Double Immunodiffusion Test Research Articles

Do anemonefishes use molecular mimicry to avoid being stung by host anemones?

Anemonefishes are known to have a protective mucous coat that allows them to contact the tentacles of their host anemone without being stung. There are two conflicting hypotheses as to the source and biochemical properties of this mucous coating. One hypothesis proposes that anemonefishes acquire anemone substances from their hosts during the behavioral process of acclimation, that protect the fish from being stung. Anemonefishes are considered to use anemone mucus as “chemical camouflage” or “macromolecular mimicry” to avoid recognition as “not-self” by the anemone, and possible subsequent stinging. Another hypothesis is that anemonefishes produce their own protective mucus coat, which lacks substances that elicit cnida (nematocyst and spirocyst) discharge by their hosts. The present study used immunological techniques to test whether the anemonefish Amphiprion clarkii (Bennett) (which was innately protected from two of the anemones used in the present study) has a mucous coat that resembles the external mucus of anemones. Polyclonal antibodies were prepared to the mucus of four species of anemones [ Heteractis crispa, (Ehrenberg) Stichodactyla haddoni (Saville-Kent), Macrodactyla doreenensis (Quoy and Gaimard), and Condylactis gigantea (Weinland)]. Ouchterlony (double immunodiffusion) tests showed that different antigens were present in the mucus of the four anemone species. Anemone antigens were not detected in the mucous coating of either naive (fish that had never before encountered sea anemones) or associated (those living with sea anemones) anemonefishes in Ouchterlony tests. However, more sensitive ELISA (enzyme-linked immunosorbent assay) tests showed that anemone mucus antigens were present in the mucous coating of associated anemonefish, but not naive fish. This showed that an innately protected A. clarkii does not produce a mucus coat that is biochemically similar to that of anemones, but that the same fish does acquire anemone substances in its mucus coat when it associates with anemones in aquaria. It remains to be shown whether these anemone substances actually provide the initial protection for those anemonefishes that must undergo acclimation behavior in order to keep from being stung by their host sea anemones, or additional protection for innately protected fishes.

Peanut Chlorotic Ringspot Virus (PCRV), a Newly Discovered Virus Infecting Peanut (Arachis hypogaea L.)

AbstractA virus causing wide chlorotic ringspot (PCRV) associated with chlorotic line pattern and motthng on an Erictoides hybrid growing in USDA‐OSU greenhouses, Stillwater, Oklahoma, was discovered. The virus was isolated and characterized and found to differ in symptomology, host range and serological properties from all the previously described viruses infecting peanut, particularly those reported in the United States to be the most important ones, peanut mottle virus, peanut stripe virus, and tomato spotted wilt virus. The virus was transmitted by both mechanical inoctilation and grafting to fourteen peanut cultivars causing identical symptoms to those originally observed on the Erictoides hybrid. In addition to peanut, the virus systemically infected Pisum sativum L. ‘Little marvel’ causing mainly mosaic and Lupinus albus L. ‘Tiftwhite’ producing severe malformation and remarkable reduction in leaflet area. The virus did not infect many other plant species of which cowpea ‘California blackeye’ (Vigna unguiculata L.) and at least five cultivars of soybean (Glydne max L.) are known to be susceptible hosts to peanut mottle virus. Phaseolus vulgaris L. ‘Topcrop’ and Chenopodium amaranticohr Coste & Reyn were found to be two useful local lesion assay and diagnostic hosts for PCRV. The virus elicited necrotic local lesions on the first and chlorotic ringspots on the second. PCRV had a dilution end point between 10−5 and 10−6, thermal inactivation point between 55°C and 60°C, and longevity in vitro up to 6 days but not 7 days. Virus particles viewed hy electron microscopy and the negative stain uranyl aceute were flexuous filamentous particles ranging in length from 750–850 nm. In both indiren PAS‐ELISA and Ouchterlony double immunodiffusion test, PCRV was serologically related to a PMV isolate from Oklahoma (PMV‐OK.) but not to bean yellow mosaic virus, peanut stripe virus, potato virus Y, watermelon mosaic virus 1, watermelon mosaic virus 2, wheat streak mosaic virus, and zucchini yellow mosaic virus.

DEVELOPMENT OF A QUANTITATIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR VITELLIN AND VITELLOGENIN OF THE BLUE CRAB CALLINECTES SAPIDUS

ABSTRACT Vitellin (Vn) and vitellogenin (Vg) were detected in the blue crab Callinectes sapidus by nondissociating polyacrylamide gel electrophoresis (PAGE). Vn was purified by gel filtration chromatography followed by PAGE, and anti-Vn immune serum was raised in rabbits. A slot blot analysis indicated that anti-Vn immune serum reacted with the ovarian homogenate from which Vn was isolated. In a double immunodiffusion test, single precipitin lines were found between the center well containing immune serum and surrounding wells containing purified Vn, ovarian homogenate, purified Vg, or female hemolymph. The precipitin lines were continuous, suggesting that the immune serum precipitated proteins of complete identity. Finally, a Western blot analysis revealed that immune serum specifically bound Vn and Vg. The IgG fraction was purified from anti-Vn immune serum and used to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The Vn standard curve was linear over the range of 62.5-1,500 ng Vn. The sensitivity of the ELISA was 148 ng/ml. Male hemolymph yielded background optical density values, confirming the specificity of the assay. Serial dilutions of ovarian homogenate or female hemolymph produced sample titration curves that were parallel to the Vn standard curve. Thus, the ELISA is suitable for quantification of Vn and Vg in ovary and hemolymph, respectively.

Immunochemical analysis of chloroplast polypeptides from maritime pine

Chloroplast polypeptides have been purified to electrophoretic homogeneity from maritime pine cotyledons ( Pinus pinaster aiton). Antibodies against the light harvesting chlorophyll a-b binding protein (LHCP) located in the thylakoid membrane and the stromatic ribulose bisphosphate carboxylase/oxygenase (Rubisco holoenzyme) were raised in rabbits by injecting pure preparations. Monospecificity of antibodies was determined by a double immunodiffusion test and further purification was achieved by immunoselection of IgGs with purified proteins immobilized on nitrocellulose membranes. Antisera were used to determine steady-state levels of polypeptides during plant development by slot-blot quantitation. Our data showed that developing Pinus pinaster seedlings accumulated chloroplast proteins in the dark, but no such accumulation was observed in dark-grown Ginkgo biloba seedlings.

Glucomannan from Candida utilis. Structural investigation.

Structure of the glucomannan isolated from the cell walls of Candida utilis has been investigated using acetolysis fragmentation, methylation analysis and NMR spectroscopy. The structure of the glucomannan resembles that of the cellular mannans of other Candida species, except that the longer tetra- and pentassacharide side-chains are terminated with a glucosyl residue. Presence of the nonreducing glucosyl groups at the ends of the side-chains caused the C. utilis not to cross-react in a double immunodiffusion test with other Candida species that possess mannan antigens and cross-react with Hansenula species with glucomannan antigens.

The presence of concanavalin A and canatoxin in Canavalia ensiformis DC tissue culture

Isolated embryos, cotyledons and embryos plusa fragment of cotyledon from seeds of Canavalia ensiformis (jack bean) were cultured in vitro. Concanavalin A and canatoxin cross-reactive material were detected by double immunodiffusion tests. Canatoxin was detectable until 30 days in cultures of embryos, embryos plus cotyledons and hypocotyls. Concanavalin A was also present in all cultures being detected until 90 days in cultures treated with 6-benzylaminopurine. No concanavalin A was detected in root cultures. Concanavalin A was present in cell suspensions until 45 days of culture; the culture medium contained neither concanavalin A nor canatoxin. Tissue cultures thus can produce Con A and CNTX and will be an important research tool for studying the biosynthesis of such substances.

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.

Detection and quantitation of antibodies against Rhizopus by enzyme-linked immunosorbent assay.

In sawmills high concentrations of spores from the mould Rhizopus microsporus may occur, causing allergic alveolitis in exposed workers. Both symptomatic and asymptomatic exposed workers may develop antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Rhizopus has been developed in order to study the relationship between antibody levels and exposure levels. The precision of the measurements of Rhizopus antibodies by ELISA carried out on the same microtiter plate was estimated to be 11%. It is therefore possible to detect changes in antibody levels of approximately 25% or more. Antibodies were studied longitudinally by ELISA in 60 wood trimmers. The observed changes in antibody levels exceeded the precision of the ELISA method substantially, indicating significant variability in antibody levels in wood trimmers. The ELISA test was compared with the double immunodiffusion test (DID). Sera from 67 wood trimmers were analyzed by both methods. Antibodies were detected by ELISA in 70% and by DID in 28% of the workers in this group, clearly demonstrating that the ELISA test is the most sensitive method for the detection of antibodies to Rhizopus.

Monoclonal antibodies to rat liver microsomal cytochrome b5

Hybridomas obtained by the fusion of spleen cells from rat cytochrome b 5-immunized mice with mouse myeloma cells produced five groups of monoclonal antibodies (MAbs) with three mouse immunoglobulin subtypes: IgG1, IgG2b and IgM. All of the MAbs bound strongly to rat cytochrome b 5 as measured by radioimmunoassay (RIA). Four clones of MAbs were also strongly immunoreactive with cytochrome b 5 when tested by Western blotting, but only one of the MAbs (1-39-2) weakly immunoprecipitated cytochrome b 5 in an Ouchterlony double-immunodiffusion test. Two of the MAbs partially inhibited cytochrome b 5-mediated NADH cytochrome c reduction catalyzed by liver microsomes (24–36%). Expression of immunodetectable cytochrome b 5 was highest in the liver, next highest in the kidney, and quite low in the other tissues examined with MAb 1-17-1 by Western blotting. This MAb recognized homologous cytochrome b 5 of human liver microsomes and in homogenates of TK − cells infected with recombinant vaccinia virus encoding human cytochrome b 5. These MAbs to cytochrome b 5 will be useful for the identification, quantification, and purification of cytochrome b 5 from animal and human tissues, and for understanding its role in cytochrome P450 catalyzed drug metabolism and carcinogen activation with respect to tissue, organ and individual differences.

Isolation and immunological characterization of three highly purified serine proteinases from Antarctic krill (Euphausia superba)

Three individual serine proteinases (I, II, III) originating from Antarctic krill (E. superba) were separated and highly purified using a combination of affinity and high resolution ion exchange chromatography. Each enzyme showed a single protein band (30 000 Daltons) in sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis indicating high purity and identical molecular weights. Moreover, each enzyme demonstrated one immunoprecipitate on crossed immunoelectrophoresis (two-dimensional agarose gel electrophoresis) using polyclonal rabbit antibodies which confirmed the high purity of the individual enzymes. A mixture of the three enzymes (I, II, III) revealed two immunoprecipitates, not one or three which should have been the case for identical or non-identical immunological properties. Double immunodiffusion test according to Ouchterlony exhibited immunological identity between enzyme II and III. Enzyme I showed only partial identity with II/III. These findings correlated well with biochemical data on the three serine proteinases. Enzyme I is able to liberate free amino acids from polypeptides in comparison with enzyme II and III (classical true endopeptidases), which do not. We suggest that these unique biochemical properties also have their immunological counterpart expressed as other antigenic determinants of the molecular structure.

Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.

Purification and Characterization of a Bovine Pregnancy-Associated Glycoprotein1

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).

Sensitive immunoradiometric assay for the detection of Paracoccidioides brasiliensis antigens in human sera

In the present study we report the standardization of an immunoradiometric assay (IRMA) for detection of Paracoccidioides brasiliensis circulating antigens that could be useful in the diagnosis and prognosis of paracoccidioidomycosis. For this purpose we studied the reactivities of P. brasiliensis and other mycotic antigens with rabbit polyclonal anti-P. brasiliensis antibodies (immunoglobulin G) in order to evaluate the sensitivity and specificity of an IRMA for detecting P. brasiliensis antigens. The results were compared with those obtained by the double immunodiffusion test, the standard technique for the serodiagnosis of paracoccidioidomycosis. By using the immunoglobulin G fraction of rabbit antisera (900 ng per well), it was possible to detect up to 3.6 ng (0.12 micrograms/ml) of cellular antigen and 360 ng (12 micrograms/ml) of metabolic antigen in contrast to the double immunodiffusion test that could detect only 12 micrograms (1.2 mg/ml) of both antigens. IRMA was shown to be feasible and very sensitive and may therefore help, together with clinical data, in establishing early diagnosis and assessing disease activity. It could also allow the study of relationships between P. brasiliensis circulating antigens and host defense mechanisms during the disease.

The use of synthetic O-antigens ofSalmonellafor improvement of serological diagnosis of enteric infections

Synthetic Salmonella O-antigens of a new copolymeric type, unlike natural antigens (lipopolysaccharides), exhibit monospecificity for groupspecific factors 0 : 3, 0 : 4, and 0 : 9 of Salmonella serogroups E, B, and D, and are, by 1-2 orders of magnitude, more sensitive in double immunodiffusion and passive hemagglutination inhibition tests. The use of the synthetic antigens for detection of antibodies in patients' sera by means of passive hemagglutination increased the specificity of this test considerably, thereby improving immunodiagnosis of salmonellosis.

The use of synthetic O-antigens of Salmonella for improvement of serological diagnosis of enteric infections

Synthetic Salmonella O-antigens of a new copolymeric type, unlike natural antigens (lipopolysaccharides), exhibit monospecificity for groupspecific factors 0:3, 0:4, and 0:9 of Salmonella serogroups E, B, and D, and are, by 1–2 orders of magnitude, more sensitive in double immunodiffusion and passive hemagglutination inhibition tests. The use of the synthetic antigens for detection of antibodies in patients' sera by means of passive hemagglutination increased the specificity of this test considerably, thereby improving immunodiagnosis of salmonellosis.

Isolation of Cysteine Protease in the Crude Mite Extract, <i>Dermatophagoides farina</i><i>e</i>

In order to study the relationship between cysteine protease and Der f I, which is one of the major allergens in the mite, Dermatophagoides farinae, isolation of cysteine protease was attempted using various column chromatographies. Both the potent cysteine protease activity and the allergenic activity were detected in the same fractions by anion exchange chromatography on a DEAE-Sephacel, gel chromatographies and chelating Sepharose 6B chromatography. In the double immunodiffusion test, the finally isolated fraction and rabbit anti-Der f I sera reacted to give a single precipitation line which fused completely with the precipitation line formed by Der f I and anti-Der f I sera. Sequence analysis for the first 10 N-terminal amino acids from cysteine protease and Der f I were identical. These results strongly suggest that cysteine protease of mites may be Der f I allergen and that measuring cysteine protease activity may possibly become a beneficial method for detecting Der f I allergens.

Mechanism of antigenic variation in an individual epitope on influenza virus N9 neuraminidase

Monoclonal antibodies which inhibit influenza virus neuraminidase (NA) and which therefore indirectly neutralize virus infectivity bind to epitopes located on the rim of the active-site crater. The three-dimensional structure of one of these epitopes, recognized by monoclonal antibody NC41, has previously been determined (W. R. Tulip, J. N. Varghese, R. G. Webster, G. M. Air, W. G. Laver, and P. M. Colman, Cold Spring Harbor Symp. Quant. Biol. 54:257-263, 1989). Nineteen escape mutants of influenza virus A/tern/Australia/G70c/75 (N9) NA selected with NC41 were sequenced. A surprising restriction was seen in the sequence changes involved. Ten mutants had a Ser-to-Phe change at amino acid 372, and six others had mutations at position 367. No escape mutants with changes at 369 or 370 were found, although these mutations were selected with other antibodies and rendered the epitope unrecognizable by antibody NC41. Another N9 NA, from A/ruddy turnstone/NJ/85, which differs by 14 amino acids from the tern virus NA, still bound antibody NC41. Epitope mapping by selecting multiple escape mutants with antibody NC41 thus identified only three of the five polypeptide loops on NA that contact the antibody. Escape mutants selected sequentially with three different monoclonal antibodies showed three sequence changes in two loops of the NC41 epitope. The multiple mutants were indistinguishable from wild-type virus by using polyclonal rabbit antiserum in double immunodiffusion tests, but NA inhibition titers were fourfold lower. The results suggest that although the NC41 epitope contains 22 amino acids, only a few of these are so critical to the interaction with antibody that a single sequence change allows selection of an escape mutant. In that case, the variety of amino acid sequence changes which can lead to polyclonal selection of new epidemic viruses during antigenic drift might be very limited.

Molecular characterization of a new hemagglutinin, subtype H14, of influenza a virus

Two influenza A viruses whose hemagglutinin (HA) did not react with any of the reference antisera for the 13 recognized HA subtypes were isolated from mallard ducks in the USSR. Antigenic analysis by hemagglutination inhibition and double immunodiffusion tests showed that the HAs of these viruses are similar to each other but distinct from the HAs of other influenza A viruses. Nucleotide sequence analysis showed that these HA genes differ from each other by only 21 nucleotides. However, they differ from all other HA subtypes at the amino acid level by at least 31 % in HAI. Thus, we propose that the HAs of these viruses [A/Mallard/Gurjev/263/82 (H14N5) and A/Mallard/Gurjev/244/82 (H14N6)] belong to a previously unrecognized subtype, and are designated H14. Unlike any other HAs of influenza viruses, the H14 HAs contained lysine at the cleavage site between HA1 and HA2 instead of arginine. Experimental infection of domestic poultry and ferrets with A/Mallard/Gurjev/263/82 (H14N5) showed that the virus is avirulent for these animals. Based on comparative sequence analysis of different HA genes, it is suggested that differences of 30% or more at the amino acid level in HA1 constitute separate subtypes. Phylogenetic analysis of representatives of each HA subtype showed that H 14 is one of the most recently diverged lineages while H8 and H 12 branched off early during the evolution of the HA subtypes. These latter two subtypes (H8 and H12) have been isolated very infrequently in recent years, suggesting that these old subtypes may be disappearing from the influenza reservoirs in nature.

Black‐pigmented, asaccharolytic Bacteroides species resembling Porphyromonas gingivalis (Bacteroides gingivalis) from beagle dogs

Black-pigmented, asaccharolytic Bacteroides strains, which positively reacted with anti-Bacteroides gingivalis (now reclassified as Porphyromonas gingivalis) serum, were isolated from beagle dogs, and their characteristics were studied and compared with those of P. gingivalis. The strains from dogs were different from P. gingivalis in their catalase activity, nutritional requirements and oxygen tolerance, and double-immunodiffusion tests showed serological dissimilarity between the strains from dogs and P. gingivalis. However, the strains from dogs had guanine-plus-cytosine contents of 49.0 to 49.3 mol %, which were very similar to P. gingivalis, and they showed strong DNA-DNA hybridization with P. gingivalis. These results indicated that beagle dogs had Porphyromonas species analogous to P. gingivalis in their oral cavities.

Obtenção de exoantígenos de Histoplasma capsulatum em meio de neopeptona, glicose, tiamina e asparagina (NGTA)

O presente trabalho teve como objetivo a produção de exoantígenos H e M das amostras 58, B-679, A-811 e O187 de Histoplasma capsulatum, utilizando o meio NGTA (neopeptona, glicose, tiamina e asparagina) em períodos de cultivo de 1, 2 e 3 meses, a 36ºC, sob agitação constante (50 v.p.m.). Os antígenos brutos foram avaliados contra anti-soro e antígeno de Histoplasma capsulatum de referência (Center for Disease Control), 4 soros de pacientes portadores de paracoccidioidomicose, 7 de histoplasmose e soro hiperimune anti-H. capsulatum produzido em coelhos, através da reação de imunodifusão dupla. Verificou-se que, com exceção de B-679 com 1 mês de crescimento, todos os demais exoantígenos apresentaram as frações H e M de precipitação. Os exoantígenos obtidos de A-811 apresentaram só a banda H. Excetuando-se os exoantígenos 58 e B-679 com 1 mês de crescimento, todos os demais exoantígenos reagiram contra soros de pacientes com histoplasmose. Em relação aos soros de pacientes com paracoccidioidomicose, somente os exoantígenos 58 e O187 não apresentaram reação cruzada. Todos os exoantígenos reagiram frente ao soro hiperimune de coelho anti-H. capsulatum. Para obtenção de exoantígenos de H. capsulatum, sugerimos que as amostras sejam cultivadas sob as condições anteriormente descritas, adotando-se o período de 3 meses de crescimento, utilizando-se exoantígenos de referência como controles da reação.