Coca's Solution Research Articles

Formulating preservatives to enhance stability of crude extracts of food allergens used for food allergy management and immunotherapy

<abstract> <p>Due to rapid degradation of food allergenic extracts, devising some optimal conditions is mandatory to boost shelf life of extracts for appropriate diagnosis of allergy and immunotherapy. In the current study, food extracts of <italic>Bos domesticus</italic> (cow) milk, <italic>Gallus domesticus</italic> (chicken) egg, <italic>Trititcum aestivum</italic> (wheat), <italic>Gallus domesticus</italic> (chicken) meat and <italic>Arachis hypogaea</italic> (peanut) were prepared and their protein estimation was evaluated by bicinchoninic acid (BCA) method. Stability of food extracts was evaluated under two groups of preservatives; storage with coca's solution (mixture of sodium chloride (NaCl) and sodium bicarbonate (NaHCO<sub>3</sub>)) and stabilizing buffer (sucrose and ethylene diamine tetra-acetic acid (EDTA)). Effect of cocktail of protease inhibitors ((phenyl methyl sulfonyl fluoride (PMSF) and dithiothreitol (DTT)) and glycerol was checked on food extracts under both groups of preservatives. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) was performed for food protein extracts after 1, 3 and 6 months under both conditions. Size of obtained protein bands were compared with allergen database (<ext-link ext-link-type="uri" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.allergen.org">http://www.allergen.org</ext-link>) in order to check their potential allergenicity. Extracts stored with coca's solution remained more potent as compared to stabilizing buffer for up to 3 months except <italic>Gallus domesticus</italic> egg, whose proteins remained more potent in stabilizing buffer as compared to coca's solution. Most of the proteins deteriorated at 6 months of storage. Glycerol had shown better results under both conditions. A formulation containing a combination of coca's solution along with cocktail of protease inhibitors and glycerol improved the shelf life of food extracts for up to 3 months, showing better potential for stabilization of allergenic food extracts and their use for immunotherapy.</p> </abstract>

Identification of wheat sensitization using an in-house wheat extract in Coca-10% alcohol solution in children with wheat anaphylaxis.

Identification of wheat sensitization by a skin prick test (SPT) is essential for children with wheat-induced anaphylaxis, since oral food challenge can cause serious adverse effects. Wheat allergens are both water/salt and alcohol soluble. The preparation of wheat extract for SPT containing both water/salt and alcohol soluble allergen is needed. To determine if a wheat extract using Coca's solution containing 10% alcohol (Coca-10% EtOH), prepared in-house, contians both water/salt and alcohol soluble allergens. Serum of children with a history of anaphylaxis after wheat ingestion was used. Wheat flour was extracted in Coca-10% alcohol solution. An SPT with both commercial and in-house wheat extracts was performed as well as specific IgE (sIgE) for wheat and omega-5 gliadin. Direct and IgE inhibition immunoblots were performed to determine serum sIgE levels against water/salt as well as alcohol soluble (gliadins and glutenins) allergens in the extracts. Six children with history of wheat anaphylaxis had positive SPT to both commercial and in-house extracts. They also had different levels of sIgE against wheat and omega-5 gliadin allergens. The results of direct immunoblotting showed all tested sera had sIgE bound to ~35 kDa wheat protein. Further IgE inhibition immunoblotting identified the ~35 kDa wheat protein as gliadin but not gluten allergen. The in-house prepared Coca-10% EtOH solution could extract both water/salt and alcohol soluble allergens. The ~35 kDa gliadin appears to be a major wheat allergen among tested individuals.

Comparison of Three Methods of Protein Extraction from Dermatophagoides Pteronyssinus for Two-dimensional Electrophoresis

To explore an effective method of Dermatophagoides pteronyssinus protein extraction suitable for two-dimensional electrophoresis (2-DE) analysis. The extracts of Dermatophagoides pteronyssinus were prepared with Coca's solution, lysis buffer of 2-DE, and Trizol reagent, respectively. Bicinchoninic acid (BCA) assay was used to determine the total protein concentration of the samples. The efficiency of different protein extraction methods were evaluated with 2-DE analysis. The concentrations of extracted protein by methods of Coca's solution, lysis buffer, and Trizol reagent were 0.63 g/L, 0.90 g/L, and 0.80 g/L, respectively. The 2-DE analysis results showed that some protein spots in low molecular weight (LMW) range could be detected with the Coca's solution method. With the lysis buffer of 2-DE method, more protein spots in LMW range could be detected, while the medium molecular weight (MMW) protein spots were absent. Several MMW protein spots (174-178 kD and 133 kD) and more LMW protein spots were detected with Trizol reagent method. Among Coca's solution, lysis buffer of 2-DE, and Trizol reagent, the concentration of extracted protein of Dermatophagoides pteronyssinus by lysis buffer of 2-DE is the highest. However, most protein components of Dermatophagoides pteronyssinus purified mite bodies can be extracted by Trizol reagent, which may generally reflect the whole profile of Dermatophagoides pteronyssinus allergens.

Antigenic and allergenic changes during storage of a pollen extract

The changes that occur in the antigens and allergens of a timothy grass pollen extract during storage were measured by radioallergosorbent test (RAST) inhibition, isoelectric focusing, (IEF), crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a dye-binding protein assay. Storage intervals varied from 7 to 92 days at temperatures of 4 °, 22 °, and 35 °C and the freeze-dried extract was rehydrated and stored in either phosphate-buffered saline with 50% glycerol (PBSG) or Coca's solution (CS). For extracts stored in CS at 35 °C, allergenic activity as measured by RAST inhibition dropped by almost one half at 7 days and certain of the IEF, CIE, and CRIE bands were no longer visible, showing that these properties correlated with allergenic activity. After 65 days, no IEF bands were visible but three CIE bands could be visualized. At 22 ° the changes were slower and at 4 °C no changes were observed for the 92-day storage time. Extracts stored in PBSG showed some of the same changes but were much more stable at all temperatures. During storage, the protein content as measured by dye binding was also reduced, showing that proteins were fragmenting. Because some of the allergenic proteins disappeared more rapidly than others, there was not only a loss in total allergenic activity but also a change in specificity. This raises serious questions concerning the use of such extracts, especially for diagnosis.

Immunotherapy in Bee-Sting Anaphylaxis

After nine months of immunotherapy with commercially prepared wholebody bee extract, a beekeeper's wife experienced anaphylaxis after a controlled bee-sting challenge. High risk of future bee stings prompted a decision to attempt desensitization with honeybee venom. This was extracted from the bee into Coca solution and administered parenterally in increasing daily doses until an equivalent of one venom sac was given per day. This maintenance dosage was continued for a month. Anaphylaxis did not occur after another bee-sting challenge. Therapy has been maintained with venom. With this treatment, there has been an increase in the level of IgG-blocking antibody and a fall in IgE titer to bee venom (radioallergosorbent test [RAST]).

Hypersensitivity to parasite proteolytic enzyme in schistosomiasis.

A proteolytic enzyme which hydrolyses hemoglobin was obtained from the supernatant fraction of homogenized Schistosoma mansoni. This enzyme elicited histaminic skin reactions in various animals, including man, which were infected with S. mansoni. It failed to induce reactions in monkeys harboring S. haematobium, S. japonicum, or S. intercalatum. In a preliminary field trial in the Caribbean, the skin test proved to be somewhat less sensitive than the customarily used extract of adult worms in Coca's solution. However, the enzyme appeared to induce fewer false positive reactions and delayed responses than did the Coca's extract. A new diagnostic test for schistosomiasis probably could be developed by using specific parasite enzymes against which the host has become sensitized in the course of infection.

Measurement of ragweed antigen E by double antibody radioimmunoassay

Antigen E (AgE), the principal allergen of short ragweed pollen, has previously been quantitated by histamine release from sensitized leukocytes or by immunodiffusion. To measure AgE activity a double antibody radioimmunoassay (RIA) was developed. With this assay as little as 7 ng. of AgE was measurable, and by logit transformation of inhibition curves AgE activity in various allergy extracts could be compared immunologically. AgE reactivity was detected in extracts of several Ambrosia and Franseria species, but only AgE from short ragweed produced inhibition curves parallel to AgE IV-C. With giant ragweed this cross-reactivity was confirmed with immunodiffusion analysis. Certain lots of extracts from botanically unrelated pollens contained small amounts of AgE with inhibition curves parallel to AgE from short ragweed. No dialyzable AgE activity was found in freshly prepared short ragweed extracts. Extraction of short ragweed pollen with 50. per cent glycerin yielded extracts with greater AgE content than extraction with Coca's solution. AgE activity from short ragweed survived freezing and thawing (10 times) and was stable for months at 4 °C. in concentrated aqueous solutions. The quantities of AgE coupled to solidphase polymers used in the radioallergosorbent test could also be estimated with RIA.

Hymenoptera sensitivity: I. Conjunctival testing in insect allergy

Conjunctival testing to a whole body mixture of bee, wasp, hornet, and yellow jacket in Coca's solution was carried out in an attempt to develop a simple clinical test which would be useful in verifying a history suggestive of stinging insect allergy. Approximately 60 per cent of 52 insect-sensitive patients undergoing hyposensitization therapy demonstrated positive reactions; whereas only 8 per cent of control subjects reacted similarly. This suggests that false positive conjunctival reactivity to Hymenoptera is unlikely, but that clinically significant numbers of patients will be falsely negative. Further implications are discussed.

EVALUATION OF HOUSE DUST ANTIGEN IN CHRONIC TONSILLITIS

To disclose the association of house dust antigen with chronic tonsillitis, studies were made on the procedure of preparing house dust extract and on its properties as an antigen. The association was confirmed by clinical, investigation of intradermal reaction conducted in cases of chronic tonsillitis along with diseases of the nose and sinuses.Extract prepared from home dust in Japanese houses contains undoubtedly an antigenic substance and intradermal reaction following to injection of the extract results from antigen-antibody reaction. Since the skin reaction may be caused also by non-specific irritant contained in the extract, the reaction due to the antigenic substance is hardly differentiated by making use of Solution No. 1 and No. 2 (containing 0.1mg and 0.02mg of nitrogen, respectively). Tne sensitization with house dust should be determined by making use of Solution No. 3 which contains only 0.01mg of nitrogen.Nitrogen content in the house dust extract may be regarded as an index of antigen titer.The extract is preserved well with little decrease in potency for a considerable period of time at room temperature, so long as it was sealed in ampule, following to the extraction from Coca's alkaline solution and neutralization with phenolphthalein as an indicator.In cases of chronic tonsillitis complicated with focal infection, the rate of positive reactors to the extract averaged 59.0 per cent, while those without complications gave a positive rate of 42.5 per cent. The difference was proved evident. As to the complications of focal infection, the skin reaction was positive in 63.6 per cent of chronic cases of tonsillitis with rheumatism and in 58.8per cent of those with nephritis. The same result was obtained by employing extract of higher dilution.As to diseases of the nose and sinuses, the rates of positive intradermal reaction in allergic rhinitis, chronic sinusitis and chronic rhinitis were 55.0 per cent, 37.3 per cent, respectively.Positive rate of skin reaction before and after tonsillectomy differed little from each other.Mechanism of focal infection has never beestudied from the standpoint of antigenic substance introduced from the outside. Although home dust antigen which was inhaled through the respiratory passages was found not to be primary causes of focal infection, the present paper suggests some relation at least to manifestation of focal infection, and elucidation of the particular relationship depends much upon further studies.

Allergenic food extracts. I. Methods of preparation: Results of a questionnaire

The majority of allergists making their own food extracts used fresh uncooked food as the source of their raw material. The food was then ground or cut into smaller particles. The majority did not defat fruits and vegetables, but did defat meats, fish, nuts, grains, and spices. The majority used a weight-by-volume method of preparing their extracts and made dilutions of same for intracutaneous testing. Either alkaline extracting fluid (Coca's solution), glycerin saline, or buffered saline was used by the allergists in general for extracting purposes. Almost one third (forty-eight) of the allergists did not concentrate food extracts. Among these were twenty-three who used 50 per cent glycerin as an extracting fluid, which rendered it difficult to concentrate. Nearly all physicians sterilized their extracts by filtration through a Seitz type of pad. Several allergists did no sterility tests subsequent to Seitz filtration. Twenty-nine physicians incubated for intervals up to three days. Three-fourths (seventy-one) of the physicians reporting did not check the pH of the extract before or after Seitz infiltration. Most of these physicians did not dialyze the extract for pH adjustment. A weight-by-volume method of standardization for food extracts was used by a majority (sixty-nine) of the allergists. There were variations in procedure in that some used a dry weight and others a wet weight as the basis for the weight-by-volume method of standardization. A few concentrated their extracts, but most did not. The strength of the testing solution showed wide variations, even among persons using the same method of extraction and standardization. The numerous variables that have been presented are not being subjected to critical analysis. Further investigation is necessary to determine the effect of each of these variables upon the allergenicity of the final extract.

The complement-fixation test in Bilharziasis II. Preparation and preservation of antigens from Schistosoma mansoni worms extracted in Coca's solution

1. Adult S. mansoni worms were extracted in Coca's solution at various temperatures by different methods. The most potent antigen was obtained by extracting a worm suspension, diluted 1:500, at 37° C. for 1 hr., with vigorous shaking at 10 min. intervals, followed by 23 hr. at 28° C. Second extraction of worm samples gave highly active material and third extractions also contained antigen.2. Complement-fixation tests, performed with the sera of twenty-five patients with chronic bilharziasis and twenty-five healthy subjects showed that the most specific results were obtained with either the first two extracts of worms, suspended in 100 parts of Coca's solution, and extracted for 1 hr. at 37° C. and 23 hr. at 28° C., or with the first extract of worms, suspended in 500 parts and extracted in the same manner.3. Samples of an extract in Coca's solution were preserved for 170 days by different methods, and their activity was tested at approximately monthly intervals. The antigen, with or without the addition of 0·1% cysteine, retained its titre for 1 month, when kept in the refrigerator. Freezing, or lyophilization of the extract caused a rapid drop in titre.4. Titration of extracts of lyophilized worms, from freshly opened ampoules, showed that a constant titre was retained for 3½ months followed by a slight drop after 5–6 months. The worm powder retained its antigenic potency when kept, thoroughly desiccated, in the refrigerator for 4 months, but lost it in 40 days when partial rehydration occurred.

The complement-fixation test in bilharziasis. I. The value of different extracts of Schistosoma mansoni and Fasciola hepatica worms as antigens.

1. Different extracts of adult worms of S. mansoni and F. hepatica have been examined in the C.F.T. for bilharziasis. Extracts of S. mansoni worms in Coca's solution, alcohol-ether and absolute alcohol after acetone extraction possess high antigenic activity, the first being the most specific but less sensitive than the others. Extracts in Coca's solution of alcohol-ether insoluble residues still showed slight activity while acetone and polysaccharide (formamide) extracts showed none.2. Using the Coca's extract of S. mansoni worms, the C.F.T. was positive in 83% of sera of untreated patients and 58% of treated. The figures using the alcohol-ether extract were 13 and 4% respectively and, for the alcoholic extract of acetone insoluble residue, 42 and 13%.3. Extracts of F. hepatica worms showed the same general trend, but even in the two extracts to show activity (alcohol-ether and alcohol extract of acetone insoluble material) the titre was too low for use in the test.4. Of rabbits immunized with three different fractions of S. mansoni worms only that given the Coca extract showed complement-fixing antibodies and then only with the homologous antigen. Skin tests on the rabbits were negative to each of the antigens used.5. The nature of the antigen in the C.F.T. is discussed, and it is suggested that the active portion is lipo-protein in nature or more than one substance is involved. For use in clinical diagnosis, an extract of S. mansoni worms in Coca's solution is recommended.Our grateful thanks are due to Mr O. D. Standen of the Wellcome Laboratories of Tropical Medicine, London, for his generous gift of worm powder, and to Professor G. Witenberg of the Hebrew University for his interest in our work.

Relation of pH of allergenic extracts to storage and skin reactivity

1. 1. Samples of a variety of stock allergenic extracts were stored in the refrigerator and tested for changes in pH over a period of six months. On the average, extracts prepared with buffered saline or Coca's solution exhibited the greatest pH stability, while those prepared with sulfoxylate showed a general decrease in pH. However, no samples were found to change beyond the limits of antigenic activity. 2. 2. Samples of dilute ragweed extract were adjusted in pH to a variety of values and then stored at both room and refrigerator temperatures. Changes in pH were observed over a period of six months. The effect of temperature on the change of pH was not significant. 3. 3. Changes in pH of ragweed extract had little effect on the skin reactivity of either allergic or nonallergic patients.

Preparation and Testing of a Specific Antigen for Diagnosis of Human Strongyloidiasis 1

Summary and Conclusions A test antigen prepared from clean filariform larve of Strongyloides cultured from the feces of the chimpanzee and extracted in isotonic (0.425 per cent) salt solution or Coca's solution, in dilution 1:100 produced 23 positive intradermal reactions and 25 positive precipitin tests in 25 chronic cases of human strongyloidiasis. Additionally there were two doubtful intradermal reactions, one in a moribund patient and one in a patient who had very severe exfoliative dermatitis. The precipitin titers ranged from 1:5,000 to 1:30,000. This antigen produced consistently negative, doubtful or weak intradermal and precipitin tests in 105 of 108 presumably uninfected control cases. The diagnostic significance of the positive intradermal and precipitin reactions has been demonstrated in these cases of Strongyloides infection. The tests provide a reliable index of infection. In cases of presumably cured Strongyloides infection the value of the intradermal and precipitin test is not so clear. It is not known how long after cure the intradermal reaction will remain positive, although it is likely that the precipitin test as carried out indicates the presence of specific antibodies.

PROTAMINE AND ALLERGY

The injection of protamine zinc insulin for the treatment of diabetes is followed in some patients by redness and swelling at the site of injection, sufficiently severe at times to lead them to discontinue the use of protamine zinc insulin. It therefore seemed desirable to investigate the nature of these reactions, especially from the standpoint of a possibly allergic mechanism. We here record our observations as to the causes and report a new observation, the induced sensitization to insulin which occasionally appears to follow the use of protamine zinc insulin. <h3>SENSITIVITY STUDIES</h3> The first part of this paper deals with the results of intracutaneous tests with protamine. The test material used was a solution of protamine in Coca's solution in a concentration containing 0.1 mg. of nitrogen per cubic centimeter. The tests were performed by the intracutaneous injection of 0.02 cc. of the test solution on the flexor surface of