THE DIAGNOSIS OF ALLERGY FROM CLADOSPORIUM HERBARUM, CLADOSPORIUM CLADOSPORIOIDES AND ALTERNARIA ALTERNATA MOLDS

TECHNICAL FIELD

The present invention relates to the test material and production method for use in the skin prick test in the diagnosis of mold allergy, produced from Cladosporium herbarum, Cladosporium cladosporioides and Alternaria alternata molds isolated from the outdoor atmosphere of Adana region and put into use as total cell extraction.

PRIOR ART

The test materials used in the diagnosis of allergies are fully imported products, and the molds used in the production of test materials are the isolations of the countries in which the test has been produced. However, because the allergen people are exposed to organisms that adapt to different ecological conditions compared to molds in different countries and even different regions, due to the fact that there are genetic differences between different strains of the same species, they exhibit biochemical and physiological phenotypic diversity such as cell component, enzyme, protein, carbohydrate, toxin, pathogenicity. Due to the genetic variation between the strains, the preparation of the test material to be used in the allergy test from the strains in the regions where people live ( exposure to the strains causing allergic reactions) will give the most accurate result for diagnosis. Therefore, the most important advantage of these products is that producing test material from regional molds, which causes an allergic reaction, gives more accurate and precise results in diagnosis.

In addition, since the standardization of ready test kits and production methodologies are under the confidentiality protection of the manufacturers, the methodology used in this production process will be a special production process for our country and the standardization of the allergen in the test material to be produced will be clearly written. Product will be produced as a total cell extract and will be presented for clinical use as a diagnostic kit. The subject of which component of the ready kits and molds are prepared is under the confidentiality protection of the companies. LIST OF FIGURES

Figure 1 . Standard Protein Curve

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the test material and production method for use in the diagnosis of allergy, produced from Cladosporium herbarum, Cladosporium cladosporioides and Alternaria alternata molds isolated from the outer atmosphere (outdoor) of Adana region and put into use as total cell extraction.

Isolated from Adana outer atmosphere (outdoor) and incubated for 14 days at 25°C by cultivation into PDA medium from stock cultures of fungal strains identified as Cladosporium and Alternaria according to their microscopic, colony morphology, biochemical and physiological properties. A small amount of scraping has been carried out to obtain a single colony from the spores animated in PDA medium, added to 1 mL of pure water and then inoculated to 200 pL of PDA medium and spread and incubated at 25°C for 14 days. As a result, single fallen colonies have been selected, inoculated into PDA medium and left for incubation at 25°C for 14 days.

The culture has been inoculated into 500 mL of Czapek Dox liquid medium and produced at 25°C in a shaker incubator for 14 days. The mycelia obtained at the end of this period has been centrifuged at 6500 rpm and precipitated at +4°C. Since the micelles have not been fully adhered to the bottom of the centrifugal tubes, they have been once filtered from the coarse filter paper and collected. The collected micelles have been closed in a bag made of filter paper and allowed to dry for 1 night at 30°C.

Preparation of Total Fungus Extracts from Cladosporium and Alternaria Strains

The samples, which are cured and removed from the water, are frozen with liquid nitrogen and crushed in the mortar and broken into smaller pieces. For further disintegration, the obtained mycelia have been disintegrated at 20500 cycle/minute with a homogenizer and then by performing 2 repetitions of thirty minutes with a sonicator. The samples that are not fully fragmented are then homogenized with the freeze-thaw (milling &thaw) application.

Based on this method:

- Samples have frozen for 3 min at -80°C,

- quickly resolved at 42°C, - they have been gently exposed to a vortex.

- These steps have been repeated in total 4 times.

Samples are filtered from the coarse filter and frozen at -20°C by adding 100 mL to 250 ml_ of conical flasks for lyophilization preparation and sterilization with a 0,2 pm injector type filter. Frozen samples are lyophilized in the lyophilizer at -82°C for 24 hours at 0,0010 mbar. Lyophilization is the process of freezing and drying of products. As it is an application at low temperatures, it is a drying technique that ensures dehydration without damaging the molecular and physical structure of the product. It is stored at +4°C until it is used for allergy tests.

Determination of the Protein Amount of Extracts

The amount of protein of fungal specimens prepared for total cell extraction has been determined by the Bradford Method. This is done by weighing lyophilized specimens at 0,5 grams, dissolving with 10 mL SF, filtered and sterilizing them with 0,2 pm injector type filter.

Solutions of 10 mg/mL Bovine serum albumin (BSA) solution to produce a standard protein curve according to Bradford method have been prepared with end concentrations of 5 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, 0,1 pg/mL.

For this purpose, 10 mg BSA is dissolved in 10 mL water. The stock BSA solution has been prepared by taking 1 mL of this solution and adding 9 mL of water (3 parallel work, so these quantities are 2 times higher and the final volume is 4 mL). The stock solution is mixed with distilled water at an appropriate rate to provide examples for the standard graph (Chart 1 ). 1 mL sample and 1 mL of Coomassie protein separator (Bradford solution) has been incubated at room temperature for 2 minutes.

Standards have been measured in the spectrophotometer at OD595nm against blank (prepared by placing 1 mL of distilled water in place of protein solution). The value of R2 has been determined by plotting the resulting absorbance values and concentration values and the XY scatter graph (Figure 1).

Chart 1. Preparation of BSA Calibration Graph

After plotting the standard protein curve, the above method has been applied to determine the protein amounts in the mold samples, and the resulting absorbance values have been averaged to be placed in the standard skew formulation, which calculated the protein amounts. Measurements are made in 3 times repetitions.

Column Chromatography

Column chromatography has been performed for each of the strains defined as Cladosporium herbarum, Cladosporium cladosporioides and Alternaria alternata used in this study. Fractionation or distillation is a process that is applied to purify or separate a non-pure liquid or liquid mixture and is based on the difference in boiling points. The “Sephadex G-100” column is used for this purpose. Preparation of Sephadex G-100 Column

It is held for one night for inflation in five grams of Sephadex 50 mL Sodium Phosphate buffer (pH 7.0). At the end of this period, the 10 mL injector has been first placed with a filter sheet on the base and the glass wool has been put on it and the gel has been slowly added with a dropper to prevent some leakage. As many as 50 mL of the buffer column has been passed to stabilize the gel on the first stage when the filling process has been completed and the gel froze.

Fractionation (splitting total cell extracts into different molecular weighted components) Once the column is ready, each sample has been weighed 0,5 grams has been thawed to 10 mL SF, filtered out of the coarse filter, sterilized with a 0,2 pm diameter injector type filter, then 500 pi sample colonized and 5 ml_ fractions have been collected. In this way, 7 tubes have been collected and protein quantities have been measured as absorbance at the spectrophotometer at 280 nm to determine the level of protein in the tubes. The determination of protein amount with the Bradford method has been performed for each fraction, which has been separated by chromatography in 10 min periods.

Determination of Molecular Weight with SDS-PAGE electrophoresis

To determine the molecular weight of samples and fractions, a 10% gel has been prepared and SDS-PAGE is delivered.

Preparing the Test Material Used for Skin PrickTest

The protein content of the total extracts produced from 3 mold strains, which are natural isolates, and their fractions separated by the Sephadex column, has been determined by the Bradford method.

Both total extracts and 5 ml_ Sephadex colonized fractions have been applied to individuals diagnosed with mold allergies in parallel to standard allergens. Lyophilized samples are weighed to 0,5 g and sterilized with a 0,2 ml_ SF (serum physiologic) dissolved, filtered and 10 pm injector type filter. The collected fractions are also sterilized with the filter, all the samples are taken to Eppendorf individually, with 2 ml_ each. 20% Glycerol-Phenol mixture (phenol concentration 0,4%) has been added to protect prepared test materials from microbial contamination.

Preparation of the Glycerol-Phenol Mixture:

First, crystal-form phenol has been liquefied at 80°C in the water bath. After the glycerol-Phenol mixture has been prepared to a ratio of 98:2 (v/v), 500 mI has been added to the 2 mL test material. The shelf life of the test material prepared in this manner, when stored at +4°C, increases with room temperature retention and is available for approximately 18 months. Each sample has been inoculated on to blood agar and sterilized before being put into use.