Ouchterlony Technique Research Articles

A simple, effective and inexpensive method to highlight antigen-antibody reaction based on liquid-semisolid phase

Identification of the immunological antigen-antibody reaction performed either in liquid phase or in agarose have several limits: antigen excess, low sensitivity, low speed reaction. A new method is described for the antigen-antibody reaction, easy, simple to perform and inexpensive. It is based on a reaction where the specific antibody is mixed with a semi-solid gel on a transparent surface and the antigen to be identified is inoculated into the gel. In this method, an immune precipitate can be shown in just few minutes, obtaining a good sensitivity compared with manual methods such as the Ouchterlony technique and other similar methods. By using albumin, IgG, RBP, transferrin, alpha-2-macroglobulin as antigen, the immune precipitate is formed within 10 min at 2–8 microgram/ml and, after 30 min, at 1–4 microgram/ml may be detected. The display of the immune precipitate is favored by oblique observation on a black background with a perpendicular light illuminating the gel from the bottom upwards. This method also allows a good control of antigen excess, a problem that is instead found in immunoturbidimetric reactions. The method here described has also been tested in serum and urine, and then it can be advantageously used as qualitative or semi-quantitative immunological test in research or diagnostic. Moreover, it could be particularly useful in low resources geographical areas, where an immunoassay that requires the use of dedicated equipment is not economically sustainable. The authors have waived the patent rights to allow the free development of new tests based on this method.

Allergic Bronchopulmonary Mycosis in Non-Atopic Patients: A Treatment Alternative

RationaleAllergic bronchopulmonary mycosis (ABPM) is a chronic hypersensitivity reaction to molds that may present a diagnostic and treatment challenge because variants of this condition deviate from standard criteria. Our goal is to increase awareness about atypical permutations of this condition and offer a possible treatment option.MethodsTwo patients were evaluated with complete histories and physical examinations. Skin testing to 55 aeroallergens including 16 mold allergens was done by the prick method. Serum precipitins were done by the Ouchterlony technique. Computed tomography (CT) of the chest, pulmonary function testing (PFTs) including lung volumes and diffusion, bronchoscopy, and bronchial washing and cultures were done in both cases. Serology was drawn to measure total IgE and specific IgE. One case underwent a methacholine challenge and transbronchial biopsy. High-dose, mono-allergen subcutaneous immunotherapy was begun.ResultsNeither case had a history of allergic asthma. Both cases had histories, physical exams, CTs of the chest, and PFTs consistent with APBM. Both cases had a mono-positive skin test, Penicillium and Aspergillus respectively, with the former mold cultured from chest secretions. Both cases had a peripheral eosinophilia. One case had an elevated total serum IgE and positive precipitins. Both have remained in remission from APBM for more than 2 years while on high-dose immunotherapy.ConclusionsVariant presentations of APBM may be difficult to diagnose. High-dose immunotherapy may be an additional approach, particularly, to mono-sensitized patients. RationaleAllergic bronchopulmonary mycosis (ABPM) is a chronic hypersensitivity reaction to molds that may present a diagnostic and treatment challenge because variants of this condition deviate from standard criteria. Our goal is to increase awareness about atypical permutations of this condition and offer a possible treatment option. Allergic bronchopulmonary mycosis (ABPM) is a chronic hypersensitivity reaction to molds that may present a diagnostic and treatment challenge because variants of this condition deviate from standard criteria. Our goal is to increase awareness about atypical permutations of this condition and offer a possible treatment option. MethodsTwo patients were evaluated with complete histories and physical examinations. Skin testing to 55 aeroallergens including 16 mold allergens was done by the prick method. Serum precipitins were done by the Ouchterlony technique. Computed tomography (CT) of the chest, pulmonary function testing (PFTs) including lung volumes and diffusion, bronchoscopy, and bronchial washing and cultures were done in both cases. Serology was drawn to measure total IgE and specific IgE. One case underwent a methacholine challenge and transbronchial biopsy. High-dose, mono-allergen subcutaneous immunotherapy was begun. Two patients were evaluated with complete histories and physical examinations. Skin testing to 55 aeroallergens including 16 mold allergens was done by the prick method. Serum precipitins were done by the Ouchterlony technique. Computed tomography (CT) of the chest, pulmonary function testing (PFTs) including lung volumes and diffusion, bronchoscopy, and bronchial washing and cultures were done in both cases. Serology was drawn to measure total IgE and specific IgE. One case underwent a methacholine challenge and transbronchial biopsy. High-dose, mono-allergen subcutaneous immunotherapy was begun. ResultsNeither case had a history of allergic asthma. Both cases had histories, physical exams, CTs of the chest, and PFTs consistent with APBM. Both cases had a mono-positive skin test, Penicillium and Aspergillus respectively, with the former mold cultured from chest secretions. Both cases had a peripheral eosinophilia. One case had an elevated total serum IgE and positive precipitins. Both have remained in remission from APBM for more than 2 years while on high-dose immunotherapy. Neither case had a history of allergic asthma. Both cases had histories, physical exams, CTs of the chest, and PFTs consistent with APBM. Both cases had a mono-positive skin test, Penicillium and Aspergillus respectively, with the former mold cultured from chest secretions. Both cases had a peripheral eosinophilia. One case had an elevated total serum IgE and positive precipitins. Both have remained in remission from APBM for more than 2 years while on high-dose immunotherapy. ConclusionsVariant presentations of APBM may be difficult to diagnose. High-dose immunotherapy may be an additional approach, particularly, to mono-sensitized patients. Variant presentations of APBM may be difficult to diagnose. High-dose immunotherapy may be an additional approach, particularly, to mono-sensitized patients.

Visceral larva migrans infections caused by toxocara species (author's transl)

A total of 4656 serum samples from suspect persons were investigated for Toxocara antibodies over the last 10 years. Using the Ouchterlony technique antibodies were demonstrated in 707 cases, and in 409 cases with the help of the microprecipitation test using secondary larvae. It was shown that the living larva test is highly specific and very sensitive so that a positive reaction can be taken as definite evidence of infection. Clinical symptoms were characterised by high eosinophilia, ophthalmologic changes, lung involvement, central nervous affection and increase of gamma-globulins. A history of contact with dogs or cats was an additional indicator. Treatment of larva migrans syndrome has to date remained unsatisfactory as no effective and well tolerated drug is known.

“Can You Hear Me Now?” Cell Communication

In the immune system cell communication is required to make sure that the appropriate cells are recruited to sites of inflammation. Communication usually occurs through secretion of specific proteins called chemokines and cytokines, which attract specific cell types. A series of laboratory investigation provide students with the understanding of how the body reacts to foreign invaders by recruiting these appropriate cell types. Students will understand how chemokines and cytokines as proteins that are secreted by macrophages, dendritic cells and helper T‐cells act as signals that induce other immune cells to perform functions that help to regulate an inflammation response. The first step of the laboratory protocol consists of students separating an “unknown” simulated blood sample by column chromatography. Once the red blood cells are separated from the plasma, the elution from the column, representing the immune cell fraction, is collected for use in the next step. This part of the protocol is called the Ouchterlony technique; it determines the interaction of chemoattractant‐interleukine 16 (IL‐16) and macrophage inflammatory proteins (MIP) in the migration of CD4 Helper T cells (which become Th1 and Th2 cells), cheek cells and an unknown. If a chemical reaction occurs between the cytokine, helper T cell, cheek cells and the unknown, a precipitation band will form demonstrating cell‐specific chemokine to cell migration therefore cell signaling. Students will analyze these results to determine if a migratory response has occurred, how these cells are communicating and also determine which chemokine attracts the migration of the unknown.

Protein composition of Cucurbita maxima and C. moschata seeds

Seeds of Cucurbita maxima, C. moschata and their interspecific hybrids were used to evaluate the intrapopulational and interpopulational variation of their protein composition. Three immunoprecipitating systems common to all the studied samples were detected by the Ouchterlony technique. Fourteen protein bands were identified by polyacrylamide gel electrophoresis (PAGE) whereas 23 bands were identified by sodiumdodecylsulfate (SDS)-PAGE. Using Western blotting (WB) also 23 bands were detected. The Jaccard's index of similarity calculated from SDS-PAGE and WB varied between 91 and 100 % for all the compared pairs of samples. These results demonstrate a high uniformity in the protein composition of all the samples and do not allow for their clear characterization.

The unexpected outcomes of medical research: serendipity and the Australia antigenBlumberg BS, Alter HJ, Visnich S. A new antigen in leukemia sera [J Am Med Assoc 1965;191:541–546

The 'Australia Antigen' is found in the sera of some normal individuals from foreign populations. The total absence of the antigen from the sera of normal United States' subjects and its relatively high frequency in acute leukemia suggests that the presence of the antigen maybe of value in the diagnosis of early acute leukemia. Whether the antigen results from or precedes the leukemia process remains to be seen. (Abstract reproduced by permission of J Am Med Assoc 1965;191:541 - 546.) Many of the major discoveries in science have been the product of chance observations that were pursued by the curious mind to their plausible and provable explanation. Mendel's peas and, at least apocryphally, Newton's apple, come to mind as paradigm shifting observations that respectively opened the fields of genetics and established the laws of gravitation. Fleming's chance observation of the inhibition of bacterial growth in the presence of bread mold ultimately produced the first antibiotic. The unexpected link between the Australia antigen and the hepatitis B virus may be less encompassing than genetics or gravitation, but nonetheless is a classic example of a serendipitous observation pursued to major unexpected payoffs, including the prevention of post-transfusion hepatitis, the development of a vaccine, the prevention of hepatocellular carcinoma and laying the foundation for

The acetylcholinesterase ichthyotoxin is a common component in the extracellular products of Vibrionaceae strains.

In previous work, it was reported that a strain of Aeromonas hydrophila (B32) produces the most potent lethal toxin with neurotoxic activity described so far for fish. In the present study, the presence and distribution of this acetylcholinesterase toxin lethal for fish were determined in extracellular products (ECP) of 42 Vibrionaceae strains using both immunological and colorimetric methods. This neurotoxin was shown to be present in the majority of the ECP from the Aeromonas and Vibrio strains tested and is responsible for the specific acetylcholinesterase activity. Also, although the Western blot and Ouchterlony techniques are valid as qualitative methods for the detection of this toxin, the Western blot procedure was 100-fold more sensitive than the Ouchterlony technique.

Allergic bronchopulmonary aspergillosis with low serum IgE levels in a child with cystic fibrosis

J Allergy Clin Immunol 1997;100:431-2.

Physical, immunological and kinetic properties of ribulose‐1,5‐bisphosphate carboxylase/oxygenase from fir (Abies alba) and spruce (Picea abies)

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from fir (Abies alba Mill.) and spruce (Picea abies [L.] Karst.) needles was purified to homogeneity. The enzyme was isolated from crude extracts through quantitative precipitation in 40‐55% and 40‐60% (NH4)2SO4 for fir and spruce. respectively, followed by linear sucrose gradient centrifugation. Using two dimensional gel electrophoresis, the isoelectric points were determined. For the large subunit (LSU) it was 6.7 for both species, and for the small subunit (SSU) it was 7.1 and 7.7 for fir and spruce, respectively. Very few differences in tryptic peptides and amino acid composition of Rubisco LSU were observed between fir and spruce. By contrast, marked differences characterized the same analyses for the Rubisco SSU of the two species. Moreover, substitution of residues was observed in the sequenced N‐terminal region when comparing fir and spruce SSU. The Ouchterlony technique showed no immu‐nochemical difference between Rubisco of fir and spruce when a rabbit antiserum to spinach Rubisco was used. The Eadie‐Hofstee plots of carboxylase activity indicated that the apparent Km(CO2) were 31 and 36 μM for the fir and spruce enzymes, respectively.

A monoclonal antibody (OPT1) to T cells which is available for paraffin-embedded materials.

A monoclonal antibody (MAb), OPT1, reactive with T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with activated T cells from peripheral blood lymphocytes (PBL). The antibody is an IgG1 antibody as demonstrated by the Ouchterlony technique. By cytofluorometric analysis, almost all CD3+ lymphocytes and only a few CD20+ lymphocytes of peripheral blood expressed the OPT1 antigen. Nonhematolymphoid cell lines were negative for OPT1 by the immunoperoxidase staining using acetone-fixed cell lines. On the contrary, peripheral T cells, cells of two T cell lines out of four and a part of the cells of one B cell line out of two were positive for OPT1. The immunoperoxidase staining of paraffin-embedded tissue sections revealed that most of lymphocytes in T cell areas of lymph nodes expressed OPT1 antigen. Some lymphocytes in both cortex and medulla of the thymus and erythroid precursors of the bone marrow were OPT1+. In the malignant lymphoma series, approximately 90% of T cell lymphomas and 6% of B cell lymphomas reacted with OPT1. None of the Reed-Sternberg cells nor Hodgkin cells in Hodgkin's disease were positive. Consequently, OPT1 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.

Protein chromatography with pyridine- and alkylthioether-based agarose adsorbents

In an attempt to identify the part of the ligand of 3-(2-pyridylthiol)-2-hydroxypropylagarose that is responsible for the specific adsorption of immunoglobulins and α 2-macroglobulin from serum, nine agarose derivatives were prepared: (I) 3-(N-2-iminopyridyl)-2-hydroxypropyl-, (II) 3-(4-pyridylthio)-2-hydroxypropyl-, (III) 3-(2-pyridylthio-N-oxide)-2-hydroxypropyl-, (IV) 3-(2-pyridylthio)-2-hydroxypropyl-, (V) 3-(ethylthio)-2-hydroxypropyl-, (VI) 3-( n-butylthio)-2-hydroxypropyl-, (VII) 3-(2-aminoethylthio)-2-hydroxypropyl-, (VIII) 3-(2-hydroxyethylthiol)-2-hydroxypropyl- and (IX) 3-(N-2-pyridyl-2-one)-2-hydroxypropylagarose. The selective adsorption of the above serum proteins to these derivatives was analysed by chromatography. The electron distributions were calculated for three of the investigated pyridine derivatives in order to establish whether there is any relationship between the electron distribution in the molecule and the absorption properties of the pyridine derivatives. By optimizing the preparation methods for the different derivatives, the possible side-reactions were minimized and checked. The adsorbed serum proteins were determined by the Ouchterlony technique and electrophoresis. The concentration of human serum albumin in the different fractions was determined by conventional radioimmunological methods. These data make it possible to classify the adsorbents according to their selectivity and hydrophobic thiophilic behaviour.

Production of anti-insulin monoclonal antibody and its clinical application

Hybridoma-produced monoclonal antibody (MoAb) against insulin is useful for insulin assays because of its specificity and plentiful supply. The spleen cells of male BALB c mice immunized against monocomponent porcine insulin were hybridized with mouse myeloma cells (P3-X63-Ag8-U1). The resulting anti-insulin antibody (Ab) was purified and characterized by radioimmunoassay (RIA) using 125I-porcine insulin and sand-wich-method enzyme immunoassay (EIA) using Ab-conjugated beads and β-galactosidase. For reference, we used anti-insulin polyclonal antibody raised in guinea pigs (PoAb). Using the Ouchterlony technique, we identified the antibody as being of subclass IgG 1. We judged this antibody to be MoAb because it did not react at all with porcine insulin during EIA, in concentrations between 0 and 12.5 ng/ml; in contrast, PoAb reacted dose-dependently. During RIA, this Ab did not cross-react with glucagon, somatostatin or pancreatic polypeptide. It did cross-react with human and bovine insulins but not with rat insulin. The proper concentration of this MoAb for RIA proved to be 1:1,500,000 and the smallest detectable level of porcine insulin by RIA using this Ab was 0.5 ng/ml. These levels were similar to those obtained with PoAb. The binding activity of this MoAb to human insulin was quite similar to that of porcine insulin. RIA insulin determinations using our MoAb correlated well with those employing PoAb.

Preliminary studies on the development of a vaccine for Pneumocystis carinii. I. Immunological and biochemical characterization

Initial progress has been made toward the development of an experimental vaccine or immunostimulant for Pneumocystis carinii. Antigen derived from cell culture propagated P. carinii proved to be a potent immunogen in the rabbit and antibody thus produced demonstrated identity with intact murine and human lung-derived P. carinii organisms. Reactivity of the antibody with P. carinii soluble antigen in the blood of rats and human subjects with P. carinii pneumonitis (PCP) was demonstrated by the Ouchterlony technique, by counterimmunoelectrophoresis (CIE) and by latex particle agglutination (LPA). Murine-derived P. carinii antigen was utilized in an enzyme-linked immunosorbent assay (ELISA) for anti- P. carinii IgG and IgM produced in immunized rabbits and for human IgG antibody against P. carinii. Preliminary biochemical analysis of whole and solubilized cysts has been carried out, as well as slab gel electrophoresis and immunoblot profiling of solubilized organisms and naturally-occurring P. carinii antigen(s) in the blood of humans and rats with PCP. These studies represent an initial step toward the development of a vaccine or immunostimulant against PCP.

Enzyme-Linked Immunosorbent Assay (ELISA) for Determining Immunological Relationships Among β-Galactosidases from Lactobacilli

Antisera were prepared against purified inducible β-galactosidase from Lactobacillus murinus ATCC 35020, and L. bulgaricus ATCC 11842, and were used to measure the immunological relations between the reference enzymes and β-galactosidase from different lactobacilli. Crude extracts from eighteen species of lactobacilli were tested against these antisera, in Ouchterlony double diffussion experiments. The conventional microcomplement fixation method used to detect immunological relationships among bacteria was compared with an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of immunological relations among β-galactosidase from different lactobacilli. The results of these assays using both antisera supported those obtained with Ouchterlony technique. From the combined results obtained with the different methods, phylogenetic maps were derived. The maps obtained with Ouchterlony and microcomplement fixation were very similar to those obtained with Ouchterlony and ELISA. We propose the use of the sensitive and rapid ELISA test as a tool to establish immunological relations among bacteria.

Bone and articular manifestations of Mixed Connective Tissue Disease

19 patients with mixed connective tissue disease (MCTD) were studied for their bone and articular manifestations by synovial scintigram, bone scintigram and bone X ray pohotogram. All of our 19 patients with MCTD had polyarthritis, Raynaud's phenomenon and swollen hands, and 11 of them had mild myositis. The sera from all patients were tested for the presence of antibody to RNP by ouchterlony technique. All of them were positive. Rheumatoid factor were found in 15 patients (78.9%). But none of them had rheumatoid nodules and articular deformities like swan nek deformity or boutonniere deformity. Abnormal hot area on synovial scintigram was present in 14 (73.7%). Location and duration of this abnormal hot area were nearly correlated to clinical arthritis. Abnormal hot area on bone scintigram was present in 4 (28.6%). It was not clear whether the location of this abnormal hot area was correlated to the location of radiographic abnormalities. Bone and articular radiographs showed abnormalities in 8 (42.1%). Osteoporosis were found in 6, erosions in 5, narrowing of joint spaces in 4, bone resorption of styloid process in 2, bone resorption of terminal phalanx in 1, juxtaarticular linear calcification in 1.In conclusion the articular abnormalities of MCTD were relatively limited, with involvement primarily of the hands and wrists.

Immunological studies on ?-amylase of Bacillus cereus

An antiserum against the β-amylase from Bacillus cereus BQ10-S1 Spo II was prepared using rabbits. The antiserum obtained was confirmed to form a specific immunoprecipitate with the purified β-amylase and showed a single band of protein with a molecular weight of 6.0x104 on the nitrocellulose sheet by the Western-Blotting method. The antiserum showed a precipitin line with the β-amylase from B. megaterium strain no. 32 by the Ouchterlony technique. However, the spur was formed on the Ouchterlony plate between the line of immunoprecipitin of the β-amylase from B. cereus BQ10-S1 Spo II and that from B. megaterium strain no. 32. On the other hand, no immune reaction occurred with the β-amylase from B. polymyxa no. 72 and those from higher plants such as soybean and barley.

A Comparative Study of the Immunologic Properties of Hoof and Nail Fibrous Proteins

We have studied the localization of epidermal (soft) and nail and hoof (hard) fibrous keratins in the various anatomic regions of bovine hoof and human nail. Indirect immunofluorescence was performed on frozen sections of various parts of hoof and nail with antibodies demonstrated to be specific for hard and soft fibrous keratins by the Ouchterlony technique. The antibody to hard fibrous keratin reacted with the upper region of hoof bed and matrix tissue but not perihoof epidermis. The antibody to soft fibrous keratin reacted with hoof bed, matrix tissue, and perihoof epidermis. Electrophoretic analysis of the fibrous proteins of hoof bed indicated they contained both soft and hard fibrous keratins while matrix tissue contained only hard keratins. Immunoblot analysis of matrix fibrous proteins indicated that most of the polypeptides reacted with both antibodies. These results indicate that the antibody to soft fibrous keratin cross-reacts with hard fibrous keratin but the antibody to hard fibrous keratin appears to be specific. Immunologic studies, therefore, must be correlated with electrophoretic studies in order to define the localization of the various types of fibrous keratins. Similar results were obtained with human nail.

Antigenic diversity of cytochromes c3 from the anaerobic, sulfate-reducing bacteria,Desulfovibrio

An indirect enzyme-linked immunoadsorption assay (ELISA) was developed for cytochrome c3 using antisera to the cytochromes fromDesulfovibrio africanus Benghazi, Desulfovibrio vulgaris Hildenborough andDesulfovibrio salexigens British Guiana. The ELISA system was used to test for cross-reactions between these antisera and the heterologous antigens. In contrast to previous experiments using the Ouchterlony technique, all of the cytochromes c3 tested exhibited some degree of cross-reaction. Considerable variation was seen in cross-reactions for cytochromes c3 from differing strains ofD. desulfuricans. This observation raises questions about the taxonomic relatedness of these strains. No cross-reaction was seen with eukaryotic cytochrome c or withD. vulgaris cytochrome c553. The data demonstrate that cytochrome c3 is capable of undergoing nonprecipitating cross-reactions, and thus may not be as immunologically unique as was once thought.

Identification of Human Urine by Immunological Techniques

ABSTRACTA rapid and inexpensive method for the identification of human urine and/or urine stains was developed. Urine could be positively identified by the presence of urinary specific Tamm-Horsfall glycoprotein (THG). Urine and extracts of urine stains were concentrated using a Millipore CX10 ultrafilter prior to analysis using the crossover electrophoresis and the Ouchterlony techniques. Human serum, human semen and sera from 10 different animal species did not contain detectable quantities of the THG. Urine specimens collected from 8 different animal species gave positive reactions with the anti-THG anti-serum. With the exception of monkey urine, urine from several animal species could be differentiated from human urine using the crossover electrophoresis technique.

Homogeneity of cell envelope protein subtypes, lipopolysaccharide serotypes, and biotypes among Haemophilus influenzae type b from patients with meningitis in The Netherlands.

Eighty strains of Haemophilus influenzae type b were randomly selected from 531 strains collected between 1975 and 1982 from patients with meningitis in The Netherlands. Subtyping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 67 of 80 isolates had identical major outer membrane protein patterns (subtype 1). Among the 13 other isolates four different polyacrylamide gel electrophoresis patterns were observed, two of which closely resembled subtype 1. Lipopolysaccharides were characterized immunologically by immunoprecipitation (Ouchterlony technique) and the gel-immuno-radio-assay. Four serotypes were found among the 80 selected strains, leaving one strain not typable. Seventy-four strains (93%) belonged to the same lipopolysaccharide serotype; 77 (97%) of 80 of the strains belonged to biotype I. Sixty strains (75%) had identical major outer membrane protein patterns (subtype 1), lipopolysaccharide serotypes (serotype 1), and biotypes (I).