISA has the methodological flexibility and the physical qualities to be an effective, inexpensive technique for quantitation of antibodies at any laboratory.

Evaluation of the gliadin antibody test for diagnosing coeliac disease.

The purpose of this study was to evaluate the diagnostic value of gliadin antibodies (GA) in coeliac disease. The test was a diffusion in gel enzyme-linked immunosorbent assay (DIG-ELISA), with combined determination of IgA and IgG. The sensitivity, specificity, and positive and negative predictive value of the test were determined, as used on a well-described, consecutive material of 100 adult patients, admitted for small-intestinal biopsy. For comparison, the positive predictive value of the test, when used on a larger Danish patient material of adults and children, was determined. One hundred and eighteen adults and 55 children with increased or borderline GA were included. For adults the positive predictive value was 71-90%, the negative predictive value 97%, sensitivity 77%, and specificity 95%. For children the positive predictive value was 74%. A graded interpretation, improving the usefulness of the test, is proposed. It is concluded that GA are useful in screening for coeliac disease (CD). It is still necessary to perform at least one small-intestinal biopsy to establish the diagnosis. Highly elevated GA levels strongly indicate CD, whereas a result well below limits makes CD unlikely.

The Diagnostic Value of the Gliadin Antibody Test in Celiac Disease in Children

Serum gliadin antibodies (IgA/IgG) were determined in 191 consecutive children (median age, 2.75 years; range, 0.33-15.5 years) admitted for a small-intestinal biopsy on suspicion of celiac disease. The test was a diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA). Of these 191, 14 (7.3%) appeared to have untreated celiac disease. Depending on the choice of cut-off value of the test (combined determination of IgA and IgG), the sensitivity was 86-100%, the specificity was 97-99%, and the positive/negative predictive values were 70-92% and 99-100%, respectively. No variation according to age was found. Gliadin antibodies were determined in 47 children who had well-treated celiac disease. Fourteen of these children were also investigated when challenged with gluten. Gliadin antibodies (IgA or IgG) decreased significantly in 13 of 13 cases when the patients shifted from a gluten-containing diet to a gluten-free one. During the gluten challenge, the IgG and IgA increased in 14 of 14 and 11 of 14 cases, respectively (two patients suffered from IgA deficiency). In eight patients who later appeared to be free of celiac disease, the gliadin antibodies were determined on gluten-free diet and during gluten challenge; no significant differences in gliadin antibodies were found. We conclude that this test is useful in selecting patients with symptoms suggesting celiac disease for a small-intestinal biopsy. The test seems to be of some value in monitoring the effects of a gluten-free diet and during gluten challenge.

The Diagnostic Value of the Gliadin Antibody Test in Celiac Disease in Children

SummarySerum gliadin antibodies (IgA/IgG) were determined in 191 consecutive children (median age, 2.75 years; range, 0.33–15.5 years) admitted for a small‐intestinal biopsy on suspicion of celiac disease. The test was a diffusion‐in‐gel enzyme‐linked immunosorbent assay (DIG‐ELISA). Of these 191, 14 (7.3%) appeared to have untreated celiac disease. Depending on the choice of cut‐off value of the test (combined determination of IgA and IgG), the sensitivity was 86–100%, the specificity was 97–99%, and the positive/negative predictive values were 70–92% and 99–100%, respectively. No variation according to age was found. Gliadin antibodies were determined in 47 children who had well‐treated celiac disease. Fourteen of these children were also investigated when challenged with gluten. Gliadin antibodies (IgA or IgG) decreased significantly in 13 of 13 cases when the patients shifted from a gluten‐containing diet to a gluten‐free one. During the gluten challenge, the IgG and IgA increased in 14 of 14 and 11 of 14 cases, respectively (two patients suffered from IgA deficiency). In eight patients who later appeared to be free of celiac disease, the gliadin antibodies were determined on gluten‐free diet and during gluten challenge; no significant differences in gliadin antibodies were found. We conclude that this test is useful in selecting patients with symptoms suggesting celiac disease for a small‐intestinal biopsy. The test seems to be of some value in monitoring the effects of a gluten‐free diet and during gluten challenge.

Inhibition of serum albumin flux across exposed dentine following conditioning with GLUMA primer, glutaraldehyde or potassium oxalates

A variety of medicaments used on dentine in various treatment procedures may cause a reduction in dentine permeability. By observing the flow of endogenous serum albumin across exposed dentine, agents known to promote dentine bonding of restorative resins or retard dentine sensitivity were assessed regarding their capacity to arrest dentinal fluid flow. Experiments were conducted in young adult macaque monkeys employing Class V cavity preparations in incisors and canines. A diffusion-in-gel-enzyme-linkedimmunosorbent-assay (DIG-ELISA) was used to quantitate serum albumin in effluents from these cavities at various time periods following either no treatment or topical application with GLUMA primer, glutaraldehyde or potassium oxalates. While in untreated cavities serum albumin continued to flow even after a period of 1 week, a substantial reduction or complete cessation of serum albumin flux was seen following topical application of the agents tested, suggesting a durable effect on dentinal fluid flow. No difference between agents was observed. Further cutting of the cavity bottom a few tenths of a millimetre resulted in renewed flow of serum albumin. Data suggest that the solutions tested are capable of reducing dentinal fluid flow onto exposed dentine surfaces.

Schistosoma mansoni antibodies in tissue specimens demonstrated by a diffusion-in-gel ELISA technique

A modification of the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) for analysis of antibodies in tissue fragments has been elaborated. In this technique, tissue specimens are placed on top of a thin gel layer covering a plastic surface coated with an antigen preparation. The gel thus serves as a medium through which diffusion of antibodies contained in tissue samples may occur. Specimens from skin, lung, liver, kidney, heart and skeletal muscle as well as blood serum and blood sampled onto filter paper were successfully used for analysis of the antibody response to two different antigens (soluble adult worm antigen and soluble egg antigen) during experimental infection of mice with the helminth parasite Schistosoma mansoni. It was concluded that skin and lung tissue reflected the serum antibody response more closely than the other tissues tested, although zone sizes were slightly smaller than those registered for corresponding serum samples. The DIG-ELISA reaction zone size was, as expected, shown to be dependent on the weight (size) of the tissue specimen. The variability of the modified technique was comparable to that found for analysis of serum by the conventional DIG-ELISA technique. It is concluded that the described technique may be used for serological analysis when blood samples cannot be easily obtained, e.g. at examination of animal carcasses or single organs.

Alterations in Bronchoalveolar Lavage Fluid but Not in Lung Function and Bronchial Responsiveness in Swine Confinement Workers

Testing of lung function and bronchial reactivity, bronchoalveolar lavage (BAL), and a skin prick test with a standard panel and six "swine" extracts obtained from swine and swine environment were performed in 20 randomly selected nonsmoking swine confinement workers. In addition, blood samples for detection of antibodies by the diffusion in gel-enzyme-linked immunosorbent assay (DIG-ELISA) technique and precipitating antibodies were drawn. Air samples for measurement of dust and endotoxin levels were collected. All the farmers regarded themselves as healthy. The results were compared with reference groups consisting of urban nonsmoking subjects who had not been exposed to pig farming environment. The pig farmers had normal lung function and the bronchial reactivity was not different from the reference group. In the BAL fluid of the farmers, the concentration of total cells and granulocytes was increased while the concentrations of lymphocytes and macrophages were normal. The BAL fluid concentrations of albumin, fibronectin, and hyaluronan were elevated in the farmers. Skin prick tests with swine extracts were negative in all farmers. Antibodies (assessed by DIG-ELISA) against swine dander, swine dust, and pig feed were increased and precipitating antibodies against swine dander were found in 14, against pig food in five, and against swine confinement dust in three of the 20 pig farmers. The concentration of airborne total dust was 7.4 mg/cu mm and the endotoxin concentration was 37 (22 to 60) ng/cu mm during tending the pigs and increased, during feeding, to 13.8 mg/cu mm and 315 (194 to 716) ng/cu mm, respectively. There was no correlation between exposure and lung function or lavage findings. In conclusion, randomly selected pig farmers had signs of airway inflammatory reaction and activation of the immune system without alteration in lung function and bronchial reactivity.

A new method for serodiagnosis of sheep fascioliasis using helminth excretory-secretory products

The diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) for antibodies to Fasciola hepatica was evaluated using adult fluke excretory-secretory products as antigen. The sensitivity and specificity of the test were compared with those of the indirect hemagglutination (IHA) technique. The results of the DIG-ELISA showed that the reaction zone diameters obtained with 42 sera from sheep with natural or experimental exposure to F. hepatica were significantly greater than those obtained with 85 sera from ovines infected with other parasites. Both sensitivity and specificity were 100% in the DIG-ELISA, whereas the sensitivity was 68.2% and the specificity was 100% in the IHA. The data suggest that the DIG-ELISA is a valuable serodiagnostic test for sheep fascioliasis.

A New Method for Diagnosis of Chagas' Disease: Diffusion-in-Gel Enzyme-Linked Immunosorbent Assay

The diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) for antibodies to Trypanosoma cruzi was evaluated using formalinized epimastigotes as antigen. The results obtained by DIG-ELISA were compared with those obtained with the indirect hemagglutination test. The results of the DIG-ELISA showed that the reaction zone diameters obtained with sera from individuals with past or present exposure to T. cruzi were significantly greater than those obtained with normal human sera. All the sera from Chagasic individuals had a positive reaction in the test, whereas sera from normal individuals and individuals with toxoplasmosis or cutaneous leishmaniasis had a negative result. A close correlation was observed between the reaction zone diameters and antibody concentration (expressed as log2 of the serum dilution). Excellent correlation was observed between results obtained by the 2 serological tests. The data suggest that the DIG-ELISA is a promising serological test for measuring antibodies to T. cruzi.

Evaluation of a solid-phase immunoassay (DIG-ELISA) for the serodiagnosis of ovine toxoplasmosis

The antibody response to Toxoplasma gondii was studied in 5 experimentally infected sheep by means of the indirect fluorescent antibody test (IFAT) and the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA). A significant increase in the antibody levels was registered by both methods as early as one week after subcutaneous inoculation with the RH strain of T.gondii . Maximal antibody levels were recorded 4 to 8 weeks after the inoculation. Analysis of a total of 135 sera obtained during different stages of experimental or natural toxoplasma infection revealed close agreement between the results of the two methods used. The expediency of regular DIG-ELISA as well as of a modified procedure for the direct analysis of blood sampled onto filter paper discs was also demonstrated. There was a close correlation between the results obtained by analysis of serum and of blood adsorbed onto filter paper.

Serum antibodies to gliadin in coeliac disease after gluten withdrawal.

Serum gliadin antibodies of the IgA and IgG classes were determined by the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) in 10 adult patients with villous atrophy of the small-intestinal mucosa. After introduction of a gluten-free diet a gradual decrease in serum gliadin antibody levels occurred, reaching statistical significance at 3 months of treatment for the IgA class (p less than 0.01) and at 6 months for the IgG class (p less than 0.05). A decrease of serum gliadin antibody levels after gluten withdrawal was related to an improvement of the intestinal mucosa and should thus be indicative of whether the patient is following the dietary recommendations. However, determination of gliadin antibody levels cannot replace small-intestinal biopsy, as there are a few patients in whom the antibody levels are not related to the morphology of the gut mucosa.

Serum IgA and IgG gliadin antibodies and small intestinal mucosal damage in children.

Serum immunoglobulin (Ig) A and IgG gliadin antibodies were determined with a simple, rapid, and inexpensive method--diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA)--and the results were related to small intestinal mucosal morphology in 234 children suspected of having malabsorption. Fifty-six of 58 children with flat intestinal mucosa had increased IgA and/or IgG gliadin antibody levels (sensitivity 97%). Fifty-four of the 58 children had celiac disease (CD) (n = 25) or probable CD (n = 29). Four children with flat mucosa had cow's milk protein and/or soy protein intolerance and three of these had increased gliadin antibody levels. Seventeen percent of 132 children with normal intestinal mucosa had increased IgA and/or IgG gliadin antibody levels. IgA and IgG gliadin antibody levels decreased significantly in the celiac children on a gluten-free diet and increased significantly after gluten challenge. Determination of serum IgA and IgG gliadin antibodies by means of DIG-ELISA is a sensitive test for small intestinal mucosal damage in children. When malabsorption is suspected, we suggest that this assay be used to select children for a small intestinal biopsy. It is also very useful for the follow-up of adherence to a gluten-free diet and to determine the effect of gluten challenge in celiac children.

Serum IgA and IgG Gliadin Antibodies and Small Intestinal Mucosal Damage in Children

Serum immunoglobulin (Ig) A and IgG gliadin antibodies were determined with a simple, rapid, and inexpensive method—diffusion‐in‐gel enzyme‐linked immunosorbent assay (DIG‐ELISA)—and the results were related to small intestinal mucosal morphology in 234 children suspected of having malabsorption. Fifty‐six of 58 children with flat intestinal mucosa had increased IgA and/or IgG gliadin antibody levels (sensitivity 97%). Fifty‐four of the 58 children had celiac disease (CD) (n = 25) or probable CD (n = 29). Four children with flat mucosa had cow's milk protein and/or soy protein intolerance and three of these had increased gliadin antibody levels. Seventeen percent of 132 children with normal intestinal mucosa had increased IgA and/or IgG gliadin antibody levels. IgA and IgG gliadin antibody levels decreased significantly in the celiac children on a gluten‐free diet and increased significantly after gluten challenge. Determination of serum IgA and IgG gliadin antibodies by means of DIG‐ELISA is a sensitive test for small intestinal mucosal damage in children. When malabsorption is suspected, we suggest that this assay be used to select children for a small intestinal biopsy. It is also very useful for the follow‐up of adherence to a gluten‐free diet and to determine the effect of gluten challenge in celiac children.

Antibodies to nontypeable Haemophilus influenzae in blood donors. A comparison between different assay methods

Antibodies to nontypeable Haemophilus influenzae were determined in sera from healthy blood donors with complement fixation (CF) test, diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA), and enzyme-linked immunosorbent assay (ELISA). In most sera, antibodies to H. influenzae could be detected with all three methods, but DIG-ELISA as well as ELISA demonstrated a greater sensitivity than the CF test. Furthermore, the ELISA methods allowed analysis of separate immunoglobulin classes, which is of great advantage when sera from infected patients are to be analysed.

Paper discs impregnated with capillary blood. A sampling technique for immunoassays by means of DIG-ELISA and DIG-TIA

A modification of the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) and the diffusion-in-gel thin layer immunoassay (DIG-TIA) using paper discs impregnated with blood or serum as source of diffusion is described. Using a BSA-anti-BSA model system the suitability of this sampling and assaying technique for simple and accurate quantitation of antibodies has been demonstrated. Paper discs impregnated with blood or serum and subsequently dried could be stored at temperatures between +4 °C and +37 °C for at least six weeks without significant loss of antibody activity. Application of paper disc DIG-ELISA was exemplified by assaying anti-BSA antibodies in rabbits immunized with BSA as well as antibodies against both soluble egg antigen and adult worm extract in mice experimentally infected with Schistosoma mansoni cercariae. The simplicity and good reproducibility of this technique should make it an attractive alternative in serological screening of large population groups. Definite advantages of this technique are the possibilities to analyse quantitatively antibodies in capillary blood, rather than serum from venous blood samples, and to store sampled material for a considerable time even under such climatic conditions as often prevail in tropical countries.

Diffusion in gel-enzyme linked immunosorbent assay (DIG-ELISA) to record the immunoglobulin response of calves vaccinated with Salmonella

A diffusion in gel-enzyme linked immunosorbent assay (DIG-ELISA) was used to record immunoglobulin development of colostrum-fed calves vaccinated with aromatic dependent (aro −) Salmonella and challenged with either the homologous or a heterologous serotype. IgG was detected by using a peroxidase conjugated rabbit antibovine IgG, whereas IgM and IgA were measured using a double sandwich technique. Although IgG levels to Salmonella endotoxins increased after exposure to Salmonella, they were found to be high in many calves prior to vaccination. However, IgM antibody levels were consistently low prior to vaccination, and their increase was a more reliable indicator of the vaccination and immune status of the calves. IgA levels were generally low and of less predictive value.

Serum gliadin antibodies for detection and control of childhood coeliac disease.

Serum gliadin antibodies of the IgA and IgG isotypes were determined by means of the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) in children during different phases of coeliac disease. Fourteen children were studied before onset of dietary treatment, 16 during a period of gluten-free diet and 16 during gluten challenge. The control groups consisted of 44 children with other gastrointestinal diseases and 14 children without gastrointestinal disorders. All of the children studied had been subjected to small-intestinal biopsy. On the basis of the results obtained in this study the diagnostic sensitivity with regard to untreated coeliac disease was found to be 100% and the diagnostic specificity 97%. In 10 coeliac children followed during the phases of diagnostic evaluation antibody levels decreased in all during dietary treatment and increased in 8 during a subsequent gluten challenge. It is suggested that determination of IgA and IgG gliadin antibodies by means of DIG-ELISA may be used as a diagnostic test for coeliac disease in children and that this test may be useful in monitoring the dietary treatment in children with known coeliac disease. Moreover, the DIG-ELISA is an inexpensive and technically simple method.

The rabbit antibody response to immunization withHaemophilus ducreyiA comparative study for immunoglobulin detection using diffusion-in-gel enzyme-linked immunosorbent assay, countercurrent immunoelectrophoresis, and double-diffusion-in-gel

Haemophilus ducreyi was used for immunization of rabbits. The serum level of specific IgG, IgM, and IgA antibodies to the same agent was determined by diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) and precipitating antibodies were detected with countercurrent immunoelectrophoresis (CIE) and double-diffusion-in-gel (DD) techniques. Antibodies to H. ducreyi could be detected in all rabbits within two weeks with all three techniques used.

Evaluation of gliadin antibodies for detection of coeliac disease.

To evaluate assay of gliadin antibodies of different immunoglobulin classes as a test for detection of coeliac disease, we analysed sera from 36 adult patients and 8 children with coeliac disease, 62 patients with other gastrointestinal diseases, and 124 blood donors with diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA). Depending on the choice of reference levels for gliadin antibodies of the IgA and IgG classes, respectively, we found a diagnostic sensitivity for coeliac disease of 93-86% and a diagnostic specificity of 95-100%. Determination of gliadin antibodies by DIG-ELISA can thus be used as a test for detection of coeliac disease and selection of patients for small-intestinal biopsy.